Desnutrin, an Adipocyte Gene Encoding a Novel Patatin Domain-containing Protein, Is Induced by Fasting and Glucocorticoids

Abstract

We have used rat cDNA microarrays to identify adipocyte-specific genes that could play an important role in adipocyte differentiation or function. Here, we report the cloning and identification of a 2.0-kb mRNA coding for a putative protein that we have designated as desnutrin. The novel gene is expressed predominantly in adipose tissue, and its expression is induced early during 3T3-L1 adipocyte differentiation. Desnutrin mRNA levels were regulated by the nutritional status of animals, being transiently induced during fasting. In vitro desnutrin gene expression was up-regulated by dexamethasone in a dose-dependent manner but not by cAMP, suggesting that glucocorticoids could mediate the increase in desnutrin mRNA levels observed during fasting. Desnutrin mRNA codes for a 486-amino acid putative protein containing a patatin-like domain, characteristic of many plant acyl hydrolases belonging to the patatin family. Confocal microscopy of enhanced green fluorescent protein-tagged desnutrin protein-transfected cells showed that the fusion protein localized in the cytoplasm. Moreover, cells overexpressing desnutrin by transfection showed an increase in triglyceride hydrolysis. Interestingly, we also found that the desnutrin gene expression level was lower in ob/ob and db/db obese mouse models. Overall, our data suggest that the newly identified desnutrin gene codes for an adipocyte protein that may function as a lipase and play a role in the adaptive response to a low energy state, such as fasting, by providing fatty acids to other tissues for oxidation. In addition, decreased expression of desnutrin in obesity models suggests its possible contribution to the pathophysiology of obesity. We have used rat cDNA microarrays to identify adipocyte-specific genes that could play an important role in adipocyte differentiation or function. Here, we report the cloning and identification of a 2.0-kb mRNA coding for a putative protein that we have designated as desnutrin. The novel gene is expressed predominantly in adipose tissue, and its expression is induced early during 3T3-L1 adipocyte differentiation. Desnutrin mRNA levels were regulated by the nutritional status of animals, being transiently induced during fasting. In vitro desnutrin gene expression was up-regulated by dexamethasone in a dose-dependent manner but not by cAMP, suggesting that glucocorticoids could mediate the increase in desnutrin mRNA levels observed during fasting. Desnutrin mRNA codes for a 486-amino acid putative protein containing a patatin-like domain, characteristic of many plant acyl hydrolases belonging to the patatin family. Confocal microscopy of enhanced green fluorescent protein-tagged desnutrin protein-transfected cells showed that the fusion protein localized in the cytoplasm. Moreover, cells overexpressing desnutrin by transfection showed an increase in triglyceride hydrolysis. Interestingly, we also found that the desnutrin gene expression level was lower in ob/ob and db/db obese mouse models. Overall, our data suggest that the newly identified desnutrin gene codes for an adipocyte protein that may function as a lipase and play a role in the adaptive response to a low energy state, such as fasting, by providing fatty acids to other tissues for oxidation. In addition, decreased expression of desnutrin in obesity models suggests its possible contribution to the pathophysiology of obesity. Triglycerides serve as the most efficient form of energy storage in times of caloric excess in many organisms. During periods of energy demand, triglycerides can be rapidly mobilized by the hydrolytic action of lipases, releasing free fatty acids that are oxidized to meet the energy requirement of the organism. In mammals, adipose tissue serves as the major lipid storage site. Whereas white adipose tissue (WAT) 1The abbreviations used are: WAT, white adipose tissue; BAT, brown adipose tissue; RACE, rapid amplification of cDNA ends; EST, expressed sequence tag; contig, group of overlapping clones; HA, hemagglutinin; EGFP, enhanced green fluorescent protein; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; MIX, methylisobutylxanthine; C/EBP, CAAT/enhancer-binding protein. stores triglycerides that can be mobilized, producing fatty acids to be used by