Carbohydrate Regulation of Hepatic Gene Expression

Hepatic expression of the genes encoding L-type pyruvate kinase (L-PK) and S14 is induced in rats upon feeding them a high carbohydrate, low fat diet. A carbohydrate response element (ChoRE) containing two CACGTG-type E boxes has been mapped in the 5′-flanking region of both of these genes. The nature of the ChoRE suggests that a member of the basic/helix-loop-helix/leucine zipper family of proteins may be responsible for mediating the response to carbohydrate. Indeed, the upstream stimulatory factor (USF), a ubiquitous basic/helix-loop-helix/leucine zipper protein, is present in hepatic nuclear extracts and binds to the ChoREs of L-PK and S14 in vitro We have conducted experiments to determine whether USF is involved in the carbohydrate-mediated regulation of L-PK and S14. For this purpose, dominant negative forms of USF that are capable of heterodimerizing with endogenous USF but not of binding to DNA were expressed in primary hepatocytes. Expression of these forms did not block either S14 or L-PK induction by glucose. In addition, we have constructed mutant ChoREs that retain their carbohydrate responsiveness but have lost the ability to bind USF. Together, these data suggest that USF is not the carbohydrate-responsive factor that stimulates S14 and L-PK expression and that a distinct hepatic factor is likely to be responsible for the transcriptional response. Hepatic expression of the genes encoding L-type pyruvate kinase (L-PK) and S14 is induced in rats upon feeding them a high carbohydrate, low fat diet. A carbohydrate response element (ChoRE) containing two CACGTG-type E boxes has been mapped in the 5′-flanking region of both of these genes. The nature of the ChoRE suggests that a member of the basic/helix-loop-helix/leucine zipper family of proteins may be responsible for mediating the response to carbohydrate. Indeed, the upstream stimulatory factor (USF), a ubiquitous basic/helix-loop-helix/leucine zipper protein, is present in hepatic nuclear extracts and binds to the ChoREs of L-PK and S14 in vitro We have conducted experiments to determine whether USF is involved in the carbohydrate-mediated regulation of L-PK and S14. For this purpose, dominant negative forms of USF that are capable of heterodimerizing with endogenous USF but not of binding to DNA were expressed in primary hepatocytes. Expression of these forms did not block either S14 or L-PK induction by glucose. In addition, we have constructed mutant ChoREs that retain their carbohydrate responsiveness but have lost the ability to bind USF. Together, these data suggest that USF is not the carbohydrate-responsive factor that stimulates S14 and L-PK expression and that a distinct hepatic factor is likely to be responsible for the transcriptional response. INTRODUCTIONThe mammalian liver is capable of converting excess dietary carbohydrate into triglycerides for storage. Feeding of a diet high in simple carbohydrates and low in fats results in the induction of many hepatic enzymes involved in lipogenesis, including liver-type pyruvate kinase (L-PK), 1The abbreviations used are: L-PKliver-type pyruvate kinaseChoREcarbohydrate response elementb/HLH/LZbasic/helix-loop-helix/leucine zipperUSFupstream stimulatory factorEMSAelectrophoretic mobility shift assayCATchloramphenicol acetyltransferasePCRpolymerase chain reactionSREBPsterol response element-binding protein. fatty acid synthase, malic enzyme, and acetyl-CoA carboxylase (for reviews see 1Hillgartner F.B. Salati L.M. Goodridge A.G. Phys. Rev. 1995; 75: 47-76Crossref PubMed Scopus (392) Google Scholar and 2Towle H.C. J. Biol. Chem. 1995; 270: 23235-23238Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). In each case, the increased enzyme production correlates with an increase in mRNA levels, and in some of these cases the increase in mRNA has been shown to be due to elevated transcription of the corresponding genes (3Vaulont S. Munnich A. Decaux J.