Fatty Aldehyde Dehydrogenase
2004; Elsevier BV; Volume: 279; Issue: 8 Linguagem: Inglês
10.1074/jbc.m312062200
ISSN1083-351X
AutoresDamien Demozay, Stéphane Rocchi, J.A. Soto Más, Sophie Grillo, Luciano Pirola, Carine Chavey, Emmanuel Van Obberghen,
Tópico(s)Diet and metabolism studies
ResumoPhosphatidylinositol 3-kinase signaling regulates the expression of several genes involved in lipid and glucose homeostasis; deregulation of these genes may contribute to insulin resistance and progression toward type 2 diabetes. By employing RNA arbitrarily primed-PCR to search for novel phosphatidylinositol 3-kinase-regulated genes in response to insulin in isolated rat adipocytes, we identified fatty aldehyde dehydrogenase (FALDH), a key component of the detoxification pathway of aldehydes arising from lipid peroxidation events. Among these latter events are oxidative stresses associated with insulin resistance and diabetes. Upon insulin injection, FALDH mRNA expression increased in rat liver and white adipose tissue and was impaired in two models of insulin-resistant mice, db/db and high fat diet mice. FALDH mRNA levels were 4-fold decreased in streptozotocin-treated rats, suggesting that FALDH deregulation occurs both in hyperinsulinemic insulin-resistant state and hypoinsulinemic type 1 diabetes models. Moreover, insulin treatment increases FALDH activity in hepatocytes, and expression of FALDH was augmented during adipocyte differentiation. Considering the detoxifying role of FALDH, its deregulation in insulin-resistant and type 1 diabetic models may contribute to the lipid-derived oxidative stress. To assess the role of FALDH in the detoxification of oxidized lipid species, we evaluated the production of reactive oxygen species in normal versus FALDH-overexpressing adipocytes. Ectopic expression of FALDH significantly decreased reactive oxygen species production in cells treated by 4-hydroxynonenal, the major lipid peroxidation product, suggesting that FALDH protects against oxidative stress associated with lipid peroxidation. Taken together, our observations illustrate the importance of FALDH in insulin action and its deregulation in states associated with altered insulin signaling. Phosphatidylinositol 3-kinase signaling regulates the expression of several genes involved in lipid and glucose homeostasis; deregulation of these genes may contribute to insulin resistance and progression toward type 2 diabetes. By employing RNA arbitrarily primed-PCR to search for novel phosphatidylinositol 3-kinase-regulated genes in response to insulin in isolated rat adipocytes, we identified fatty aldehyde dehydrogenase (FALDH), a key component of the detoxification pathway of aldehydes arising from lipid peroxidation events. Among these latter events are oxidative stresses associated with insulin resistance and diabetes. Upon insulin injection, FALDH mRNA expression increased in rat liver and white adipose tissue and was impaired in two models of insulin-resistant mice, db/db and high fat diet mice. FALDH mRNA levels were 4-fold decreased in streptozotocin-treated rats, suggesting that FALDH deregulation occurs both in hyperinsulinemic insulin-resistant state and hypoinsulinemic type 1 diabetes models. Moreover, insulin treatment increases FALDH activity in hepatocytes, and expression of FALDH was augmented during adipocyte differentiation. Considering the detoxifying role of FALDH, its deregulation in insulin-resistant and type 1 diabetic models may contribute to the lipid-derived oxidative stress. To assess the role of FALDH in the detoxification of oxidized lipid species, we evaluated the production of reactive oxygen species in normal versus FALDH-overexpressing adipocytes. Ectopic expression of FALDH significantly