FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2150767656> ?p ?o ?g. }

• W2150767656 endingPage "36660" @default.
• W2150767656 startingPage "36652" @default.
• W2150767656 abstract "Sterol regulatory element-binding proteins (SREBPs) are a family of membrane-bound transcription factors that regulate cholesterol and fatty acid homeostasis. In mammals, three SREBP isoforms designated SREBP-1a, SREBP-1c, and SREBP-2 have been identified. SREBP-1a and SREBP-1c are derived from the same gene by virtue of alternatively spliced first exons. SREBP-1a has a longer transcriptional activation domain and is a more potent transcriptional activator than SREBP-1c in cultured cells and liver. Here, we describe the physiologic consequences of overexpressing the nuclear form of SREBP-1a (nSREBP-1a) in adipocytes of mice using the adipocyte-specific aP2 promoter (aP2-nSREBP-1a). The transgenic aP2-nSREBP-1a mice developed markedly enlarged white and brown adipocytes that were fully differentiated. Adipocytes isolated from aP2-nSREBP-1a mice had significantly increased rates of fatty acid synthesis and enhanced fatty acid secretion. The increased production and release of fatty acids from adipocytes led, in turn, to a fatty liver. Overexpression of the alternative SREBP-1 isoform, nSREBP-1c, in adipose tissue inhibits adipocyte differentiation; as a result, the transgenic nSREBP-1c mice develop a syndrome resembling human lipodystrophy, which includes a loss of peripheral white adipose tissue, diabetes, and fatty livers (Shimomura, I., Hammer, R. E., Richardson, J. A., Ikemoto, S., Bashmakov, Y., Goldstein, J. L., and Brown, M. S. (1998) Genes Dev. 12, 3182–3194). In striking contrast, nSREBP-1a overexpression in fat resulted in the hypertrophy of fully differentiated adipocytes, no diabetes, and mild hepatic steatosis. These results suggest that nSREBP-1a and nSREBP-1c have distinct roles in adipocyte fat metabolism in vivo. Sterol regulatory element-binding proteins (SREBPs) are a family of membrane-bound transcription factors that regulate cholesterol and fatty acid homeostasis. In mammals, three SREBP isoforms designated SREBP-1a, SREBP-1c, and SREBP-2 have been identified. SREBP-1a and SREBP-1c are derived from the same gene by virtue of alternatively spliced first exons. SREBP-1a has a longer transcriptional activation domain and is a more potent transcriptional activator than SREBP-1c in cultured cells and liver. Here, we describe the physiologic consequences of overexpressing the nuclear form of SREBP-1a (nSREBP-1a) in adipocytes of mice using the adipocyte-specific aP2 promoter (aP2-nSREBP-1a). The transgenic aP2-nSREBP-1a mice developed markedly enlarged white and brown adipocytes that were fully differentiated. Adipocytes isolated from aP2-nSREBP-1a mice had significantly increased rates of fatty acid synthesis and enhanced fatty acid secretion. The increased production and release of fatty acids from adipocytes led, in turn, to a fatty liver. Overexpression of the alternative SREBP-1 isoform, nSREBP-1c, in adipose tissue inhibits adipocyte differentiation; as a result, the transgenic nSREBP-1c mice develop a syndrome resembling human lipodystrophy, which includes a loss of peripheral white adipose tissue, diabetes, and fatty livers (Shimomura, I., Hammer, R. E., Richardson, J. A., Ikemoto, S., Bashmakov, Y., Goldstein, J. L., and Brown, M. S. (1998) Genes Dev. 12, 3182–3194). In striking contrast, nSREBP-1a overexpression in fat resulted in the hypertrophy of fully differentiated adipocytes, no diabetes, and mild hepatic steatosis. These results suggest that nSREBP-1a and nSREBP-1c have distinct roles in adipocyte fat metabolism in vivo. Obesity is an ever-increasing public health concern that it is now estimated to afflict at least one-third of the United States population (2Mokdad A.H. Bowman B.A. Ford E.S. Vinicor F. Marks J.S. Koplan J.P. J. Am. Med. Assoc. 