The transcription of fatty acid synthase (FAS), a central enzyme in de novo lipogenesis, is dramatically induced by fasting͞refeeding and insulin. We reported that upstream stimulatory factor binding to the ؊65 E-box is required for induction of the FAS transcription by insulin in 3T3-L1 adipocytes. On the other hand, we recently found that two upstream 5 regions are required for induction in vivo by fasting͞refeeding and insulin; one at ؊278 to ؊131 albeit at a low level, and the other at ؊444 to ؊278 with an E-box at ؊332 where upstream stimulatory factor functions for maximal induction. Here, we generated double transgenic mice carrying the chloramphenicol acetyltransferase reporter driven by the various 5 deletions of the FAS promoter region and a truncated active form of the sterol regulatory element (SRE) binding protein (SREBP)-1a. We found that SREBP participates in the nutritional regulation of the FAS promoter and that the region between ؊278 and ؊131 bp is required for SREBP function. We demonstrate that SREBP binds the ؊150 canonical SRE present between ؊278 and ؊131, and SREBP can function through the ؊150 SRE in cultured cells. These in vivo and in vitro results indicate that SREBP is involved in the nutritional induction of the FAS promoter via the ؊278͞؊131 region and that the ؊150 SRE is the target sequence. F atty acid synthase (FAS) plays a central role in de novo lipogenesis in mammals (1). By the action of its seven active sites, FAS catalyzes all of the reaction steps in the conversion of acetyl-CoA and malonyl-CoA to palmitate. FAS activity is not known to be regulated by allosteric effectors or covalent modification. However, FAS concentration is exquisitely sensitive to nutritional and hormonal status in lipogenic tissues, liver, and adipose tissue (1-3). FAS mRNA is not detectable in livers of fasted mice but refeeding a high-carbohydrate, fat-free diet increases FAS mRNA levels dramatically, because of the changes in transcription (4, 5). Increased circulating insulin and decreased glucagon levels may participate in the regulation of FAS expression. FAS gene transcription was not detectable in fasted or refed streptozotocin-diabetic mice but increased by insulin administration (5). We mapped an insulin response sequence to Ϫ71 to Ϫ50 bp that contains a core E-box (CAT-GTG) by transfection of serial 5Ј deletions of the FAS promoter fused to the luciferase (LUC) reporter gene into 3T3-L1 adipocytes (6, 7). Binding to the Ϫ65 E-box by upstream stimulatory factor (USF)1 and USF2, which belong to the basic helix-loophelix leucine zipper family of transcription factors, was required for the insulin-mediated induction of the FAS gene (6-8). We also demonstrated that the 2.1-kb 5Ј flanking sequence is sufficient for the tissue-specific and hormonal͞nutritional regulation of the FAS gene in the in vivo context. Recently, by generating transgenic mice bearing various 5Ј deletion FAS promoter-chloramphenicol acetyltransferase (CAT) genes, we found that, unlike in cultured cell system, two 5Ј prom...

Pref-1 is an epidermal growth factor-like domain-containing transmembrane protein that is cleaved to generate a soluble factor. It is abundant in 3T3-L1 preadipocytes but absent in mature adipocytes. Constitutive expression of pref-1 or the addition of its ectodomain inhibits adipogenesis. We find that the pref-1 gene is an early target of dexamethasone, a component of the dexamethasone/methylisobutylxanthine differentiation mixture used routinely for adipoconversion. The time course of the decrease in pref-1 mRNA by dexamethasone reflected the pref-1 mRNA halflife determined by actinomycin D treatment. Nuclear run-on assays showed that dexamethasone attenuates pref-1 transcription. We demonstrate a correlation between pref-1 down-regulation and adipoconversion by varying the time period and concentration of dexamethasone. Increasing the dexamethasone treatment from 2 to 4 days resulted in a time-dependent pref-1 down-regulation and increased differentiation as measured by adipocyte marker mRNAs. The dexamethasone concentration between 1 and 10 nM showed a dose-dependent decrease in pref-1 mRNA and an enhancement of adipogenesis. To test the hypothesis that dexamethasone initiation of adipoconversion may be via down-regulation of pref-1, we lowered endogenous pref-1 mRNA levels by stably transfecting 3T3-L1 preadipocytes with antisense pref-1. At 1 M, antisense cells had enhanced adipose conversion; a similar degree of differentiation occurred with 2 nM dexamethasone, a concentration that does not support differentiation of control 3T3-L1 cells. We conclude that dexamethasone-mediated repression of pref-1 contributes to the mechanisms whereby glucocorticoids promote adipogenesis.Preadipocyte factor-1 (pref-1) 1 is an epidermal growth factor (EGF) repeat domain-containing transmembrane protein with an anti-adipogenic function (1-4). Pref-1 is highly expressed in 3T3-L1 preadipocytes and is totally absent after their differentiation to mature adipocytes. Interfering with this normal repression of pref-1 during adipogenesis by constitutive expression of pref-1 in 3T3-L1 preadipocytes or by the addition of soluble pref-1 ectodomain markedly decreases adipocyte conversion (1-4); pref-1 expression is also abolished during the adipose conversion of primary rat preadipocytes in cultures, and their differentiation is inhibited by the pref-1 ectodomain (5). Taken together, these data, and the detection of soluble pref-1 in circulation (6), support a functional role for pref-1 in adipocyte differentiation in vivo. In this regard, pref-1 mRNA levels were recently shown to be elevated by adipose-specific expression of SREBP-1c in a transgenic mouse model of congenital generalized lipodystrophy, a condition characterized by poorly developed white and brown adipose tissue (7). Pref-1 belongs to that class of proteins that can act as either transmembrane or soluble molecules; membrane-associated pref-1 is cleaved at two sites in the extracellular domain, thereby extending its potential range of function (4). Therefore, pref-1 i...

Human studies support the relationship between high intake of fructose-sweetened beverages and type 2 diabetes, but there is a debate on whether this effect is fructose-specific or it is merely associated to an excessive caloric intake. Here we investigate the effects of 2 months’ supplementation to female rats of equicaloric 10% w/v fructose or glucose solutions on insulin sensitivity in target tissues. Fructose supplementation caused hepatic deposition of triglycerides and changed the fatty acid profile of this fraction, with an increase in monounsaturated and a decrease in polyunsaturated species, but did not cause inflammation and oxidative stress. Fructose but not glucose-supplemented rats displayed an abnormal glucose tolerance test, and did not show increased phosphorylation of V-akt murine thymoma viral oncogene homolog-2 (Akt) in white adipose tissue and liver after insulin administration. In skeletal muscle, phosphorylation of Akt and of Akt substrate of 160 kDA (AS160) was not impaired but the expression of the glucose transporter type 4 (GLUT4) in the plasma membrane was reduced only in fructose-fed rats. In conclusion, fructose but not glucose supplementation causes fatty liver without inflammation and oxidative stress and impairs insulin signaling in the three major insulin-responsive tissues independently from the increase in energy intake.