decreased reactive oxygen species production in cells treated by 4-hydroxynonenal, the major lipid peroxidation product, suggesting that FALDH protects against oxidative stress associated with lipid peroxidation. Taken together, our observations illustrate the importance of FALDH in insulin action and its deregulation in states associated with altered insulin signaling. Phosphatidylinositol 3-kinase (PI3K) 1The abbreviations used are: PI3K, phosphatidylinositol 3-kinase; FAS, fatty-acid synthase; FALDH, fatty aldehyde dehydrogenase; HFD, high fat diet; RAP-PCR, RNA arbitrarily primed PCR; ROS, reactive oxygen species; SREBP-1c, sterol-regulatory element-binding protein 1c; STZ, streptozotocin; S-V, stroma vascular fraction; 4-HNE, 4-hydroxynonenal; PKB, protein kinase B; MGO, methylglyoxal; CMDCFDA, chloromethyl-2′,7′-dichlorofluorescein diacetate; DMEM, Dulbecco's modified Eagle's medium; GFP, green fluorescent protein; Ad, adenovirus; BSA, bovine serum albumin; RT-PCR, reverse transcriptase-PCR; PPARγ, peroxisome proliferator-activated receptor γ; EGF, epidermal growth factor; PBS, phosphate-buffered saline; HPRT, hydroxy-phospho-ribosyl transferase. is a key component of the intracellular insulin signaling machinery. PI3K activation occurs after binding of the Src homology 2 domains of its p85 regulatory subunit to specific tyrosine-phosphorylated sites of the insulin receptor substrates (1Van Obberghen E. Baron V. Delahaye L. Emanuelli B. Filippa N. Giorgetti-Peraldi S. Lebrun P. Mothe-Satney I. Peraldi P. Rocchi S. Sawka-Verhelle D. Tartare-Deckert S. Giudicelli J. Eur J. Clin. Investig. 2001; 31: 966-977Crossref PubMed Scopus (78) Google Scholar, 2White M.F. Curr. Opin. Genet. Dev. 1994; 4: 47-54Crossref PubMed Scopus (92) Google Scholar). By phosphorylating the D3 position of the inositol ring of phosphoinositides, PI3K generates the second messenger phosphatidylinositol 3,4,5-triphosphate (3Alessi D.R. Kozlowski M.T. Weng Q.P. Morrice N. Avruch J. Curr. Biol. 1998; 8: 69-81Abstract Full Text Full Text PDF PubMed Scopus (516) Google Scholar, 4Shepherd P.R. Withers D.J. Siddle K. Biochem. J. 1998; 333: 471-490Crossref PubMed Scopus (838) Google Scholar, 5Kotani K. Ogawa W. Hino Y. Kitamura T. Ueno H. Sano W. Sutherland C. Granner D.K. Kasuga M. J. Biol. Chem. 1999; 274: 21305-21312Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) that participates in the recruitment and/or activation of downstream kinases such as 3-phosphoinositide-dependent protein kinase-1, protein kinase B (PKB), and atypical protein kinases C λ and ζ (3Alessi D.R. Kozlowski M.T. Weng Q.P. Morrice N. Avruch J. Curr. Biol. 1998; 8: 69-81Abstract Full Text Full Text PDF PubMed Scopus (516) Google Scholar, 4Shepherd P.R. Withers D.J. Siddle K. Biochem. J. 1998; 333: 471-490Crossref PubMed Scopus (838) Google Scholar). By activating these kinases, PI3K generates several insulin-dependent actions on metabolism such as glucose transport, glycogen synthesis, glycolysis, and lipogenesis. Moreover, PI3K acts by modulating the expression of a number of genes involved in lipid and glucose homeostasis, including phosphoenolpyruvate carboxykinase (5Kotani K. Ogawa W. Hino Y. Kitamura T. Ueno H. Sano W. Sutherland C. Granner D.K. Kasuga M. J. Biol. Chem. 1999; 274: 21305-21312Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 6Liao J. Barthel A. Nakatani K. Roth R.A. J. Biol. Chem. 1998; 273: 27320-27324Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 7Agati J.M. Yeagley D. Quinn P.G. J. Biol. Chem. 1998; 273: 18751-18759Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) and glucose-6-phosphatase (8Dickens M. Svitek C.A. Culbert A.A. O'Brien R.M. Tavare J.M. J. Biol. Chem. 1998; 273: 