2001; 286: 1195-1200Crossref PubMed Scopus (2241) Google Scholar). Understanding how progenitor cells differentiate into adipocytes may be critical for the development of anti-obesity therapies.Sterol regulatory element-binding proteins (SREBPs) 1The abbreviations used are: SREBPs, sterol regulatory element-binding proteins; nSREBP, nuclear sterol regulatory element-binding protein; WAT, white adipose tissue; BAT, brown adipose tissue; C/EBPα, cCAAT/enhancer-binding protein-α; PPAR, peroxisome proliferator-activated receptor; UCP-1, uncoupling protein-1; FFA, free fatty acid; Pref-1, preadipocyte factor-1; TNF-α, tumor necrosis factor-α.1The abbreviations used are: SREBPs, sterol regulatory element-binding proteins; nSREBP, nuclear sterol regulatory element-binding protein; WAT, white adipose tissue; BAT, brown adipose tissue; C/EBPα, cCAAT/enhancer-binding protein-α; PPAR, peroxisome proliferator-activated receptor; UCP-1, uncoupling protein-1; FFA, free fatty acid; Pref-1, preadipocyte factor-1; TNF-α, tumor necrosis factor-α. are a family of membrane-bound transcription factors that principally regulate lipid synthesis (3Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (2940) Google Scholar), but they have also been implicated in adipocyte differentiation (1Shimomura I. Hammer R.E. Richardson J.A. Ikemoto S. Bashmakov Y. Goldstein J.L. Brown M.S. Genes Dev. 1998; 12: 3182-3194Crossref PubMed Scopus (673) Google Scholar, 4Tontonoz P. Kim J.B. Graves R.A. Spiegelman B.M. Mol. Cell. Biol. 1993; 13: 4753-4759Crossref PubMed Scopus (533) Google Scholar, 5Kim J.B. Spiegelman B.M. Genes Dev. 1996; 10: 1096-1107Crossref PubMed Scopus (834) Google Scholar). SREBPs belong to the larger basic helix-loop-helix leucine zipper family of transcription factors, but are unique because they are synthesized as ∼1150-amino acid precursors bound to the endoplasmic reticulum and nuclear envelope (3Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (2940) Google Scholar). To be active, SREBPs undergo a sequential two-step cleavage process to release the transcriptionally active NH2-terminal portion of the protein, designated the nuclear form. Once the nuclear form is released from the membrane, it can enter the nucleus and bind to the promoters of target genes to activate transcription.Three SREBP isoforms (designated SREBP-1a, SREBP-1c, and SREBP-2) have been identified in humans and animals (3Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (2940) Google Scholar). SREBP-1a and SREBP-1c are encoded by the same gene that undergoes alternative splicing in which the two transcripts are driven by different promoters (6Yokoyama C. Wang X. Briggs M.R. Admon A. Wu J. Hua X. Goldstein J.L. Brown M.S. Cell. 1993; 75: 187-197Abstract Full Text PDF PubMed Scopus (783) Google Scholar, 7Hua X. Wu J. Goldstein J.L. Brown M.S. Hobbs H.H. Genomics. 1995; 25: 667-673Crossref PubMed Scopus (246) Google Scholar). SREBP-1a and SREBP-1c differ only in the first exon, which encodes a portion of an acidic transcriptional activation domain (6Yokoyama C. Wang X. Briggs M.R. Admon A. Wu J. Hua X. Goldstein J.L. Brown M.S. Cell. 1993; 75: 187-197Abstract Full Text PDF PubMed Scopus (783) Google Scholar, 7Hua X. Wu J. Goldstein J.L. Brown M.S. Hobbs H.H. Genomics. 1995; 25: 667-673Crossref PubMed Scopus (246) Google Scholar). SREBP-1a has a longer activation domain and is much more potent than SREBP-1c in transcriptionally activating known target genes in cultured cells (8Pai J.-t. Guryev O. Brown M.S. Goldstein J.L. J. Biol. Chem. 1998; 273: 26138-26148Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar) and liver (9Shimano H. Horton J.D. Shimomura I. Hammer R.E. Brown M.S. Goldstein J.L. J. Clin. Invest. 1997; 99: 846-854Crossref PubMed Scopus (675) Google Scholar). The relative amounts of the SREBP-1a and SREBP-1c transcripts differ among cells and organs. SREBP-1a is the predominant transcript in most cultured cells. In most animal tissues, including liver and adipose tissue, the SREBP-1c transcript is predominant (10Shimomura I. Shimano H. Horton J.D. Goldstein J.L. Brown M.S. J. Clin. Invest. 1997; 99: 838-845Crossref PubMed Scopus (634) Google Scholar, 11Shimomura I. Bashmakov Y. Shimano H. Horton J.D. Goldstein J.L. Brown M.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12354-12359Crossref PubMed Scopus (126) Google Scholar, 12Shimomura I. Bashmakov Y. Ikemoto S. Horton J.D. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13656-13661Crossref PubMed Scopus (619) Google Scholar). The third SREBP isoform, designated SREBP-2, is produced by a different gene, and it contains a long acidic activation domain resembling that of SREBP-1a (13Hua X. Yokoyama C. Wu J. Briggs M.R. Brown M.S. Goldstein J.L. Wang X. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11603-11607Crossref PubMed Scopus (499) Google Scholar).In liver, all SREBP isoforms are capable of activating the same families of genes, but they do so with varying relative efficiencies. SREBP-2 preferentially activates multiple genes in the cholesterol biosynthetic pathway, whereas SREBP-1a and SREBP-1c preferentially activate genes involved in the synthesis of fatty acids and triglycerides (14Horton J.D. Goldstein J.L. Brown M.S. J. Clin. Invest. 2002; 109: 1125-1131Crossref PubMed Scopus (3683) Google Scholar). The function and transcriptional activation properties of SREBPs in tissues other than liver have been only partially characterized. Of particular interest is the in vivo function of SREBPs in adipocytes owing to the potential role SREBP-1 isoforms may have in adipogenesis (4Tontonoz P. Kim J.B. Graves R.A. Spiegelman B.M. Mol. Cell. Biol. 1993; 13: 4753-4759Crossref PubMed Scopus (533) Google Scholar).Overexpression of transcriptionally active nuclear SREBP-1c (nSREBP-1c) in adipocytes of transgenic mice using the fat-specific aP2 promoter inhibits adipocyte differentiation and produces a syndrome in mice that shares the general features of congenital generalized lipodystrophy in humans (1Shimomura I. Hammer R.E. Richardson J.A. Ikemoto S. Bashmakov Y. Goldstein J.L. Brown M.S. Genes Dev. 1998; 12: 3182-3194Crossref PubMed Scopus (673) Google Scholar). The amount of white adipose tissue (WAT) in the lipodystrophic mice is markedly reduced, and many of the residual cells have the histologic appearance of immature adipocytes. These changes are associated with a marked reduction in many mRNAs encoding genes involved in adipogenesis such as C/EBPα, PPARγ, and adipsin. The majority of adipocytes in brown adipose tissue (BAT) are histologically similar to immature white adipocytes. The mRNAs for uncoupling protein-1 (UCP-1) and the additional adipogenic genes listed above are also markedly reduced. The transgenic mice also exhibit profound insulin resistance, hyperglycemia, and enlarged fatty livers.To investigate the physiologic function of the SREBP-1a isoform in adipocytes, we have produced transgenic mice that overexpress transcriptionally active nSREBP-1a in WAT and BAT using the aP2 enhancer/promoter (aP2-nSREBP-1a mice). The phenotype of aP2-nSREBP-1a mice differs completely from that observed in aP2-nSREBP-1c mice (1Shimomura I. Hammer R.E. Richardson J.A. Ikemoto S. Bashmakov Y. Goldstein J.L. Brown M.S. Genes Dev. 1998; 12: 3182-3194Crossref PubMed Scopus (673) Google Scholar). Overexpression of nSREBP-1a in adipose tissue did not inhibit adipocyte differentiation, but rather activated the entire pathway of cholesterol and fatty acid biosynthetic genes, leading to increased lipid accumulation in the adipocytes. The white adipocytes from the transgenic mice were massively enlarged and contained excess triglyceride. The brown adipocytes contained large unilocular lipid droplets that produced a histologic appearance resembling mature white adipocytes. Isolated adipocytes from aP2-nSREBP-1a mice manifested significantly increased rates of fatty acid synthesis, which were associated with increased fatty acid secretion into the medium. In vivo, this led to a higher influx of free fatty acids (FFAs) from adipose tissues into liver, resulting in a fatty liver.EXPERIMENTAL PROCEDURESGeneral Materials and Methods—DNA manipulations were performed using standard molecular techniques (15Sambrook J. Russell D.W. Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press, New York2001Google Scholar). [9,10-3H]Oleic acid and [stearoyl-1-14C]stearoyl-CoA were purchased from American Radio-labeled Chemicals (St. Louis, MO), and potassium [1-14C]palmitate was purchased from PerkinElmer Life Sciences. All other chemicals used were from Sigma unless otherwise stated. The plasma concentration of cholesterol, triglycerides, insulin, glucose, and free fatty acids and the contents of liver cholesterol and triglyceride were measured as described previously (1Shimomura I. Hammer R.E. Richardson J.A. Ikemoto S. Bashmakov Y. Goldstein J.L. Brown M.S. Genes Dev. 1998; 12: 3182-3194Crossref PubMed Scopus (673) Google Scholar, 16Ishibashi S. Brown M.S. Goldstein J.L. Gerard R.D. Hammer R.E. Herz J. J. Clin. Invest. 