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© 2004 The American Society for Nutritional Sciences J. Nutr. 134:2205-2210, September 2004

Nutrient-Gene Interactions

Starvation and Feeding a High-Carbohydrate, Low-Fat Diet Regulate the Expression Sterol Regulatory Element-Binding Protein-1 in Chickens1

Yanqiao Zhang and F. Bradley Hillgartner2

Department of Biochemistry and Molecular Pharmacology, School of Medicine, West Virginia University, Morgantown, WV 265061

2To whom correspondence should be addressed. E-mail: fbhillgartner{at}hsc.wvu.edu.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
In mammalian liver, the mature form of sterol regulatory element-binding protein-1c (SREBP-1c) is an important activator of a wide array of genes involved in triacylglycerol biosynthesis. Starvation and feeding a high-carbohydrate, low-fat diet modulate the concentration of mature SREBP-1c primarily by a pretranslational mechanism. It is not known whether alterations in nutritional status regulate the concentration of SREBPs in nonmammalian species. In this study, we found that in previously starved chicks, feeding a high-carbohydrate, low-fat diet stimulated a robust increase (14-fold at 5 h of feeding) in the concentration of mature SREBP-1 in liver. Feeding a high-carbohydrate, low-fat diet also increased the concentration of precursor SREBP-1 and SREBP-1 messenger RNA in chick liver; however, the magnitude of this effect was substantially lower than that observed for mature SREBP-1. DNA binding experiments demonstrated that 3 protein complexes containing SREBP bound the acetyl-CoA carboxylase-{alpha} (ACC{alpha}) sterol regulatory element (SRE) in chick liver and that the binding activity of 2 of these complexes was increased by consumption of a high-carbohydrate, low-fat diet. Additional analyses showed that feeding a high-carbohydrate, low-fat diet had no effect on the concentration of mature SREBP-2 and the binding of SREBP-2 to the ACC{alpha} SRE in chick liver. These results indicate that alterations in the concentration of mature SREBP-1 play a role in mediating the effects of starvation and feeding a high-carbohydrate, low-fat diet on ACC{alpha} transcription in chick liver and that diet-induced changes in mature SREBP-1 concentration in chick liver are mediated primarily by a posttranslational mechanism.

KEY WORDS: • transcription • fatty acid synthesis • acetyl-CoA carboxylase • insulin • thyroid hormone

Sterol regulatory element binding proteins (SREBP)3 are basic helix-loop-helix-leucine zipper (bHLH-Zip) transcription factors that play an important role in controlling genes involved in the synthesis of cholesterol and fatty acids (1). SREBPs are synthesized as inactive precursor proteins that are embedded in endoplasmic reticulum membranes (2,3). To become transcriptionally active, precursor SREBP is escorted to the Golgi apparatus, where it undergoes a sequential 2-step proteolytic cleavage catalyzed by site-1 protease and site-2 protease. This process releases an amino-terminal SREBP fragment that is referred to as the mature form. Mature SREBP is transported into the nucleus, where it binds sterol regulatory elements (SRE) of genes involved in lipid biosynthesis. Three isoforms of SREBP have been identified in mammals. Two of these isoforms, designated SREBP-1a and SREBP-1c, are expressed from the same gene. They vary in sequence at their amino termini due to the use of alternative promoters and leading exons. The third isoform, designated SREBP-2, is expressed from a separate gene. SREBP-1c and SREBP-2 are the major isoforms of SREBP expressed in mammalian liver (4). Results from studies using transgenic mice indicate that SREBP-1c is more effective than SREBP-2 in modulating expression of genes involved in fatty acid synthesis, whereas SREBP-2 is more effective than SREBP-1c in modulating genes involved in cholesterol synthesis (5–7).

In mammals, alterations in nutritional status regulate the concentration of SREBP-1c in liver. For example, feeding previously starved mice a high-carbohydrate, low-fat diet increases the hepatic concentration of mature SREBP-1c (5,6,8). This effect is mediated primarily by changes in the abundance of SREBP-1c messenger RNA (mRNA). Currently, there is no information on whether alterations in nutritional status modulate the concentration of SREBPs in nonmammalian species.

One gene whose transcription is controlled by SREBP is acetyl-CoA carboxylase-{alpha} (ACC{alpha}). ACC{alpha} catalyzes the ATP-dependent carboxylation of acetyl-CoA to malonyl CoA and is considered a key control point in the de novo fatty acid synthesis pathway (9). In livers of avians and mammals, transcription of ACC{alpha} is low during starvation and is high during consumption of a high-carbohydrate, low-fat diet (10). The more downstream promoter (promoter 2) of the chicken ACC{alpha} gene contains an SRE that acts in concert with an adjacent triiodothyronine (T3) response element to mediate the stimulatory effects of insulin and T3 and the inhibitory effects of glucagon on ACC{alpha} transcription in chick hepatocyte cultures (11,12). Because insulin, T3, and glucagon are humoral factors communicating changes in dietary status to the liver, the ACC{alpha} SRE has been proposed to play a role in mediating the effects of starvation and feeding a high-carbohydrate, low-fat diet on ACC{alpha} transcription in liver. Information on whether alterations in nutritional status modulate the binding of SREBP to the ACC{alpha} SRE is currently lacking.