20144-20149Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar), two enzymes that control key steps of gluconeogenesis in liver and the expression of which is repressed by insulin via activation of PI3K. In contrast, insulin up-regulates hexokinase-2 in skeletal muscle and glucokinase in liver in a PI3K-dependent manner (9Osawa H. Sutherland C. Robey R.B. Printz R.L. Granner D.K. J. Biol. Chem. 1996; 271: 16690-16694Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 10Foretz M. Guichard C. Ferré P. Foufelle F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12737-12742Crossref PubMed Scopus (594) Google Scholar). PI3K also plays a role in lipogenesis by increasing fatty-acid synthase (FAS) gene expression (11Wang D. Sul H.S. J. Biol. Chem. 1998; 273: 25420-25426Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). These effects in the liver are mediated by the transcription factor sterol-regulatory element-binding protein 1c (SREBP-1c) (10Foretz M. Guichard C. Ferré P. Foufelle F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12737-12742Crossref PubMed Scopus (594) Google Scholar, 12Foretz M. Pacot C. Dugail I. Lemarchand P. Guichard C. Le Liepvre X. Berthelier-Lubrano C. Spiegelman B. Kim J.B. Ferré P. Foufelle F. Mol. Cell. Biol. 1999; 19: 3760-3768Crossref PubMed Scopus (452) Google Scholar). Finally, recent studies suggest that PI3K up-regulates the expression of the glucose transporter Glut-4 in muscle and in brown adipose tissue and of p85α in muscle, suggesting a positive feedback effect of PI3K on its own expression (13Laville M. Auboeuf D. Khalfallah Y. Vega N. Riou J.P. Vidal H. J. Clin. Investig. 1996; 98: 43-49Crossref PubMed Scopus (128) Google Scholar, 14Valverde A.M. Navarro P. Teruel T. Conejo R. Benito M. Lorenzo M. Biochem. J. 1999; 337: 397-405Crossref PubMed Scopus (58) Google Scholar, 15Roques M. Vidal H. J. Biol. Chem. 1999; 274: 34005-34010Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Development of insulin resistance plays an important role in the etiology of type 2 diabetes and reflects the impairment of insulin action at the cellular level. Several studies (16Anai M. Funaki M. Ogihara T. Terasaki J. Inukai K. Katagiri H. Fukushima Y. Yazaki Y. Kikuchi M. Oka Y. Asano T. Diabetes. 1998; 47: 13-23Crossref PubMed Scopus (174) Google Scholar, 17Andreelli F. Laville M. Ducluzeau P.H. Vega N. Vallier P. Khalfallah Y. Riou J.P. Vidal H. Diabetologia. 1999; 42: 358-364Crossref PubMed Scopus (59) Google Scholar, 18Bjornholm M. Kawano Y. Lehtihet M. Zierath J.R. Diabetes. 1997; 46: 524-527Crossref PubMed Scopus (0) Google Scholar, 19Cusi K. Maezono K. Osman A. Pendergrass M. Patti M.E. Pratipanawatr T. DeFronzo R.A. Kahn C.R. Mandarino L.J. J. Clin. Investig. 2000; 105: 311-320Crossref PubMed Scopus (910) Google Scholar) have described a decrease in expression and/or activation of PI3K in response to insulin in different insulin-resistant rodent models as well as in patients with type 2 diabetes. We hypothesized that defects in the regulation of gene expression controlled by PI3K may participate in the pathogenesis of type 2 diabetes and insulin resistance. To identify genes involved in glucose homeostasis, which are specifically controlled by PI3K in response to insulin, we performed RNA arbitrarily primed (RAP)-PCR on freshly isolated rat adipocytes in the presence or absence of insulin and wortmannin, a pharmacological inhibitor of PI3K. By using this approach, we isolated several clones corresponding to both previously identified and novel genes. One RAP-PCR product corresponds to fatty aldehyde dehydrogenase (FALDH), a member of the aldehyde dehydrogenase family that oxidizes aliphatic and aromatic aldehydes to the corresponding carboxylic acids (20Miyauchi K. Masaki R. Taketani S. Yamamoto A. Akayama M. Tashiro Y. J. Biol. Chem. 1991; 266: 19536-19542Abstract Full Text PDF PubMed Google Scholar). These enzymes are considered to be important for the detoxification of both exogenous and endogenous aldehydes such as those derived from lipid peroxidation of membrane phospholipids (21Vasiliou V. Pappa A. Petersen D.R. Chem. Biol. Interact. 