1993; 92: 883-893Crossref PubMed Scopus (1262) Google Scholar).Transgene Construction and Generation of Transgenic aP2nSREBP-1a Mice—An expression plasmid containing the mouse aP2 promoter fused to amino acids 1–460 of the human SREBP-1a cDNA was constructed as follows. A plasmid containing 5.4 kb of the aP2 gene promoter in pBluescript II SK(+) (designated paP2-Pro) was a generous gift from Dr. B. M. Spiegelman. A 1.5-kb EcoRI-SalI cDNA fragment of human SREBP-1a encoding amino acids 1–460 was excised from the pPEPCK-SREBP-1a460 plasmid (17Shimano H. Horton J.D. Hammer R.E. Shimomura I. Brown M.S. Goldstein J.L. J. Clin. Invest. 1996; 98: 1575-1584Crossref PubMed Scopus (695) Google Scholar) and ligated into the EcoRI and SalI sites of the pCMV5 vector. 2D. W. Russell, unpublished construct. The resulting plasmid was designated pCMV5-SREBP-1a460. A 1.7-kb EcoRI-SphI fragment containing SREBP-1a (amino acids 1–460) and a 3′-polyadenylation signal were excised from pCMV5-SREBP-1a460 and blunted using DNA polymerase I (Klenow fragment). The resulting fragment was designated SREBP-1a460-poly(A). SREBP-1a460-poly(A) was subcloned into the SmaI site of paP2-Pro to yield the final plasmid designated paP2-SREBP-1a460. The integrity of this plasmid was confirmed by DNA sequencing.Techniques used for generating transgenic mice were as described previously (18Hofmann S.L. Russell D.W. Brown M.S. Goldstein J.L. Hammer R.E. Science. 1988; 239: 1277-1281Crossref PubMed Scopus (84) Google Scholar). A 7.1-kb NotI-ClaI fragment containing the aP2 promoter, SREBP-1a460, and poly (A+) tail from paP2-SREBP-1a460 was gel-purified as described (17Shimano H. Horton J.D. Hammer R.E. Shimomura I. Brown M.S. Goldstein J.L. J. Clin. Invest. 1996; 98: 1575-1584Crossref PubMed Scopus (695) Google Scholar). A total of 613 fertilized eggs were microinjected with the aP2-SREBP-1a construct and survived to the two-cell stage. Among the 74 offspring, 19 (26%) had integrated the transgene as determined by dot-blot hybridization of DNA from tail homogenates. Of 19 founder mice subjected to partial lipectomy, 10 (53%) expressed the human SREBP-1a transcript as determined by Northern blotting (17Shimano H. Horton J.D. Hammer R.E. Shimomura I. Brown M.S. Goldstein J.L. J. Clin. Invest. 1996; 98: 1575-1584Crossref PubMed Scopus (695) Google Scholar). Mice with high levels of expression were bred to (C57BL/6J × SJL)F1 mice (Jackson Laboratories, Bar Harbor, ME), and four independent transgenic lines were established. The transgenic lines were designated aP2-nSREBP-1a lines A, B, C, and D for lines 854-3, 951-4, 855-3, and 852-3 in order of the relative transgene mRNA expression in WAT from highest to lowest. All mice were housed in colony cages, maintained on a 14-h light/10-h dark cycle, and fed Teklad mouse/rat diet 7002 from Harlan Teklad Premier Laboratory Diets (Madison, WI).Immunoblot Analysis—Nuclear extracts and membrane fractions were prepared from mouse livers and adipose tissue as described previously (19Sheng Z. Otani H. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 935-938Crossref PubMed Scopus (275) Google Scholar). Immunoblot analysis of mouse SREBP-1 and SREBP-2 was carried out exactly as described previously (17Shimano H. Horton J.D. Hammer R.E. Shimomura I. Brown M.S. Goldstein J.L. J. Clin. Invest. 1996; 98: 1575-1584Crossref PubMed Scopus (695) Google Scholar, 20Shimano H. Shimomura I. Hammer R.E. Goldstein J.L. Brown M.S. Horton J.D. J. Clin. Invest. 1997; 100: 2115-2124Crossref PubMed Scopus (352) Google Scholar).Blot Hybridization of RNA—All cDNA probes for Northern blot analysis have been described previously (12Shimomura I. Bashmakov Y. Ikemoto S. Horton J.D. Brown M.S. Goldstein J.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13656-13661Crossref PubMed Scopus (619) Google Scholar, 17Shimano H. Horton J.D. Hammer R.E. Shimomura I. Brown M.S. Goldstein J.L. J. Clin. Invest. 1996; 98: 1575-1584Crossref PubMed Scopus (695) Google Scholar, 20Shimano H. Shimomura I. Hammer R.E. Goldstein J.L. Brown M.S. Horton J.D. J. Clin. Invest. 