The aims of the present study were to identify and to characterize the chicken homolog of SREBP-1; to determine the effects of starvation and feeding a high-carbohydrate, low-fat diet on the abundance of SREBP-1 in chicken liver; to assess the role of pretranslational processes in mediating diet-induced changes in SREBP-1 levels; and to ascertain the effects of starvation and feeding a high-carbohydrate, low-fat diet on the binding of SREBPs to the ACC{alpha} SRE in chicken liver.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
    Isolation of the cDNA encoding chicken SREBP-1. A chicken liver cDNA library (lambda ZAP, Stratagene) was screened by using a 32P-labeled cDNA probe containing the basic helix-loop-helix domain (nucleotides 721 to 1103 relative to the translation start site) of human SREBP-1a. pBluescript phagemids were excised from the lambda ZAP vector of 8 positive clones, and the insert DNAs were subjected to dideoxy sequencing. Analysis of the cDNA inserts only partially identified the chicken SREBP-1 (cSREBP-1) sequence. To isolate sequences from the 5' end of cSREBP-1, rapid amplification of 5'-cDNA ends (5' RACE) was carried out by using the FirstChoice RLM-RACE kit (Ambion). PCR products obtained from 5' RACE were subcloned into pCR II (Invitrogen) and then were sequenced. A continuous, full-length cDNA for cSREBP-1 was developed by joining the cDNA fragment obtained from 5' RACE with those obtained from cDNA library screening. The full-length cDNA for cSREBP-1 was subcloned into pSV-SPORT1 (Invitrogen) to form pSV-SPORT1–cSREBP-1. To construct an expression plasmid that expresses the mature form of cSREBP-1, DNA sequences encoding mature cSREBP-1 (amino acids 1 to 464) were amplified by using PCR. The upstream primer contained a sequence 5' of the start codon that allowed for optimal translation initiation. The downstream primer contained a stop codon immediately 3' of the codon encoding amino acid 464. The PCR-generated cDNA fragment encoding mature cSREBP-1 was subcloned into pSV-SPORT1 to form pSV-SPORT1–cSREBP-1 (1–464). A cDNA encoding a dominant negative form of cSREBP-1 lacking amino acids 86 to 107 (cSREBP-1 {Delta}22) was also subcloned into pSV-SPORT1 to form pSV-SPORT1–cSREBP-1 {Delta}22.

    Animals. Newly hatched white leghorn chicks (CBL Farms) were housed in brooders with thermostatically controlled heaters. They had water ad libitum and were fed a commercial chick starter diet (Buckeye Starter Grower; analysis by weight: 71% carbohydrate, 18% protein, 3% fat, 5% crude fiber). A 12-h light–dark cycle (lights on at 0600) was maintained throughout the studies. Ten-day-old chicks were starved for 24 h and then were refed a high-carbohydrate, low-fat diet that contained, by weight, 65% glucose, 25% isolated soy protein (ICN Biochemicals), 1% safflower oil, 6% mineral mix (Briggs chicks salts A, Teklad) (13), 1% vitamin mix (A.O.A.C., Teklad) (14), 2% cellulose, and 0.4% DL-methionine. This protocol was approved by the Animal Care and Use Committee of West Virginia University.

    Cell culture and transient transfection. Primary cultures of chick embryo hepatocytes were prepared as described previously (15) and were maintained in serum-free Waymouth’s medium MD705/1 (Invitrogen) containing 50 nmol/L insulin (gift from Lilly) and 1 µmol/L corticosterone. Cells were transfected 6 h after plating by using 20 µg lipofectin (Invitrogen), 1 µg SRE-TKCAT (11), and varying amounts of pSV-SPORT1–cSREBP-1 (1–464), pSV-SPORT1–cSREBP-1 {Delta}22, and/or pSV-SPORT1 (empty expression plasmid). The total molar concentration of expression plasmid was the same in each transfection. pBluescript KS(+) was added to bring the total amount of transfected DNA to 2.02 µg/plate. At 18 h of incubation, the transfection medium was replaced with fresh medium. At 66 h of incubation, cells were harvested and cell extracts were prepared as described by Baillie et al. (16). CAT (chloramphenicol acetyltransferase) activity (17) and protein (18) were assayed by the indicated methods.