-F. Kahn A. J. Biol. Chem. 1986; 261: 7621-7625Abstract Full Text PDF PubMed Google Scholar, 4Paulauskis J.D. Sul H.S. J. Biol. Chem. 1989; 264: 574-577Abstract Full Text PDF PubMed Google Scholar, 5Ntambi J.M. J. Biol. Chem. 1992; 267: 10925-10930Abstract Full Text PDF PubMed Google Scholar, 6Kim K.-S. Park S.-W. Kim Y.-S. Biochem. Biophys. Res. Commun. 1992; 189: 264-271Crossref PubMed Scopus (22) Google Scholar, 7Rongnoparut P. Verdon C.P. Gehnrich S.C. Sul H.S. J. Biol. Chem. 1991; 266: 8086-8091Abstract Full Text PDF PubMed Google Scholar, 8Shin D.-H. Paulauskis J.D. Moustaid N. Sul H.S. J. Biol. Chem. 1991; 266: 23834-23839Abstract Full Text PDF PubMed Google Scholar). Little is known about the molecular mechanisms responsible for this transcriptional response.In our efforts to characterize the regulation of lipogenic gene expression, we have been studying the genes encoding L-PK and S14 in rat liver. These genes are transcriptionally induced upon carbohydrate feeding (3Vaulont S. Munnich A. Decaux J.-F. Kahn A. J. Biol. Chem. 1986; 261: 7621-7625Abstract Full Text PDF PubMed Google Scholar, 9Jacoby D.B. Zilz N.D. Towle H.C. J. Biol. Chem. 1989; 264: 17623-17626Abstract Full Text PDF PubMed Google Scholar, 10Jump D.B. Bell A. Santiago V. J. Biol. Chem. 1990; 265: 3474-3478Abstract Full Text PDF PubMed Google Scholar). This induction can be reproduced in cultured primary hepatocytes by varying the glucose concentrations in the media (9Jacoby D.B. Zilz N.D. Towle H.C. J. Biol. Chem. 1989; 264: 17623-17626Abstract Full Text PDF PubMed Google Scholar, 11Mariash C.N. Seelig S. Schwartz H.L. Oppenheimer J.H. J. Biol. Chem. 1986; 261: 9583-9586Abstract Full Text PDF PubMed Google Scholar, 12Decaux J.-F. Antoine B. Kahn A. J. Biol. Chem. 1989; 264: 11584-11590Abstract Full Text PDF PubMed Google Scholar). Increased glucose metabolism is thought to generate the intracellular signal in the regulatory pathway. Although the exact nature of this pathway remains unknown, several possible intracellular signals have been proposed (13Foufelle F. Gouhot B. Pegorier J.-P. Perdereau D. Girard J. Ferre P. J. Biol. Chem. 1992; 267: 20543-20546Abstract Full Text PDF PubMed Google Scholar, 14Mariash C.N. Schwartz H.L. Metabolism. 1986; 35: 452-456Abstract Full Text PDF PubMed Scopus (12) Google Scholar, 15Doiron B. Cuif M.-H. Chen R. Kahn A. J. Biol. Chem. 1996; 271: 5321-5324Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). While insulin is necessary for the regulation, it has been shown to play only a permissive role in promoting effective glucose metabolism (16Doiron B. Cuif M.-H. Kahn A. Diaz-Guerra M.-J.M. J. Biol. Chem. 1994; 269: 10213-10216Abstract Full Text PDF PubMed Google Scholar, 17Lefrançois-Martinez A.-M. Diaz-Guerra M.-J.M. Vallet V. Kahn A. Antoine B. FASEB J. 1994; 8: 89-96Crossref PubMed Scopus (61) Google Scholar). Sequences responsible for mediating the carbohydrate response have been mapped in the 5′-flanking region of both the S14 and L-PK genes (18Thompson K.S. Towle H.C. J. Biol. Chem. 1991; 266: 8679-8682Abstract Full Text PDF PubMed Google Scholar, 19Bergot M.-O. Diaz-Guerra M.-J.M. Puzenat N. Raymondjean M. Kahn A. Nucleic Acids Res. 1992; 20: 1871-1878Crossref PubMed Scopus (153) Google Scholar, 20Shih H. Towle H.C. J. Biol. Chem. 1992; 267: 13222-13228Abstract Full Text PDF PubMed Google Scholar, 21Shih H. Towle H.C. J. Biol. Chem. 1994; 269: 9380-9387Abstract Full Text PDF PubMed Google Scholar, 22Liu Z. Thompson K.S. Towle H.C. J. Biol. Chem. 1993; 268: 12787-12795Abstract Full Text PDF PubMed Google Scholar). Both genes contain a common regulatory element that consists of two 5′-CACGTG-type E box motifs separated by 5 base pairs (19Bergot M.-O. Diaz-Guerra M.-J.M. Puzenat N. Raymondjean M. Kahn A. Nucleic Acids Res. 1992; 20: 1871-1878Crossref PubMed Scopus (153) Google Scholar, 23Shih H.