peripheral tissues, brown adipose tissue (BAT) itself uses the accumulated lipids to generate heat, a process known as adaptative thermogenesis (1Rosen E.D. Spiegelman B.M. Annu. Rev. Cell Dev. Biol. 2000; 16: 145-171Crossref PubMed Scopus (1057) Google Scholar). In addition, WAT plays an important role as an endocrine organ, secreting a wide variety of factors that are involved in various aspects of physiology, including appetite control, peripheral metabolism, immune response, and vascular function (2Gregoire F.M. Smas C.M. Sul H.S. Physiol. Rev. 1998; 78: 783-809Crossref PubMed Scopus (1873) Google Scholar, 3MacDougald O.A. Mandrup S. Trends Endocrinol. Metab. 2002; 13: 5-11Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar, 4Fruhbeck G. Gomez-Ambrosi J. Muruzabal F.J. Burrell M.A. Am. J. Physiol. 2001; 280: E827-E847Crossref PubMed Google Scholar). The function of adipose tissue is dependent on the energy requirements and is tightly controlled by nutrient, neural, and hormonal signals. The crucial contribution of adipose tissue to the energy metabolism and function of an organism is manifested by pathological conditions in which dysregulation of adipose function leads to the development of severe diseases such as insulin resistance, diabetes mellitus, and cardiovascular disease (5Unger R.H. Orci L. FASEB J. 2001; 15: 312-321Crossref PubMed Scopus (375) Google Scholar). The adipocyte is the main cell type present in adipose tissue. During adipose tissue development, precursor cells differentiate to generate adipocytes fully equipped with the enzymatic machinery and regulatory proteins that are needed to carry out their function in controlling fat metabolism and energy homeostasis. The adipogenic process entails dramatic morphological and biochemical changes and alteration in the expression of hundreds of genes (6Burton G.R. Nagarajan R. Peterson C.A. McGehee Jr., R.E. Gene (Amst.). 2004; 329: 167-185Crossref PubMed Scopus (85) Google Scholar, 7Guo X. Liao K. Gene (Amst.). 2000; 251: 45-53Crossref PubMed Scopus (110) Google Scholar, 8Sadowski H.B. Wheeler T.T. Young D.A. J. Biol. Chem. 1992; 267: 4722-4731Abstract Full Text PDF PubMed Google Scholar). This includes increases in the expression of genes involved in the specialized role of adipose tissue in lipid metabolism, hormone responsiveness, the extracellular matrix, and secretion of endocrine and regulatory factors (2Gregoire F.M. Smas C.M. Sul H.S. Physiol. Rev. 1998; 78: 783-809Crossref PubMed Scopus (1873) Google Scholar). Moreover, some of the genes are expressed only in adipose tissue and constitute a unique functional trait of this tissue. Identification of such genes or gene products is of crucial importance in unraveling the regulatory mechanisms of adipocyte differentiation and function. It is also a requirement for fully understanding the etiology of the pathologies associated with adipose tissue malfunction. To identify novel genes that have a role in adipocyte differentiation or function, we have used several approaches based on differential gene expression. By differential screening and differential display techniques using the adipogenic 3T3-L1 cell line and primary preadipocytes, we previously cloned two regulatory proteins involved in the control of adipocyte differentiation, Pref-1 (9Smas C.M. Sul H.S. Cell. 1993; 73: 725-734Abstract Full Text PDF PubMed Scopus (564) Google Scholar) and ENC-1 (10Zhao L. Gregoire F. Sul H.S. J. Biol. Chem. 2000; 275: 16845-16850Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), respectively (10Zhao L. Gregoire F. Sul H.S. J. Biol. Chem. 2000; 275: 16845-16850Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 11Villena J.A. Kim K.-H. Sul H.S. Horm. Metab. Res. 2002; 34: 664-670Crossref PubMed Scopus (53) Google Scholar). We recently employed the microarray technique to clone ADSF/resistin (12Kim K.-H. Lee K. Moon Y.S. Sul H.S. J. Biol. Chem. 2001; 276: 11252-11256Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar), an adipocyte-specific secreted factor that inhibits adipocyte differentiation in vitro and in vivo (12Kim K.-H. Lee K. Moon Y.S. Sul H.S. J. Biol. Chem. 2001; 276: 11252-11256Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar, 13Kim K.