2000; 129: 1-19Crossref PubMed Scopus (307) Google Scholar). FALDH is a microsomal NAD/NADP-dependent enzyme that acts on long chain aliphatic substrates. The cDNA for FALDH encodes a protein of 485 amino acids (22Rizzo W.B. Lin Z. Carney G. Chem. Biol. Interact. 2001; 130-132: 297-307Crossref PubMed Scopus (26) Google Scholar). This protein has a hydrophobic carboxyl-terminal amino acid sequence that is necessary for microsomal membrane anchoring (23Masaki R. Yamamoto A. Tashiro Y. J. Cell Biol. 1994; 126: 1407-1420Crossref PubMed Scopus (68) Google Scholar). In the present work, we characterized the regulation of FALDH gene expression by insulin, and we addressed the role of FALDH in insulin action in normal, insulin-resistant, and type 1 diabetes conditions. Materials—Recombinant human insulin was from Novo-Nordisk (Copenhagen, Denmark). Recombinant human epidermal growth factor was from Strathmann Biotec AG (Hamburg, Germany). LY294002 was from Calbiochem. Wortmannin and methylglyoxal (MGO) were from Sigma. Chloromethyl-2′,7′-dichlorofluorescein diacetate (CM-DCFDA) was from Molecular Probes (Eugene, OR). 4-Hydroxynonenal (4-HNE) was from Merck. Antibodies to phospho-Ser-473 PKB were from New England Biolabs (Beverly, MA), and anti-Myc was from Santa Cruz Biotechnology (Santa Cruz, CA). Secondary anti-mouse or anti-rabbit antibodies conjugated to horseradish peroxidase were purchased from Jackson Immuno-Research (Copenhagen, Denmark). Animal Treatment and Protocols—Care of animals was performed in accordance with institutional guidelines. Male Wistar rats (150-200 g), 8-10 weeks of age, were purchased from Janvier Laboratory (Laval-LeGenest, France). Male C57BL/6J and db/db mice were obtained at 6 weeks of age from Janvier Laboratory. Animals were maintained in a temperature-controlled facility (22 °C) on a 12-h light/dark cycle and were given free access to food (standard laboratory chow diet from UAR, Epinay-S/Orge, France) unless otherwise indicated. In some experiments, rats or mice were deprived of food for 20 h. Fasted animals were given an intraperitoneal injection with insulin at 1 IU/kg (Actrapid 100 IU/ml, Novo-Nordisk, Denmark) for 6 h or refed with chow diet for 8 h. Animals were euthanized by cervical dislocation, and tissues were rapidly harvested, weighed, and processed for preparation of total RNA or cell culture. High fat diet male C57BL/6J mice were generated by giving high fat and high sucrose diets (D12327, Research Diet, New Brunswick, NJ) over 15 weeks. To generate type 1 diabetic animals, Sprague-Dawley rats were given an intraperitoneal injection of streptozotocin (65-70 mg/kg) as described previously (24Hauguel-de Mouzon S. Peraldi P. Alengrin F. Van Obberghen E. Endocrinology. 1993; 132: 67-74Crossref PubMed Google Scholar). Adipose Tissue Culture—Epididymal fat pads from Wistar rats were dissected, minced finely, and digested with Liberase Blendzyme 3 (Roche Applied Science) by shaking at 37 °C for 45 min. Isolated adipocytes were separated from stromal-vascular cells (S-V) by filtration through a 30-μm nylon mesh. The cells were then washed twice with Krebs-Ringer bicarbonate Hepes pH 7.4 (KRBH) buffer and processed for RNA preparation. For primary adipocyte culture, isolated adipocytes were placed in Falcon 2059 tubes in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal calf serum, 50 units/ml