-M. Liu Z. Towle H.C. J. Biol. Chem. 1995; 270: 21991-21997Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). Linking multiple copies of this element to a basal promoter is sufficient to induce carbohydrate-responsive activity; this element is thus referred to as a carbohydrate response element (ChoRE). However, in the context of the natural promoter, each gene contains a distinct accessory factor site adjacent to the ChoRE that is necessary for the full extent of the response (22Liu Z. Thompson K.S. Towle H.C. J. Biol. Chem. 1993; 268: 12787-12795Abstract Full Text PDF PubMed Google Scholar, 23Shih H.-M. Liu Z. Towle H.C. J. Biol. Chem. 1995; 270: 21991-21997Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 24Diaz Guerra M.-J.M. Bergot M.-O. Martinez A. Cuif M.-H. Kahn A. Raymondjean M. Mol. Cell. Biol. 1993; 13: 7725-7733Crossref PubMed Scopus (97) Google Scholar).The presence of the CACGTG-like sites within the ChoRE suggests that a member of the basic/helix-loop-helix/leucine zipper (b/HLH/LZ) family of transcription factors may be involved in the carbohydrate-mediated regulation (25Murre C. McGaw P.S. Vaessin H. Caudy M. Jan L.Y. Lan Y.N. Cabrera C.V. Buskin J.N. Hauschka S.D. Lassar A.B. Weintraub H. Baltimore D. Cell. 1989; 58: 537-544Abstract Full Text PDF PubMed Scopus (1293) Google Scholar, 26Prendergast G.C. Ziff E.B. Science. 1991; 251: 186-189Crossref PubMed Scopus (428) Google Scholar). These proteins all contain a basic DNA binding region followed by helix-loop-helix and leucine zipper dimerization domains. The b/HLH/LZ class includes Myc and its related family members, along with other proteins such as TFE3, TFEB, SREBP1/ADD1, and the upstream stimulatory factor (USF) (27Blackwell T.K. Kretzner L. Blackwood E.M. Eisenman R.N. Weintraub H. Science. 1990; 250: 1149-1151Crossref PubMed Scopus (697) Google Scholar, 28Beckmann H. Su L.-K. Kadesch T. Genes 4: 167-179Crossref PubMed Scopus (352) Google Scholar, 29Carr C.S. Sharp P.A. Mol. Cell. Biol. 1990; 10: 4384-4388Crossref PubMed Scopus (189) Google Scholar, 30Ogawa N. Oshima Y. Mol. Cell. Biol. 1990; 10: 2224-2236Crossref PubMed Scopus (104) Google Scholar, 31Yokoyama 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 (781) Google Scholar, 32Tontonoz P. Kim J.M. Graves R.A. Spiegelman B.M. Mol. Cell. Biol. 1993; 13: 4753-4759Crossref PubMed Scopus (533) Google Scholar, 33Gregor P.D. Sawadogo M. Roeder R.G. Genes 4: 1730-1740Crossref PubMed Scopus (433) Google Scholar). Of these, USF appears to be the predominant b/HLH/LZ factor in hepatic nuclear extracts. USF was first identified by its ability to bind to the upstream stimulatory element of the adenovirus major late promoter (34Carthew R.W. Chodosh L.A. Sharp P.A. Cell. 1985; 43: 439-448Abstract Full Text PDF PubMed Scopus (404) Google Scholar, 35Sawadogo M. Roeder R. Cell. 1985; 43: 165-175Abstract Full Text PDF PubMed Scopus (716) Google Scholar). In vitro electrophoretic mobility shift assays (EMSAs) using hepatic nuclear extracts produced one major shifted complex that bound specifically to the S14 and L-PK ChoREs and which was “supershifted” by the addition of an antibody against USF (21Shih H. Towle H.C. J. Biol. Chem. 1994; 269: 9380-9387Abstract Full Text PDF PubMed Google Scholar, 22Liu Z. Thompson K.S. Towle H.C. J. Biol. Chem. 1993; 268: 12787-12795Abstract Full Text PDF PubMed Google Scholar, 24Diaz Guerra M.-J.M. Bergot M.-O. Martinez A. Cuif M.