-H. Zhao L. Moon Y.S. Kang C. Sul H.S. Proc. Natl. Sci. U. S. A. 2004; 101: 6780-6785Crossref PubMed Scopus Google Scholar) and that may be a factor to insulin J. A. C.M. S. L. M.A. 2004; PubMed Scopus Google Scholar). Here, we report the cloning and identification of the cDNA coding for a putative protein that we desnutrin. This novel gene is induced early during 3T3-L1 adipocyte differentiation and is expressed predominantly in adipose tissue. Desnutrin mRNA levels are the control of the nutritional status of animals, being transiently induced during fasting. In desnutrin gene expression was regulated by but not by cAMP, suggesting that glucocorticoids could mediate the increase in desnutrin mRNA during fasting. Desnutrin mRNA codes for a 486-amino acid protein containing a patatin-like domain, characteristic of many plant acyl hydrolases that to the patatin family. We found that of desnutrin triglyceride in the suggesting the function of desnutrin as a lipase and its role in the adaptive response of adipose tissue to the low energy of fasting, releasing for to meet the energy requirement for other of genes expressed in adipose tissue was by the gene expression of mouse tissues using rat as previously (12Kim K.-H. Lee K. Moon Y.S. Sul H.S. J. Biol. Chem. 2001; 276: 11252-11256Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar). were with by using of WAT, and found in with WAT were of Desnutrin cDNA and of cDNA J. Res. PubMed Scopus Google Scholar) using the identified rat expressed sequence clone sequence as a in the mouse data of a on the mouse The gene was identified as the and we to as desnutrin. The mRNA sequence of the gene was used to for cloning the cDNA containing the of WAT was with and in a of of the was used as the and desnutrin cDNA was by using and The was cloned the and the was fully We to the of desnutrin cDNA using the and WAT cDNA as the was out for of for for and for The was cloned the and of a desnutrin expression was out using the cDNA containing the coding as the and and The was cloned the and the of the for expression. fusion protein the coding of desnutrin was by a of desnutrin the with enhanced green fluorescent protein was using 3T3-L1 adipocyte cDNA as the and and The was on with and and cloned the of the tissue of desnutrin mRNA and its by and were In the were for a of to which were and tissues were for were a for of being Desnutrin mRNA levels was also by and in adipose tissue ob/ob and db/db for of the and of WAT was with phosphate-buffered and to with and in Dulbecco's modified Eagle's was out for tissue was The cell was a to tissue and adipocytes were the vascular by The vascular was with a and cells were by for were for Cell and cells were in containing To differentiation of 3T3-L1 cells were with dexamethasone and for the cells were to differentiation with and for which of the cells the adipocyte the the of glucocorticoids on desnutrin were with MIX, cAMP, or the cells were in containing transfection of cells with or expression was out by the as previously F.M. R. R.E. J.A. K. in Scholar). and mouse tissues or cells was using to the of was to on and was out in using for adipocyte fatty protein CAAT/enhancer-binding and Confocal cells were transiently with of as and on cells were with for The were with PBS, and were with for The were several times with and on using and to the a control, the only was were using a by and cells with an expression for desnutrin were times with and with and acid were in and and using a The was by for The was for to the and the was for to the the of protein were to and to a for using of cells were with the expression or the control as cells were for in containing fatty and acid of were with and for lipid or with for an and cells and were by the of and J. Sci. Scholar) and by using acid to lipids or to lipids were by To the lipids to the by of cell was the of and with of and the was by of Desnutrin microarray using cDNA microarrays to identify genes expressed in adipose tissue. We the gene expression in and adipose tissue using rat cDNA and identified a of genes that showed only on the that were with WAT were and adipose expression was by using and WAT not of showed expression in the sequence of the clone as a the of the mouse data of identified a on the of the in We used the sequence of the gene mRNA for the of a of to the coding of the gene using mouse cDNA with In addition, the was by out of amplification products were and the sequence was used for to generate the desnutrin cDNA of Desnutrin a protein with a of acids and an of The cDNA a of and a of with a The the of proteins in a wide of including and In two proteins with a of to desnutrin have and to be proteins and have in the and secretion of the In addition, two other putative proteins with desnutrin have protein and the In two proteins can be a protein that to be the mouse of the protein and recently cloned S. F. J. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar) as an adipose protein expression is up-regulated during adipocyte differentiation, but during in proteins are found in and in Interestingly, putative proteins some of are present in such as the protein and in including and The of desnutrin and other proteins in such a variety of suggests that desnutrin to a of proteins are expressed in and this a function of proteins that is not of organisms. Desnutrin mRNA in was to the expression of desnutrin in various of mouse of mouse with a desnutrin a mRNA of in The of a is in with the of a in the desnutrin cDNA the we that our desnutrin cDNA sequence in desnutrin mRNA was expressed in various of WAT as as but desnutrin levels were also found in other tissues such as and desnutrin mRNA was found a level in and desnutrin is expressed in adipose tissues It is to that the desnutrin expression levels on the type or of adipose tissue. Overall, showed desnutrin mRNA levels the white adipose fat showed the level of expression with and a gene known to be expressed in adipose tissue, showed a of expression to that of desnutrin in the adipose with a level of expression in and WAT We desnutrin gene expression in fat the obese mouse models ob/ob and db/db by as as by not We observed lower desnutrin mRNA levels in db/db and lower levels in ob/ob with suggesting a possible of desnutrin function in obesity. To identify the cell type that is for the expression level of desnutrin mRNA in adipose tissue, of WAT were adipocytes and a vascular containing as as cells and in the desnutrin was not in the vascular but was found in the adipocyte with adipocyte such as adipocyte fatty and The adipocyte-specific expression of desnutrin mRNA was in 3T3-L1 an cell line that can be induced to differentiate adipocytes hormone and used as a for Desnutrin mRNA was found only in but not in or 3T3-L1 We desnutrin gene expression during the of of 3T3-L1 to and in desnutrin mRNA was not in its expression was rapidly cells were induced to The level of desnutrin mRNA was cells were fully as by of lipids and expression of adipocyte such adipocyte fatty and Interestingly, a level of desnutrin mRNA could be observed in 3T3-L1 cells as early as of differentiation, the expression of the various adipocyte desnutrin is an adipocyte gene induced early during 3T3-L1 adipocyte differentiation. of Desnutrin mRNA by and by of the which is to is by fasting, and its levels are S. F. J. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). of the the two putative we to desnutrin gene expression could also be regulated by the nutritional status of the organism. WAT to was for desnutrin expression. in the levels of desnutrin mRNA were in in that for the and were for of nutritional of desnutrin gene expression. To desnutrin expression is during or up-regulated by a of were in which were for and or for of fasting. in with desnutrin mRNA levels rapidly of fasting, a level and to levels was the expression of the desnutrin gene was with that of the expression were but was induced The known to be tightly regulated by and H.S. Annu. Rev. 1998; PubMed Scopus Google Scholar), was used as a mRNA levels were not during fasting, but during the that desnutrin gene expression is rapidly and transiently induced in WAT by fasting. During fasting, and glucocorticoids levels are and serve as of the adaptive response to by changes in gene expression to the desnutrin mRNA is induced during fasting, we cAMP, the of or glucocorticoids can desnutrin expression. 