penicillin, 50 μg/ml streptomycin for primary culture. The cells were incubated at 37 °C, 5% CO2. Cells were depleted overnight in DMEM supplemented with 5% (w/v) bovine serum albumin (BSA) before insulin treatment. The samples were collected, and total RNA was isolated and was analyzed by RT-PCR. For adenoviral infection, isolated adipocytes in culture were infected for 12 h, and the medium was then replaced for 12 h before starvation. Isolation and Culturing of Hepatocytes—Hepatocytes were isolated from male Wistar rats by collagenase dissociation of the liver as described by Fehlmann et al. (25Fehlmann M. Le Cam A. Freychet P. J. Biol. Chem. 1979; 254: 10431-10437Abstract Full Text PDF PubMed Google Scholar). Freshly isolated hepatocytes were incubated in Krebs-Ringer bicarbonate (KRb) buffer, pH 7.4, containing defatted BSA (10 mg/ml), gentamycin (0.05 mg/ml) and were gassed with a mixture of 5% CO2, 95% O2. Primary cultures of hepatocytes were performed essentially as described by Morin et al. (26Morin O. Fehlmann M. Freychet P. Mol. Cell. Endocrinol. 1982; 25: 339-352Crossref PubMed Scopus (24) Google Scholar). Cells were plated at a final concentration of 106 cells/ml in Waymouth's medium supplemented with 10% (w/v) fetal calf serum. After 4 h at 37 °C, the medium was replaced with serum-free Waymouth's medium containing defatted BSA (2 mg/ml), penicillin, and streptomycin, and cells were incubated overnight before insulin treatment. For adenoviral infection, hepatocytes were infected with adenovirus for 12 h, and culture medium was replaced, and cells were incubated for 24 h at 37 °C. RAP-PCR—RAP-PCR was performed with the RAP-PCR kit (Stratagene, Amsterdam, The Netherlands), according to the method of Welsh et al. (27Welsh J. Chada K. Dalal S.S. Cheng R. Ralph D. McClelland M. Nucleic Acids Res. 1992; 20: 4965-4970Crossref PubMed Scopus (495) Google Scholar). Isolated adipocytes were treated or not with insulin (10-7m) or cotreated with insulin and wortmannin (100 nm) for 6 h. After extraction of total RNA, poly(A+) RNA was purified using a Poly(A) Tract mRNA Isolation System (Promega, Charbonnières, France). The RT reaction was performed using 100 ng of RNA poly(A+) and different arbitrary primers (1.25 μm) for 1 h at 37 °C by adding to the First Strand Buffer 125 μm of each dNTP, 40 units of RNase (Stratagene), and 25 units of Moloney murine leukemia virus-reverse transcriptase (Stratagene). The reaction was stopped by heating to 90 °C for 5 min. To perform RAP-PCRs, 1× Taq Reaction Buffer, 1 μm arbitrary primers, 50 μm of each dNTP, 1 unit of Taq polymerase, and 0.2 μCi/ml of [α-33P]ATP were added to 1 μl of the cDNA. The PCR was started by incubation at 37 °C for 5 min and 72 °C for 5 min, followed by 40 cycles at 94 °C for 1 min, 60 °C for 2 min, and 72 °C for 2 min. To visualize the RAP-PCR products, 5 μl from each reaction was mixed with 10 μl of stop buffer containing 80% (v/v) formamide, 50 mm Tris-HCl, pH 8.8, 1 mm EDTA, 0.1% (w/v) xylene cyanol, 0.1% (w/v) bromphenol blue, and heated at 80 °C for 2 min. 4 μl of each reaction was loaded on a 4% acrylamide, 7 m urea gel. Electrophoresis was performed at 55 watts in 1× TBE buffer, and the gel was autoradiographed. The RAP-PCR gel and the autoradiogram were aligned, and individual bands representing differentially expressed products were cut and removed from the gel. Each isolated band was incubated in 70 μl of elution buffer at 60 °C for 1 h and at room temperature for 12 h. Eluted samples were collected and reamplified by PCR with the same primer used for the RAP-PCR. The RAP-PCR products were then sub-cloned in pCR 2.1 TOPO (Invitrogen) and sequenced using M13 reverse and forward primers. FALDH Assay—Hepatocytes were washed with PBS