-H. Kahn A. Raymondjean M. Mol. Cell. Biol. 1993; 13: 7725-7733Crossref PubMed Scopus (97) Google Scholar). However, the USF binding site from the adenovirus major late promoter was unable to substitute for the ChoRE in either the L-PK or the S14 gene, indicating that USF alone is not capable of mediating the response (21Shih H. Towle H.C. J. Biol. Chem. 1994; 269: 9380-9387Abstract Full Text PDF PubMed Google Scholar). Additionally, the ubiquitous nature of USF (36Sirito M. Lin Q. Maity T. Sawadogo M. Nucleic Acids Res. 1994; 22: 427-433Crossref PubMed Scopus (291) Google Scholar), with its ability to with in other genes that are not by glucose D. C.V. J. Biol. Chem. 1991; 266: Full Text PDF PubMed Google Scholar, R.W. Chodosh L.A. Sharp P.A. 267: 13222-13228Abstract Full Text PDF PubMed Google Scholar). hepatocytes were from rats a with to a were using the in E and with and glucose for were for in containing either or glucose and for For using dominant negative forms of was to the a of the This has been shown to increase the that cultured hepatocytes to elevated glucose concentrations H.-M. Towle H.C. 1995; Google Scholar). the addition of were in low glucose for the with low or high glucose. of to its forms was by followed by with and and and were using to the ChoRE E boxes and the accessory factor site Mol. Cell. Biol. 1991; PubMed Scopus Google Scholar). The exact of each is shown in and were used for each one upstream from the site and one These pairs either an site or a site in of the to be The chain was using (9Jacoby D.B. Zilz N.D. Towle H.C. J. Biol. Chem. 1989; 264: 17623-17626Abstract Full Text PDF PubMed Google as a were with and or and to generate from the and from the to These were into the and sites of Sequences of the were by DNA Expression and which and L.A. R.W. Sharp P.A. Science. PubMed Scopus (97) Google Scholar), were by Sawadogo D. of were into the S. H. H. J. Biol. Chem. 1989; 264: Full Text PDF PubMed Google Scholar). This was by a to the site and the into the site of A containing was by the chain the using with or sites to generate a encoding and The was with and and into A expression was by and Roeder The was by using to the and of were upstream and from either of the basic each an encoding the basic region containing an The or were with and or and and into the and sites of by The was by first the to the and it with a The was with and and into a containing the of which was by The was from this by with and and into into which ChoRE were has been (18Thompson K.S. Towle H.C. J. Biol. Chem. 1991; 266: 8679-8682Abstract Full Text PDF PubMed Google Scholar). were containing the shown in each by a These were into the site in upstream of the L-PK basal were to to determine the and of the ChoRE were containing the The of the E box motifs are and in from the in each These were into (18Thompson K.S. Towle H.C. J. Biol. Chem. 1991; 266: 8679-8682Abstract Full Text PDF PubMed Google upstream of the L-PK basal and were and a high carbohydrate, low fat diet for extracts were by the of Genes PubMed Scopus Google Scholar). were as H. Towle H.C. J. Biol. Chem. 1992; 267: 13222-13228Abstract Full Text PDF PubMed Google Scholar). A of and of nuclear were to in a H. Towle H.C. J. Biol. Chem. 1992; 267: 13222-13228Abstract Full Text PDF PubMed Google Scholar). For were to the to the addition of nuclear have identified by a carbohydrate response element and in the 5′-flanking region of the S14 gene H.-M. Liu Z. Towle H.C. J. Biol. Chem. 1995; 270: 21991-21997Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). This is to the ChoRE of the L-PK gene the response element (19Bergot M.-O. Diaz-Guerra M.-J.M. Puzenat N. Raymondjean M. Kahn A. Nucleic Acids Res. 1992; 20: 1871-1878Crossref PubMed Scopus (153) Google In either case, two CACGTG-like E boxes separated by 5 base pairs are In the of the S14 the has only a with the of the of this into the E box that it a to the in to glucose H.-M. Liu Z. Towle H.C. J. Biol. Chem. 1995; 270: 21991-21997Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). we have shown that of the E box in the context of the natural S14 gene the ability of the S14 promoter to to glucose. of the E box the response. has shown that both E boxes of the L-PK gene are for (19Bergot M.-O. Diaz-Guerra M.