1997; 100: 2115-2124Crossref PubMed Scopus (352) Google Scholar, 21Shimomura I. Shimano H. Korn B.S. Bashmakov Y. Horton J.D. J. Biol. Chem. 1998; 273: 35299-35306Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). Total RNA was prepared using an RNA STAT-60™ (Tel-Test Inc., Friendswood, TX). For Northern gel analysis, equal aliquots of total RNA made from adipose tissue from each mouse listed in Table I (line A) were pooled (total, 10 μg), subjected to electrophoresis on a 1% formaldehyde-agarose gel, and transferred to Hybond N+ membrane (Amersham Biosciences). Hybridization conditions and cDNA probe preparations were carried out as described (1Shimomura I. Hammer R.E. Richardson J.A. Ikemoto S. Bashmakov Y. Goldstein J.L. Brown M.S. Genes Dev. 1998; 12: 3182-3194Crossref PubMed Scopus (673) Google Scholar). The resulting bands were quantified by exposing the filter to a BAS 1000 Bio Imaging Analyzer (Fuji Medical Systems, Stamford, CT). The -fold change was calculated after the signal was normalized to the signal generated by glyceraldehyde-3-phosphate dehydrogenase.Table IPhenotypic comparison of wild-type and aP2-nSREBP-1a miceLine ALine BParameter5-Week-old10-Week-old4-Week-oldWild-typeTransgenicWild-typeTransgenicWild-typeTransgenicSex5 males5 males6 males8 males4 males4 malesBody weight (g)19.4 ± 0.420.1 ± 1.323.7 ± 0.825.4 ± 1.217.2 ± 0.318.4 ± 0.9Liver weight (g)1.3 ± 0.11.8 ± 0.1ap < 0.001 (Student's t test).1.4 ± 0.072.2 ± 0.18ap < 0.001 (Student's t test).0.88 ± 0.071.32 ± 0.03Epididymal fat weight (g)0.13 ± 0.020.13 ± 0.020.22 ± 0.030.17 ± 0.03bp < 0.05 (Student's t test).0.086 ± 0.0120.096 ± 0.010Brown fat weight (g)0.042 ± 0.0100.149 ± 0.0200.054 ± 0.0040.240 ± 0.0470.049 ± 0.0070.147 ± 0.008ap < 0.001 (Student's t test).Liver cholesterol content (mg/g)2.3 ± 0.072.1 ± 0.061.9 ± 0.031.9 ± 0.082.4 ± 0.202.5 ± 0.10Liver triglyceride content (mg/g)8.4 ± 0.5516.2 ± 1.2ap < 0.001 (Student's t test).4.2 ± 0.610.5 ± 0.4ap < 0.001 (Student's t test).7.3 ± 0.5012.5 ± 1.1ap < 0.001 (Student's t test).Plasma cholesterol (mg/dl)80 ± 566 ± 583 ± 2.279 ± 3.792 ± 1187 ± 7Plasma triglycerides (mg/dl)199 ± 32178 ± 22158 ± 25148 ± 20127 ± 29149 ± 13Plasma insulin (ng/ml)1.1 ± 0.30.8 ± 0.21.5 ± 0.33.2 ± 1.20.7 ± 0.20.9 ± 0.3Plasma glucose (mg/dl)148 ± 13153 ± 11164 ± 19155 ± 18139 ± 23151 ± 19Plasma FFA (mm)752 ± 1021320 ± 159ap < 0.001 (Student's t test).602 ± 1361096 ± 161bp < 0.05 (Student's t test).686 ± 1061042 ± 141bp < 0.05 (Student's t test).a p < 0.001 (Student's t test).b p < 0.05 (Student's t test). Open table in a new tab RNase Protection Assay—RNase protection assays for preadipocyte factor-1 (Pref-1) and mouse tumor necrosis factor-α (TNF-α) were done using the cDNA template described previously (1Shimomura I. Hammer R.E. Richardson J.A. Ikemoto S. Bashmakov Y. Goldstein J.L. Brown M.S. Genes Dev. 1998; 12: 3182-3194Crossref PubMed Scopus (673) Google Scholar). Antisense cRNA was transcribed with [α-32P]CTP (20 mCi/ml) using bacteriophage T7 RNA polymerase (Ambion Inc., Austin, TX). The specific activities of the transcribed RNAs were measured in each experiment and were in the range of 1.7–2.6 × 109 cpm/μg for all RNAs except for β-actin RNA, which was 5.3–8.1 × 108 cpm/μg. Aliquots of total RNA (20 μg) from each sample were subjected to the RNase protection assay using a HybSpeed™ RPA kit (Ambion Inc.). Each assay tube contained a cRNA probe for the mRNA to be tested plus a cRNA probe complementary to the mRNA of β-actin. In preparing the probes, we adjusted the specific activity of [α-32P]CTP to give a β-actin signal comparable to the test mRNAs. After RNase A/T1 digestion, protected fragments were separated on 8 m urea and 4.8% polyacrylamide gels. The gels were then dried and subjected to autoradiography using reflection film and intensifying screens (DuPont). The dried gels were also analyzed quantitatively with a Bio Imaging Analyzer using BAS 1000 MacBAS software (Fuji Medical Systems). The level of β-actin mRNA in each RNA sample was used to normalize signals obtained for the test mRNAs.Acyl-CoA Synthetase and Stearoyl-CoA Activity Measurements— Liver acyl-CoA synthetase activity was measured in 8-week-old nonfasted male mice (five wild-type and five aP2-nSREBP-1a, line A) during the early light cycle. Acyl-CoA synthetase activity was measured as described (22Bar-Tana J. Rose G. Shapiro B. Biochem. J. 1971; 122: 353-380Crossref PubMed Scopus (132) Google Scholar) with minor modifications. Briefly, the tissues were washed with 5 mm Tris-HCl buffer (pH 