and homogenized in 2 ml of homogenization buffer (25 mm Tris-HCl, pH 8.0, 250 mm sucrose) with a Teflon glass Potter homogenizer. Final protein concentration was assayed by bicinchoninic acid technique (BCA protein assay kit, Interchim, Montluçon, France). FALDH assay was performed as described previously by Kelson et al. (28Kelson T.L. Secor McVoy J.R. Rizzo W.B. Biochim. Biophys. Acta. 1997; 1335: 99-110Crossref PubMed Scopus (118) Google Scholar) using 100 μg of crude homogenate with a mixture containing 193 μl of deionized water, 100 μl of 200 mm glycine-NaOH buffer, pH 9.5, 0.4% v/v Triton X-100, 40 μl of 100 mm pyrazole, 20 μl of 10 mg/ml bovine serum albumin (fatty acid free), 24 μl of 25 mm NAD+. Each sample was incubated at 37 °C, and the reaction was initiated by adding 3 μl of 160 μm dodecanol. FALDH activity was monitored by measuring the increase in absorbance at 340 nm at different times. Control reactions were run for each sample by substituting 3 μl of 100% v/v ethanol for the aldehyde solution and running the reaction as above. FALDH-dependent enzyme activity was calculated by subtracting the absorbance measured in the absence of aldehyde from that seen in its presence and dividing by 60 min to express the final result as units/min. Adipocyte Differentiation—3T3-L1 preadipocytes (American Type Culture Collection, Manassas, VA) were grown in DMEM supplemented with 10% (v/v) fetal calf serum, 50 units/ml penicillin, 50 μg/ml streptomycin and allowed to reach confluence as described previously. At 2 days post-confluence (day 0), differentiation was initiated by the addition of 100 nm insulin, 1 μm dexamethasone, and 0.25 mm isobutylmethylxanthine in DMEM with 10% (v/v) fetal bovine serum. Three days later (day 3), the induction medium was replaced by DMEM supplemented with 10% (v/v) fetal bovine serum and insulin only and was changed every 2 days. Adipogenesis was assessed by analysis of the expression of adipocyte-specific genes (aP2 or PPARγ) and by lipid accumulation using microscopic analysis. Total RNA Extraction and Northern Blot Analysis—Total cellular RNA from tissues (liver, muscle, or white adipose tissue) or cells was isolated using the Trizol reagent (Invitrogen) following the manufacturer's instructions. For Northern blot analysis, 10 μg of total RNA was denatured in formamide and formaldehyde at 60 °C and separated by electrophoresis on 1.2% w/v agarose gels. RNA was then transferred to positively charged Hybond-N membranes (Amersham Biosciences) and cross-linked to the membrane by heating to 80 °C. Specific cDNA probes were labeled with [α-32P]dCTP by random priming using the Rediprime kit (Amersham Biosciences) and purified with the Probequant kit (Amersham Biosciences). Blots were hybridized with labeled cDNA probes overnight at 42 °C in NorthernMax hybridization buffer (Ambion, Inc., Austin, TX). Membranes were then washed in 1× SSC, 0.5% (w/v) SDS, and exposed to PhosphorImager for 4-24 h. The signals were scanned (Storm 840) and quantified using ImageQuant 5.0 software (Amersham Biosciences). Blots were stripped and rehybridized with a 5′-32P-labeled 18 S oligonucleotide probe in order to normalize the signal. Real Time Quantitative PCR—Total RNA was treated with DNase (Ambion), and 1 μg was reverse-transcribed for 60 min at 42 °C using the Reverse Transcription System kit (Promega) in the presence of random primers and oligo(dT)15. Quantitative PCR was performed by monitoring in real time the increase in fluorescence of the SYBR Green dye on an ABI PRISM 7000 Sequence Detector System (Applied Biosystems, Courtaboeuf, France) according to the manufacturer's instructions. PCR primers for each gene were designed using Primer Express software (Applied