-J.M. Puzenat N. Raymondjean M. Kahn A. Nucleic Acids Res. 1992; 20: 1871-1878Crossref PubMed Scopus (153) Google Scholar, 22Liu Z. Thompson K.S. Towle H.C. J. Biol. Chem. 1993; 268: 12787-12795Abstract Full Text PDF PubMed Google Scholar). the presence of two E box motifs is a for mediating the glucose response of these two of the lipogenic enzyme S14 and L-PK ChoREs are sufficient to a response to glucose in multiple copies to a basal promoter or to an binding However, in either an adjacent accessory factor site is for the full extent of the response in the context of the natural For the L-PK promoter, the factor is capable of binding to the ChoRE and its (22Liu Z. Thompson K.S. Towle H.C. J. Biol. Chem. 1993; 268: 12787-12795Abstract Full Text PDF PubMed Google Scholar, 24Diaz Guerra M.-J.M. Bergot M.-O. Martinez A. Cuif M.-H. Kahn A. Raymondjean M. Mol. Cell. Biol. 1993; 13: 7725-7733Crossref PubMed Scopus (97) Google Scholar). In the of the S14 gene, the accessory site to the ChoRE binds an as nuclear factor H.-M. Liu Z. Towle H.C. J. Biol. Chem. 1995; 270: 21991-21997Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). this element in the context of the natural S14 gene the glucose response. this site appears to play an role in A has been in several other genes to transcriptional regulation by and For the gene encoding the low is are low by the of the transcriptional factor M.R. C. Wang X. Brown M.S. Goldstein J.L. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar). The of by is but it is by binding of the ubiquitous factor to adjacent sites within this gene H.C. Oppenheimer J.H. S. A. PubMed Scopus Google Scholar, L. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). with in the of the fatty acid and A carboxylase J.M. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar, J.M. J.M. S. A. 1996; PubMed Scopus Google Scholar). and Kim S. Kim J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google have that binding is for glucose of the A carboxylase this binding site is as an accessory factor to an ChoRE or whether it is the binding site for the carbohydrate-responsive factor remains to be the S14 the L-PK promoter appears to contain binding sites for presence of CACGTG-type E boxes within the ChoRE suggests that a member of the b/HLH/LZ family of transcription factors is involved in In USF has from this family as a for the carbohydrate-responsive its ability to bind to the S14 or L-PK ChoREs in vitro (22Liu Z. Thompson K.S. Towle H.C. J. Biol. Chem. 1993; 268: 12787-12795Abstract Full Text PDF PubMed Google Scholar, S. Puzenat N. F. M. Kahn A. Raymondjean M. J. Mol. Biol. 1989; PubMed Scopus Google Scholar). However, we have that USF is involved in the glucose regulation of the L-PK or S14 genes in The expression of dominant negative forms of USF did not the carbohydrate response of either ChoRE in primary the proteins were to block the of This is in with that of A.-M. Martinez A. Antoine B. Raymondjean M. Kahn A. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). This that a dominant negative of to block the L-PK glucose response in the is present to this in the be The of the primary hepatocytes a While the extent of the these two is are not For as to the primary the glucose response in the is This is due to the that a other is responsible for the Although not specifically it is possible that the contain endogenous USF the primary this were dominant negative USF be to the endogenous USF. in the is the of the dominant negative USF forms and of the glucose induction While we used in the present a may be that is to the endogenous USF and thus USF the other it is that the effective of USF to expression of transcription factor or that is for transcription of the L-PK In this the of the dominant negative of USF the glucose response be these we an in which the role of USF in the glucose response be The USF binding and glucose responsiveness was in a of ChoRE The of that USF binding and against a role for USF. In of the of the motifs to in an increased response to glucose in the context of S14 ChoRE This was not by in experiments and bound USF the element containing motifs by This binding was both for nuclear which consists of (36Sirito M. Lin Q. Maity T. Sawadogo M. Nucleic Acids Res. 1994; 22: 427-433Crossref PubMed Scopus (291) Google Scholar, B. A.