3T3-L1 preadipocytes, which not have levels of desnutrin were for with MIX, cAMP, or in of the induced expression of the desnutrin that and are not involved in the of desnutrin mRNA during fasting. a was to increase desnutrin mRNA levels in The of dexamethasone and to have a on desnutrin gene of the of adipocyte differentiation by Overall, that glucocorticoids not desnutrin gene expression in 3T3-L1 increase in desnutrin mRNA was also observed fully adipocytes were with dexamethasone The increase not to be as as that in preadipocytes, of the levels of desnutrin mRNA in The of desnutrin mRNA by dexamethasone was and dose-dependent and for with of dexamethasone to showed levels of desnutrin mRNA as low as and a increase in with the of The response in the in with the for dexamethasone with the of 3T3-L1 cells Google Scholar), suggesting that the of dexamethasone on desnutrin mRNA levels was by to its Moreover, desnutrin expression was dexamethasone of Desnutrin of the desnutrin acid sequence a of the protein. To the desnutrin protein in in the we transiently an expression for a desnutrin fusion protein cells and its by fusion protein was an fusion protein in an to of putative in showed a in the cells the suggesting a of the desnutrin protein. the other in with a report S. F. J. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar), of expression showed a of in the not To the of the desnutrin cells were with an desnutrin expression and by was was not in the or but a was in the was also in the but this was to the of as by microscopy not we that putative desnutrin is a protein. of of Desnutrin on function to of the desnutrin protein in In an to identify a functional in we its acid sequence with the protein data patatin present in the acids of desnutrin was The its to a storage protein found in that lipid acyl and Interestingly, of desnutrin and the proteins showed that the of in this patatin the proteins in could also be found in proteins of as as and not could be identified a a and a containing a The and constitute a that is for the lipase of patatin F. PubMed Scopus Google Scholar). The and that proteins to the and the in suggests a function. The of the protein by the desnutrin gene to the patatin-like protein to of desnutrin could of triglycerides to its lipase We lipid with acid for in cells or control cells with and the lipid was by in the cells desnutrin or cells with a control showed the lipid and the of lipids was observed control and This that of lipids was not by desnutrin The triglyceride levels not the control cells were in for an the other in the triglyceride levels decreased by and lower were in the and other lipid that expression of only triglyceride hydrolysis. The in triglycerides observed in cells was and of their levels in the cells were by in control was only a in that the triglycerides in cells were a in control The free fatty acid levels decreased during the of in and control the lower levels of triglycerides in the we could not levels of free fatty suggesting that the fatty acids triglyceride were to the or rapidly in the We the lipids in the of the cells of lipids the showed that the lipids found in the were free fatty acids in the free fatty acids in the the cells were in to cells showed levels of free fatty acids to the with control the in triglyceride levels was for in by an increase in free fatty acids to the providing that the cells have a of In our for genes that could play a crucial role in adipocyte differentiation or function, we previously have used cDNA microarrays to identify genes that are expressed in adipose tissue (12Kim K.-H. Lee K. Moon Y.S. Sul H.S. J. Biol. Chem. 2001; 276: 11252-11256Abstract Full Text Full Text PDF PubMed Scopus (555) Google Scholar). the we have identified and cloned adipose tissue a cDNA a 486-amino acid putative protein that we have desnutrin. The acid sequence that desnutrin to a of proteins that have and that are regulated by the nutritional of the the genes the two of this desnutrin and have cloned and identified to be predominantly Whereas mRNA expression is to adipose tissue, desnutrin expressed predominantly a level in adipose tissue, is also found a low level in a variety of This expression in adipose tissue suggests the of the desnutrin gene in a function to but not of adipose tissue. The increase in triglyceride associated with desnutrin and the of desnutrin gene expression by are with this most tissues their triglycerides for and during energy the energy is not and cells to on the by adipose tissue, an specialized in energy storage in the form of It is that desnutrin mRNA levels on the adipose tissue type and The differential expression of the desnutrin gene could be a factor to the observed adipose J. PubMed Scopus Google Scholar, F. J. PubMed Scopus Google Scholar). Interestingly, desnutrin gene expression is lower in ob/ob and db/db obese mouse suggesting a role of desnutrin in the pathophysiology of