Biosystems, Courtaboeuf, France), with a melting temperature at 58-60 °C and a resulting product of ∼100 bp. Each PCR was carried out in triplicate in a 20-μl volume using SYBR Green I Master Mix Plus (Eurogentec, Seraing, Belgium) for 15 min at 95 °C for initial denaturing, followed by 40 cycles of 95 °C for 30 s and 60 °C for 30 s in the ABI Prism 7000 sequence Detector System (Applied Biosystems). To exclude the contamination of nonspecific PCR products such as primer dimers, melting curve analysis was applied to all final PCR products after the cycling protocols. Values for each gene were normalized to expression levels of HPRT mRNA in rat tissue and 36B4 mRNA in mouse tissue. Each RT-PCR quantification experiment was performed in triplicate. Relative quantification of FALDH gene was calculated by using 2-ΔCt formula, as recommended by the manufacturer (Applied Biosystems). Results were expressed relative to the control condition, which was arbitrary assigned a value of 1. Primers sequences used to quantify FALDH mRNA by real time RT-PCR were designed by using the Primer Express software from Applied Biosystems. Oligonucleotides used were as follows: rat FALDH sense, 5′-AGCCCAGCTACATTGACAGAGA-3′, and antisense, 5′-ACACAGGATATAGTCAGAGCAATACA-3′; mouse FALDH sense, 5′-CAGCATTTCCTGGAGCAATG-3′, and antisense, 5′-AGCTTGGAATTACCCTTTCGTTCT-3′; HPRT sense, 5′-AGCCTGGTCATGTTGCCTTT-3′, and antisense, 5′-AAAGAACTTATAGCCCCCCTTGA-3′; and 36B4 sense, 5′-CTTTATCAGCTGCACATCACTCAGA, and antisense, 5′-TCCAGGCTTTGGGCATCA-3′. Western Blot—Western blotting was performed on whole cell lysates from isolated adipocytes. Protein extracts were obtained by mixing 200 μl of fat cell suspension with 200 μl of Laemmli buffer (3% (w/v) SDS, 70 mm Tris-HCl, pH 7, 11% (v/v) glycerol). The samples were incubated for 5 min at 90 °C, and protein concentration was assayed with the bicinchoninic acid technique (BCA protein assay kit, Interchim, Monluçon, France). Proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes (Hybond C, Amersham Biosciences), and analyzed by immunoblotting. Immunoreactive proteins were detected by enhanced chemiluminescence (Amersham Biosciences). Generation of Recombinant Adenoviruses—Adenoviruses expressing FALDH were generated by homologous recombination in Escherichia coli BJ 5183 as described previously (29Pirola L. Bonnafous S. Johnston A.M. Chaussade C. Portis F. Van Obberghen E. J. Biol. Chem. 2003; 278: 15641-15651Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Briefly, co-transformation of E. coli BJ 5183 led to recombination between FALDH (cloned in pCDNA3) and a viral vector recombinogenic with the pCDNA3 cytomegalovirus promoter and poly(A) sequence (VmcDNA, provided by S. Rusconi, University of Fribourg, Switzerland) (29Pirola L. Bonnafous S. Johnston A.M. Chaussade C. Portis F. Van Obberghen E. J. Biol. Chem. 2003; 278: 15641-15651Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Recombinants were screened by PCR analysis using a pair of primers that annealed to the viral vector and to the cytomegalovirus promoter sequence, respectively. A positive clone harboring FALDH was further amplified in E. coli DH5-α, digested with PacI, and transfected by the calcium phosphate method into helper 293 cells to produce viral particles. Adenoviruses were stored in 0.1 m Tris, 0.25 m NaCl, 1 mg/ml BSA, 50% (v/v) glycerol, pH 7.5, at -20 °C. Adenoviruses expressing p110α CAAX (Ad p110α CAAX, where AA is aliphatic amino acid) and GFP (Ad-GFP) were described previously (29Pirola L. Bonnafous S. Johnston A.M. Chaussade C. Portis F. Van Obberghen E. J. Biol. Chem. 2003; 278: 15641-15651Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Viral titer of stocks was >108 plaque-forming units/ml. Assay for