-M. A. Kahn A. Raymondjean M. Martinez A. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar), and which be present as of each E box to in a ChoRE that as as the by T.K. J. A. Kretzner L. Eisenman R.N. Weintraub H. Mol. Cell. Biol. 1993; 13: PubMed Scopus Google Scholar), this did not bind USF either in assays or by a role of USF or in glucose appears is that from both our and has shown that USF can bind to the ChoRE (22Liu Z. Thompson K.S. Towle H.C. J. Biol. Chem. 1993; 268: 12787-12795Abstract Full Text PDF PubMed Google Scholar, S. Puzenat N. F. M. Kahn A. Raymondjean M. J. Mol. Biol. 1989; PubMed Scopus Google Scholar). This is with the that USF expression of L-PK promoter into that this binding is of that to the is to a binding with a to transcription USF is the carbohydrate-responsive While we the that USF may as a complex with factor that its for ChoRE with USF we this to be likely that other of the b/HLH/LZ family have been to with is that and 5 the exact E box motifs but in to each These two in their to a glucose that the of the two E boxes is this we have a in which the E box is present in a with mutant this was of a glucose we that the the ChoRE E boxes is for regulation H.-M. Liu Z. Towle H.C. J. Biol. Chem. 1995; 270: 21991-21997Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). Together, these results that a two binding factors for an effective glucose USF is not the carbohydrate-responsive we not see factor capable of binding to the ChoRE in we that such a factor or complex may be present in liver but a such that this complex in nuclear extracts of liver may not be A was in the for which binds to the response element of the low Although proteins are present in nuclear extracts that bind the response to be the factor mediating the response to X. Briggs M.R. Hua X. C. Goldstein J.L. Brown M.S. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar). upon was the We are using the element which is not bound by as a to for such a complex involved in carbohydrate INTRODUCTIONThe mammalian liver is capable of converting excess dietary carbohydrate into triglycerides for storage. Feeding of a diet high in simple carbohydrates and low in fats results in the induction of many hepatic enzymes involved in lipogenesis, including liver-type pyruvate kinase (L-PK), 1The abbreviations used are: L-PKliver-type pyruvate kinaseChoREcarbohydrate response elementb/HLH/LZbasic/helix-loop-helix/leucine zipperUSFupstream stimulatory factorEMSAelectrophoretic mobility shift assayCATchloramphenicol acetyltransferasePCRpolymerase chain reactionSREBPsterol response element-binding protein. fatty acid synthase, malic enzyme, and acetyl-CoA carboxylase (for reviews see 1Hillgartner F.B. Salati L.M. Goodridge A.G. Phys. Rev. 1995; 75: 47-76Crossref PubMed Scopus (392) Google Scholar and 2Towle H.C. J. Biol. Chem. 1995; 270: 23235-23238Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). In each case, the increased enzyme production correlates with an increase in mRNA levels, and in some of these cases the increase in mRNA has been shown to be due to elevated transcription of the corresponding genes (3Vaulont S. Munnich A. Decaux J.-F. Kahn A. J. Biol. Chem. 1986; 261: 7621-7625Abstract Full Text PDF PubMed Google Scholar, 4Paulauskis J.D. Sul H.S. J. Biol. Chem. 1989; 264: 574-577Abstract Full Text PDF PubMed Google Scholar, 5Ntambi J.M. J. Biol. Chem. 1992; 267: 10925-10930Abstract Full Text PDF PubMed Google Scholar, 6Kim K.-S. Park S.-W. Kim Y.-S. Biochem. Biophys. Res. Commun. 