7.4) containing 0.25 m sucrose, and the adventitia was carefully removed. The tissues were then minced, homogenized with 10 volumes of 5 mm Tris-HCl buffer (pH 7.4) containing 0.25 m sucrose, and centrifuged at 800 × g for 5 min. Five μg of protein from the supernatant was added to a reaction mixture that contained 150 mm Tris-HCl buffer (pH 7.4), 0.1% Triton X-100, 50 mm MgCl2,20mm ATP, 200 mm potassium [1-14C]palmitate (56 mCi/mmol), 200 mm CoASH, and 2.25 mm glutathione in a total volume of 250 μl. This mixture was incubated for 5 min at 37 °C. The reaction was terminated by adding 1 ml of isopropyl alcohol, heptane, and 1 n H2SO4 (40:10:1). Following thorough mixing, 0.35 ml of water and 0.6 ml of heptane were added and mixed by shaking for 5 min. The upper organic phase was removed and discarded. The lower water phase was washed twice with 0.6 ml of heptane, and the radioactivity in 0.7 ml of the lower phase was counted. Hepatic stearoyl-CoA desaturase activity was measured in 8-week-old non-fasted male mice (seven wild-type and six aP2-nSREBP-1a, line A) during the early light cycle as described previously (21Shimomura I. Shimano H. Korn B.S. Bashmakov Y. Horton J.D. J. Biol. Chem. 1998; 273: 35299-35306Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar).Primary Adipocytes—Adipocytes were prepared and pooled from epididymal and brown fat pads from four 12-week-old male wild-type and four aP2-nSREBP-1a line A littermate mice. Fat depots were resected under aseptic conditions, and adipocytes were isolated by collagenase digestion according to the procedure of Rodbell (23Rodbell M. J. Biol. Chem. 1964; 239: 375-380Abstract Full Text PDF PubMed Google Scholar) with minor modifications as described below. The fat pads were minced in Krebs-Ringer HEPES buffer (pH 7.4) containing 5 mmd-glucose, 2% bovine serum albumin, 135 mm NaCl, 2.2 mm CaCl2·2H2O, 1.25 mm MgSO4·7H2O, 0.45 mm KH2PO4, 2.17 mm Na2HPO4, and 10 mm HEPES. Adipose tissue fragments were digested in Krebs-Ringer HEPES buffer with 1.25 mg/ml rat type II collagenase at 37° C with gentle shaking at 60 cycles/min for 45 min. The resultant cell suspension was diluted in 13 ml of cold Krebs-Ringer HEPES buffer. Isolated adipocytes were separated from undigested tissue by filtration through four layers of cheesecloth and washed three times with cold Krebs-Ringer HEPES buffer. For washing, cells were resuspended in 50 ml of buffer and centrifuged at 400 × g for 5 min. The final wash was with 13 ml of the culture medium (Dulbecco's modified Eagle's medium containing 10% fetal bovine serum). Floating cells were collected as primary adipocytes, plated on 60-mm collagen-treated dishes (catalog no. 40416, BD Biosciences), and cultured at 37 °C in 9% CO2. Adipocytes were incubated with Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 0.1 mg/ml gentamycin, and 0.5 mm sodium [14C]acetate (18 dpm/pmol) for 5 h. Incorporation of [14C]acetate into fatty acids and cholesterol was determined as described previously (8Pai J.-t. Guryev O. Brown M.S. Goldstein J.L. J. Biol. Chem. 1998; 273: 26138-26148Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). The data are expressed as nanomoles of [14C]acetate incorporated into fatty acids and picomoles of [14C]acetate incorporated into cholesterol per μg of cellular DNA.RESULTSUnregulated overexpression of nSREBP-1a exclusively in adipose tissue was achieved by the construction of a transgene encoding only the transcriptionally active fragment of human SREBP-1a driven by the adipose-specific enhancer/promoter of the aP2 gene (24Ross S.R. Graves R.A. Greenstein A. Platt K.A. Shyu H.-L. Mellovitz B. Spiegelman B.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9590-9594Crossref PubMed Scopus (184) Google Scholar). The truncated SREBP-1a transgene product terminates prior to the membrane attachment domain and therefore enters the nucleus directly without a requirement for proteolysis (3Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (2940) Google Scholar).We studied four lines of transgenic mice that were derived from independent founders (lines A–D). Fig. 1A shows the results from Northern blot analysis of mRNA derived from the aP2-nSREBP-1a transgene in line A mice, confirming that the transgene mRNA was expressed abundantly in both WAT and BAT. The transgene mRNA