Reactive Oxygen Species Production—The intracellular formation of reactive oxygen species was detected using the fluorescent probe CM-DCFDA (30Maziere C. Floret S. Santus R. Morliere P. Marcheux V. Maziere J.C. Free Radic. Biol. Med. 2003; 34: 629-636Crossref PubMed Scopus (40) Google Scholar). Starved cells were treated with MGO or 4-HNE at times and concentrations indicated below. The cells were washed two times in PBS and exposed to 5 × 10-5m CM-DCFDA in phosphate-buffered saline for 1 h at 37 °C. The cells were washed two times in PBS, solubilized in water, and sonicated. The fluorescence was determined at 488/525 nm, normalized on a protein basis, and expressed as percentage of control. Identification of Differentially Expressed Genes by Insulin-induced PI3K Activation in Isolated Rat Adipocytes—To identify novel genes controlled specifically by PI3K in response to insulin, we performed RAP-PCR on freshly isolated rat adipocytes treated or not with insulin (10-7m) and with or without exposure to wortmannin (100 nm), an inhibitor of PI3K. Briefly, poly(A) RNA was extracted for each condition and reverse-transcribed. RAP-PCR was performed using several combinations of arbitrary primers. RAP-PCR products were then separated by electrophoresis. We found 27 clones showing PI3K-dependent modulation in mRNA expression. Subsequent Northern blot analysis identified and confirmed 10 differentially expressed genes in isolated adipocytes, corresponding to both previously identified and unidentified genes. Among these clones, we isolated a RAP-PCR product corresponding to FAS cDNA. The transcriptional control of this gene by insulin via PI3K activation has been described previously (11Wang D. Sul H.S. J. Biol. Chem. 1998; 273: 25420-25426Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar), thus validating our approach. Characterization of FALDH mRNA Expression in Isolated Adipocytes—Another RAP-PCR product, recognizing a 4-kb mRNA by Northern blot analysis, corresponds to the FALDH gene. By using real time quantitative PCR, we observed a 4-fold increase in FALDH gene expression after stimulation of isolated adipocytes with insulin (10-7m, 6 h) (Fig. 1A). When adipocytes were incubated simultaneously with insulin and wortmannin (100 nm), the increase in expression was abolished, suggesting a PI3K-specific control on the FALDH gene. In parallel, we tested another growth factor, epidermal growth factor (EGF), which also activates the PI3K pathway in adipocytes (Fig. 1B). As expected, we observed the same increase in FALDH gene expression in cells stimulated with EGF and insulin, suggesting that both factors are able to control FALDH expression via a PI3K-dependent pathway. To confirm PI3K activation, a phosphoserine PKB Western blot was performed (Fig. 1B, bottom). Both insulin and EGF increase the phosphorylation of PKB on serine 473, which is compatible with PI3K-dependent PKB activation. Finally, to confirm the role of PI3K in the control of FALDH expression, we performed an adenovirus infection experiment (Fig. 1C) in which isolated adipocytes were infected with Ad-GFP as negative control or with Ad-p110α CAAX, a constitutively active form of PI3K (31Khwaja A. Rodriguez-Viciana P. Wennstrom S. Warne P.H. Downward J. EMBO J. 1997; 16: 2783-2793Crossref PubMed Scopus (939) Google Scholar). As expected, FALDH expression was increased upon insulin stimulation of adipocytes expressing GFP (4.19-fold). When cells were infected with Ad-p110α CAAX, we observed an insulin-independent increase in FALDH mRNA expression (3.32-fold). Moreover, FALDH expression was not modified in adipocytes ectopically expressing p110α CAAX and treated with the PI3K inhibitor, LY294002. These results confirm that FAL
Referência(s)