1992; 189: 264-271Crossref PubMed Scopus (22) Google Scholar, 7Rongnoparut P. Verdon C.P. Gehnrich S.C. Sul H.S. J. Biol. Chem. 1991; 266: 8086-8091Abstract Full Text PDF PubMed Google Scholar, 8Shin D.-H. Paulauskis J.D. Moustaid N. Sul H.S. J. Biol. Chem. 1991; 266: 23834-23839Abstract Full Text PDF PubMed Google Scholar). Little is known about the molecular mechanisms responsible for this transcriptional response.In our efforts to characterize the regulation of lipogenic gene expression, we have been studying the genes encoding L-PK and S14 in rat liver. These genes are transcriptionally induced upon carbohydrate feeding (3Vaulont S. Munnich A. Decaux J.-F. Kahn A. J. Biol. Chem. 1986; 261: 7621-7625Abstract Full Text PDF PubMed Google Scholar, 9Jacoby D.B. Zilz N.D. Towle H.C. J. Biol. Chem. 1989; 264: 17623-17626Abstract Full Text PDF PubMed Google Scholar, 10Jump D.B. Bell A. Santiago V. J. Biol. Chem. 1990; 265: 3474-3478Abstract Full Text PDF PubMed Google Scholar). This induction can be reproduced in cultured primary hepatocytes by varying the glucose concentrations in the media (9Jacoby D.B. Zilz N.D. Towle H.C. J. Biol. Chem. 1989; 264: 17623-17626Abstract Full Text PDF PubMed Google Scholar, 11Mariash C.N. Seelig S. Schwartz H.L. Oppenheimer J.H. J. Biol. Chem. 1986; 261: 9583-9586Abstract Full Text PDF PubMed Google Scholar, 12Decaux J.-F. Antoine B. Kahn A. J. Biol. Chem. 1989; 264: 11584-11590Abstract Full Text PDF PubMed Google Scholar). Increased glucose metabolism is thought to generate the intracellular signal in the regulatory pathway. Although the exact nature of this pathway remains unknown, several possible intracellular signals have been proposed (13Foufelle F. Gouhot B. Pegorier J.-P. Perdereau D. Girard J. Ferre P. J. Biol. Chem. 1992; 267: 20543-20546Abstract Full Text PDF PubMed Google Scholar, 14Mariash C.N. Schwartz H.L. Metabolism. 1986; 35: 452-456Abstract Full Text PDF PubMed Scopus (12) Google Scholar, 15Doiron B. Cuif M.-H. Chen R. Kahn A. J. Biol. Chem. 1996; 271: 5321-5324Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). While insulin is necessary for the regulation, it has been shown to play only a permissive role in promoting effective glucose metabolism (16Doiron B. Cuif M.-H. Kahn A. Diaz-Guerra M.-J.M. J. Biol. Chem. 1994; 269: 10213-10216Abstract Full Text PDF PubMed Google Scholar, 17Lefrançois-Martinez A.-M. Diaz-Guerra M.-J.M. Vallet V. Kahn A. Antoine B. FASEB J. 1994; 8: 89-96Crossref PubMed Scopus (61) Google Scholar). Sequences responsible for mediating the carbohydrate response have been mapped in the 5′-flanking region of both the S14 and L-PK genes (18Thompson K.S. Towle H.C. J. Biol. Chem. 1991; 266: 8679-8682Abstract Full Text PDF PubMed Google Scholar, 19Bergot M.-O. Diaz-Guerra M.-J.M. Puzenat N. Raymondjean M. Kahn A. Nucleic Acids Res. 1992; 20: 1871-1878Crossref PubMed Scopus (153) Google Scholar, 20Shih H. Towle H.C. J. Biol. Chem. 1992; 267: 13222-13228Abstract Full Text PDF PubMed Google Scholar, 21Shih H. Towle H.C. J. Biol. Chem. 1994; 269: 9380-9387Abstract Full Text PDF PubMed Google Scholar, 22Liu Z. Thompson K.S. Towle H.C. J. Biol. Chem. 1993; 268: 12787-12795Abstract Full Text PDF PubMed Google Scholar). Both genes contain a common regulatory element that consists of two 5′-CACGTG-type E box motifs separated by 5 base pairs (19Bergot M.-O. Diaz-Guerra M.-J.M. Puzenat N. Raymondjean M. Kahn A. Nucleic Acids Res. 1992; 20: 1871-1878Crossref PubMed Scopus (153) Google Scholar, 23Shih H.-M. Liu Z. Towle H.C. J. Biol. Chem. 1995; 270: 21991-21997Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). Linking multiple copies of this element to a basal promoter is sufficient to induce carbohydrate-responsive activity; this element is thus referred to as a carbohydrate response element (ChoRE). However, in the context of the natural promoter, each gene contains a distinct accessory factor site adjacent to the ChoRE that is necessary for the full extent of the response (22Liu Z. Thompson K.S. Towle H.C. J. Biol. Chem. 1993; 268: 12787-12795Abstract Full Text PDF PubMed Google Scholar, 23Shih H.