levels in WAT from lines B–D were 70, 35, and 30% of that measured in line A (data not shown). By Northern blotting, there was no evidence of transgene mRNA expression in liver (Fig. 1A) or in brain, spleen, and kidney (data not shown). As shown in Fig. 1B, transgenic aP2nSREBP-1a line A mice had abundant nSREBP-1a protein expressed in adipose tissue.Phenotypic Parameters in aP2-nSREBP-1a Mice— Table I lists the relevant phenotypic parameters measured in aP2nSREBP-1a mice (lines A and B) and their respective wild-type littermates. The most profound abnormalities were in aP2nSREBP-1a line A mice. Similar but less profound changes were observed in the line B mice, which expressed lower levels of nSREBP-1a. The significant abnormalities noted in aP2nSREBP-1a mice were as follows: 1) enlarged livers, 2) elevated hepatic triglyceride content, 3) enlarged interscapular brown fat pads, and 4) increased concentration of plasma FFAs. Plasma levels of cholesterol, triglyceride, insulin, and glucose were not significantly altered. Of note, the epididymal fat pad weights were not significantly different from those in wild-type littermates.Histologic Changes in Fat of Transgenic Mice—The histology of WAT and BAT from representative wild-type and aP2nSREBP-1a line A mice is shown in Fig. 2. The epididymal fat pad from a 40-day-old wild-type mouse had mature adipocytes of uniform size containing unilocular fat droplets (Fig. 2A). The epididymal fat pad from a 40-day-old transgenic littermate mouse also showed mature adipocytes with distinct unilocular vacuoles; however, a small percentage of adipocytes were significantly larger than the wild-type adipocytes (Fig. 2B). This phenotype became more prominent as the mice aged. Fig. 2C shows WAT from a 70-day-old transgenic mouse. The diameter of most adipocytes from aP2-nSREBP-1a mice was markedly greater than that of adipocytes from wild-type mice. Many fat pads from mice of this age also showed an inflammatory response, characterized by a monocytic infiltration.Fig. 2Representative histologic sections of WAT and BAT. A and B, epididymal fat pads from 40-day-old wild-type and transgenic aP2-nSREBP-1a line A littermate mice, respectively; C, epididymal fat pad from a 70-day-old transgenic aP2-nSREBP-1a line A mouse; D and E, interscapular brown fat pads from 40-day-old wild-type and transgenic littermate mice, respectively. Note that the adipocytes of the white and brown fat pads from the transgenic mice were markedly enlarged (hematoxylin and eosin stain; magnification ×112).View Large Image Figure ViewerDownload (PPT)Equally dramatic histologic changes were observed in BAT from aP2-nSREBP-1a mice. BAT from a 40-day-old wild-type mouse consisted of small multilocular adipocytes, as shown in Fig. 2D. In contrast, most adipocytes in BAT from aP2nSREBP-1a mice were markedly enlarged and contained predominantly unilocular fat droplets. This resulted in a histologic appearance similar to that of normal WAT (Fig. 2E).Expression of mRNAs for Target Genes in Fat— Fig. 3 shows multiple Northern blots measuring mRNAs for genes encoding proteins involved in lipid metabolism and adipogenesis in WAT (Fig. 3A) and BAT (Fig. 3B) from wild-type and aP2nSREBP-1a line A mice. Significant increases were measured in the mRNAs for the low density lipoprotein receptor as well as enzymes involved in cholesterol biosynthesis in WAT and BAT from transgenic mice. In particular, the mRNA encoding 3-hydroxy-3-methylglutaryl-CoA reductase, a rate-controlling enzyme in the cholesterol biosynthetic pathway, was increased by >20-fold in WAT and BAT. The mRNAs for the fatty acid and triglyceride biosynthetic enzymes acetyl-CoA carboxylase-1, fatty-acid synthase, and" @default.
• W2150767656 created "2016-06-24" @default.
• W2150767656 creator A5018425993 @default.
• W2150767656 creator A5026744789 @default.
• W2150767656 creator A5037246488 @default.
• W2150767656 creator A5043003801 @default.
• W2150767656 creator A5044891277 @default.
• W2150767656 date "2003-09-01" @default.
• W2150767656 modified "2023-10-18" @default.
• W2150767656 title "Overexpression of Sterol Regulatory Element-binding Protein-1a in Mouse Adipose Tissue Produces Adipocyte Hypertrophy, Increased Fatty Acid Secretion, and Fatty Liver" @default.
• W2150767656 cites W1492605893 @default.