obesity. The data a not to the role of desnutrin. the other a protein data identified the of a patatin-like in the of desnutrin. is a of a of proteins found in and other PubMed Scopus Google Scholar). It for of the protein and is to be a storage protein. In addition, patatin lipid acyl J. 2001; PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). This enzymatic on a by a in the characteristic of and a belonging to the F. PubMed Scopus Google Scholar). The of desnutrin to including the of the that constitute the and the increase in triglyceride observed in cells overexpressing the desnutrin our that the desnutrin protein may be a desnutrin to have on triglycerides in or was observed in the lipid The a protein with and a patatin with the the J. J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the other plant on a of including and and but not J. 2001; PubMed Scopus Google Scholar). Interestingly, the type a recently lipase containing a patatin domain, triglycerides and other lipids its K. A. J. J. PubMed Scopus Google Scholar). This in the of in the that form the domain, suggests that the for may on that are the containing a patatin This differential can also have important functional The of by is associated with and in models 2000; PubMed Scopus Google Scholar). pathologies to be by in the of the cells to the lipase of the K. A. J. J. PubMed Scopus Google Scholar). In the lipase associated with a function, the of some by their J.A. Physiol. PubMed Scopus Google Scholar). the other the of and the fatty acids can function as or of that mediate D.A. 2000; PubMed Scopus Google Scholar). The of the desnutrin protein for its tissue and the of expression by suggest that the desnutrin gene could play a role in the response of the organism to of triglycerides and providing free fatty acids to other tissues to be oxidized in of energy In this lipase is known to be a in the of fatty acids in adipocytes as in C. PubMed Scopus Google Scholar). In lipase and fatty acid is levels of in lipase and in the of that are not and that are triglyceride Interestingly, as desnutrin gene expression is regulated by but not by We that desnutrin may function as a triglyceride our present was by transfection of the of the desnutrin of the protein using as as approaches in adipocytes this The nutritional of the patatin-like genes to be to of the gene expression is by and up-regulated by The desnutrin an of being transiently up-regulated by fasting. The expression of desnutrin and genes may a differential hormonal during and this suggests for We have that desnutrin gene expression is induced by of the involved in the response of an organism to fasting, but not by the other of gene expression by to be by and which action by the The of action of glucocorticoids on desnutrin gene expression is the that low of to the for the can desnutrin expression suggests that its is by the we have not found response in the of the desnutrin It is known that the can its not only by to the response but also by with other factors such as R. S. J. Cell. Full Text PDF PubMed Scopus Google Scholar) and S. Cell. Biol. 1993; 13: PubMed Google Scholar) by a Interestingly, several for that could mediate the are present in the desnutrin not is an action of glucocorticoids on desnutrin gene expression an factor that is induced by This may the of desnutrin gene expression by we not in desnutrin mRNA by dexamethasone in the of not In with the cloning of desnutrin we a of genes coding for proteins by the of a patatin-like as a of the two of the identified and to be hormonal and nutritional We suggest this gene of desnutrin and the protein the control nutritional status on its expression in adipose tissue. desnutrin and genes are expressed in adipose tissue, and desnutrin protein to be involved in triglyceride by in energy homeostasis. In this obesity models lower levels of desnutrin suggesting its contribution to the pathophysiology of obesity. in vitro biochemical including of the are needed for for the function of desnutrin and It be to which we found localized in the could be to lipid which could be regulated by as in the of lipase C. G. J.A. C. J. Cell Biol. PubMed Scopus Google Scholar). in vivo on the or of function using the role of desnutrin in the response of the organism to caloric as as its possible in pathologies associated with adipose function or lipid metabolism such as and We L. for with