-M. Liu Z. Towle H.C. J. Biol. Chem. 1995; 270: 21991-21997Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 24Diaz Guerra M.-J.M. Bergot M.-O. Martinez A. Cuif M.-H. Kahn A. Raymondjean M. Mol. Cell. Biol. 1993; 13: 7725-7733Crossref PubMed Scopus (97) Google Scholar).The presence of the CACGTG-like sites within the ChoRE suggests that a member of the basic/helix-loop-helix/leucine zipper (b/HLH/LZ) family of transcription factors may be involved in the carbohydrate-mediated regulation (25Murre C. McGaw P.S. Vaessin H. Caudy M. Jan L.Y. Lan Y.N. Cabrera C.V. Buskin J.N. Hauschka S.D. Lassar A.B. Weintraub H. Baltimore D. Cell. 1989; 58: 537-544Abstract Full Text PDF PubMed Scopus (1293) Google Scholar, 26Prendergast G.C. Ziff E.B. Science. 1991; 251: 186-189Crossref PubMed Scopus (428) Google Scholar). These proteins all contain a basic DNA binding region followed by helix-loop-helix and leucine zipper dimerization domains. The b/HLH/LZ class includes Myc and its related family members, along with other proteins such as TFE3, TFEB, SREBP1/ADD1, and the upstream stimulatory factor (USF) (27Blackwell T.K. Kretzner L. Blackwood E.M. Eisenman R.N. Weintraub H. Science. 1990; 250: 1149-1151Crossref PubMed Scopus (697) Google Scholar, 28Beckmann H. Su L.-K. Kadesch T. Genes 4: 167-179Crossref PubMed Scopus (352) Google Scholar, 29Carr C.S. Sharp P.A. Mol. Cell. Biol. 1990; 10: 4384-4388Crossref PubMed Scopus (189) Google Scholar, 30Ogawa N. Oshima Y. Mol. Cell. Biol. 1990; 10: 2224-2236Crossref PubMed Scopus (104) Google Scholar, 31Yokoyama 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 (781) Google Scholar, 32Tontonoz P. Kim J.M. Graves R.A. Spiegelman B.M. Mol. Cell. Biol. 1993; 13: 4753-4759Crossref PubMed Scopus (533) Google Scholar, 33Gregor P.D. Sawadogo M. Roeder R.G. Genes 4: 1730-1740Crossref PubMed Scopus (433) Google Scholar). Of these, USF appears to be the predominant b/HLH/LZ factor in hepatic nuclear extracts. USF was first identified by its ability to bind to the upstream stimulatory element of the adenovirus major late promoter (34Carthew R.W. Chodosh L.A. Sharp P.A. Cell. 1985; 43: 439-448Abstract Full Text PDF PubMed Scopus (404) Google Scholar, 35Sawadogo M. Roeder R. Cell. 1985; 43: 165-175Abstract Full Text PDF PubMed Scopus (716) Google Scholar). In vitro electrophoretic mobility shift assays (EMSAs) using hepatic nuclear extracts produced one major shifted complex that bound specifically to the S14 and L-PK ChoREs and which was “supershifted” by the addition of an antibody against USF (21Shih H. Towle H.C. J. Biol. Chem. 1994; 269: 9380-9387Abstract Full Text PDF PubMed Google Scholar, 22Liu Z. Thompson K.S. Towle H.C. J. Biol. Chem. 1993; 268: 12787-12795Abstract Full Text PDF PubMed Google Scholar, 24Diaz Guerra M.-J.M. Bergot M.-O. Martinez A. Cuif M.-H. Kahn A. Raymondjean M. Mol. Cell. Biol. 1993; 13: 7725-7733Crossref PubMed Scopus (97) Google Scholar). However, the USF binding site from the adenovirus major late promoter was unable to substitute for the ChoRE in either the L-PK or the S14 gene, indicating that USF alone is not capable of mediating the response (21Shih H. Towle H.C. J. Biol. Chem. 1994; 269: 9380-9387Abstract Full Text PDF PubMed Google Scholar). Additionally, the ubiquitous nature of USF (36Sirito M. Lin Q. Maity T. Sawadogo M. Nucleic Acids Res. 1994; 22: 427-433Crossref PubMed Scopus (291) Google Scholar), with its ability to with in other genes that are not by glucose D. C.V. J. Biol. Chem. 1991; 266: Full Text PDF PubMed Google Scholar, R.W. Chodosh L.A. Sharp P.A. & Dev. PubMed Scopus Google Scholar, L.A. R.W. Sharp P.A. Science. PubMed Scopus (97) Google Scholar), the as to the of the response is We thus to determine whether or not USF is involved in the regulatory pathway induced by carbohydrate.