• W2150767656 cites W1513078811 @default.
• W2150767656 cites W1570662124 @default.
• W2150767656 cites W1992864925 @default.
• W2150767656 cites W2001195445 @default.
• W2150767656 cites W2002711503 @default.
• W2150767656 cites W2004920315 @default.
• W2150767656 cites W2009616432 @default.
• W2150767656 cites W2023719883 @default.
• W2150767656 cites W2024217191 @default.
• W2150767656 cites W2027874598 @default.
• W2150767656 cites W2030610610 @default.
• W2150767656 cites W2037094605 @default.
• W2150767656 cites W2038381362 @default.
• W2150767656 cites W2039999119 @default.
• W2150767656 cites W2040641918 @default.
• W2150767656 cites W2077971415 @default.
• W2150767656 cites W2078600472 @default.
• W2150767656 cites W2081393029 @default.
• W2150767656 cites W2086072924 @default.
• W2150767656 cites W2090895117 @default.
• W2150767656 cites W2102161788 @default.
• W2150767656 cites W2107735993 @default.
• W2150767656 cites W2119390770 @default.
• W2150767656 cites W2123652234 @default.
• W2150767656 cites W2124615340 @default.
• W2150767656 cites W2129138669 @default.
• W2150767656 cites W2134243830 @default.
• W2150767656 cites W2143873575 @default.
• W2150767656 cites W2155846381 @default.
• W2150767656 cites W2157817725 @default.
• W2150767656 cites W2220565486 @default.
• W2150767656 cites W4252021281 @default.
• W2150767656 doi "https://doi.org/10.1074/jbc.m306540200" @default.
• W2150767656 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12855691" @default.
• W2150767656 hasPublicationYear "2003" @default.
• W2150767656 type Work @default.
• W2150767656 sameAs 2150767656 @default.
• W2150767656 citedByCount "206" @default.
• W2150767656 countsByYear W21507676562012 @default.
• W2150767656 countsByYear W21507676562013 @default.
• W2150767656 countsByYear W21507676562014 @default.
• W2150767656 countsByYear W21507676562015 @default.
• W2150767656 countsByYear W21507676562016 @default.
• W2150767656 countsByYear W21507676562017 @default.
• W2150767656 countsByYear W21507676562018 @default.
• W2150767656 countsByYear W21507676562019 @default.
• W2150767656 countsByYear W21507676562020 @default.
• W2150767656 countsByYear W21507676562021 @default.
• W2150767656 countsByYear W21507676562022 @default.
• W2150767656 countsByYear W21507676562023 @default.
• W2150767656 crossrefType "journal-article" @default.
• W2150767656 hasAuthorship W2150767656A5018425993 @default.
• W2150767656 hasAuthorship W2150767656A5026744789 @default.
• W2150767656 hasAuthorship W2150767656A5037246488 @default.
• W2150767656 hasAuthorship W2150767656A5043003801 @default.
• W2150767656 hasAuthorship W2150767656A5044891277 @default.
• W2150767656 hasBestOaLocation W21507676561 @default.
• W2150767656 hasConcept C104317684 @default.
• W2150767656 hasConcept C126322002 @default.
• W2150767656 hasConcept C134018914 @default.
• W2150767656 hasConcept C167414201 @default.
• W2150767656 hasConcept C171089720 @default.
• W2150767656 hasConcept C185592680 @default.
• W2150767656 hasConcept C24972589 @default.
• W2150767656 hasConcept C2776175234 @default.
• W2150767656 hasConcept C2776834966 @default.
• W2150767656 hasConcept C2776941171 @default.
• W2150767656 hasConcept C2778163477 @default.
• W2150767656 hasConcept C2778718757 @default.
• W2150767656 hasConcept C2778772119 @default.
• W2150767656 hasConcept C2779134260 @default.
• W2150767656 hasConcept C49039625 @default.
• W2150767656 hasConcept C543025807 @default.
• W2150767656 hasConcept C55493867 @default.
• W2150767656 hasConcept C71924100 @default.
• W2150767656 hasConcept C85051948 @default.
• W2150767656 hasConcept C86803240 @default.
• W2150767656 hasConcept C95444343 @default.
• W2150767656 hasConceptScore W2150767656C104317684 @default.
• W2150767656 hasConceptScore W2150767656C126322002 @default.
• W2150767656 hasConceptScore W2150767656C134018914 @default.
• W2150767656 hasConceptScore W2150767656C167414201 @default.
• W2150767656 hasConceptScore W2150767656C171089720 @default.
• W2150767656 hasConceptScore W2150767656C185592680 @default.
• W2150767656 hasConceptScore W2150767656C24972589 @default.
• W2150767656 hasConceptScore W2150767656C2776175234 @default.
• W2150767656 hasConceptScore W2150767656C2776834966 @default.

• next