    Western blot analysis. Membrane and nuclear extracts were prepared from liver cells as described (11). Immunoblot analysis was carried out by using mouse monoclonal antibodies against SREBP-1 (IgG-2A4) and SREBP-2 (IgG-1D2) (American Type Culture Collection). IgG-2A4 and IgG-1D2 have been shown to react with the chicken forms SREBP-1 and SREBP-2, respectively (11).

    RNase protection assay. RNA probes for chicken SREBP-1, SREBP-2, ß-actin, and 18S ribosomal RNA (rRNA) were generated as previously described (11,12). RNA was extracted from chick tissues by the guanidinium thiocyanate–phenol–chloroform method (19). RNase protection assays were performed as previously described (11).

    Gel mobility shift assay. Mature forms of chicken SREBP-1 and SREBP-2 were translated in vitro by using the TNT SP6 coupled reticulocyte lysate system (Promega). Nuclear extracts were prepared as described in (12). A double-stranded oligonucleotide containing the ACC{alpha} SRE (–84 to –66 bp relative to the transcription initiation site of ACC{alpha} promoter 2) was labeled by filling in overhanging 5' ends by using a Klenow fragment of Escherichia coli DNA polymerase in the presence of [{alpha}-32P]dCTP. Gel mobility shift assays were carried out as previously described (12).

    Statistical methods. Differences between pairs of means were determined by the Student’s t test and were considered significant when P < 0.05. All data are reported as means ± SEM.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
    Identification and characterization of chicken SREBP-1. The full length cDNA for chicken SREBP-1 was obtained by screening a chicken liver cDNA library and by 5' RACE (Fig. 1). Chicken SREBP-1 encoded a 1115 amino acid protein (GenBank accession number AY029224) with a domain structure similar to that of mammalian SREBPs. The bHLH-Zip domain exhibited the highest degree of sequence identity. Sumoylation (20), acetylation (21), and ubiquitination (22) sites were conserved in chicken SREBP-1. The recognition sequence for site-2 protease was also well conserved (23). However, the putative recognition sequence for site-1 protease deviated from that observed in mammals (24). The leucine in the site-1 protease recognition motif, RXXL, was replaced by a methionine. MAP kinase phosphorylation sites in human SREBP-1 were also not conserved in chicken SREBP-1. Of the 2 SREBP-1 isoforms described in mammalian species, the amino-terminal sequence of chicken SREBP-1 more closely resembled that of the 1a isoform. Other forms of chicken SREBP-1 that vary in the amino-terminal coding region were not detected by 5' RACE and RNase protection assays when using probes to the 5' end of chicken SREBP-1 (data not shown). Thus, chickens do not appear to express a second SREBP-1 isoform that corresponds to 1c isoform in mammals. Previous work has shown that Drosophila also express a single form of SREBP-1 (25).



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  		FIGURE 1 Comparison of the structure of chicken SREBP-1 with mammalian SREBPs. Top, schematic overview of the domain structures of chicken SREBP-1 and human SREBP-1a. Numbers correspond to amino acids that define domain boundaries. The percentage identities between chicken and human sequences are indicated for each domain. Bottom, alignment of the N-terminal amino acid sequences of chicken SREBP-1, mouse SREBP-1a, and human SREBP-1a. Identical residues in the 3 sequences are denoted by an asterisk. The black square and the black circle denote conserved lysine residues that are targets of sumoylation and acetylation/ubiquitination, respectively. Arrows indicate the cleavage sites for site-1 protease (S1P) and site-2 protease (S2P).

 
To confirm that chicken SREBP-1 functions as a transcriptional activator, we determined the effects of expression of exogenous chicken SREBP-1 on transcription directed by an SRE. Chick embryo hepatocyte cultures were transiently transfected with a reporter plasmid containing the chicken ACC{alpha} SRE (SRE–TKCAT) and expression plasmids containing or lacking the coding region for the mature form of chicken SREBP-1. Overexpression of mature chicken SREBP-1 from pSV-SPORT1–cSREBP-1 (1–464), caused a marked increase in SRE–TKCAT activity but had no effect on the activity of a reporter plasmid lacking a SRE (TKCAT) (Fig. 2). As an additional control, a dominant negative form of chicken SREBP-1 lacking 22 amino acids in a proline-rich region (amino acids 86–107) was expressed in chick hepatocytes. This mutant, referred to as SREBP-1 {Delta}22, is unable to dimerize with itself and bind DNA but is able to dimerize with wild-type SREBP-1 and inhibit its DNA binding activity (data not shown). The structure of SREBP-1 {Delta}22 differs from that of a previously described SREBP dominant negative construct, which contains a mutation in the bHLH-Zip domain (26). Expression of SREBP-1 {Delta}22 reversed the stimulatory effects of exogenous wild-type SREBP-1 on SRE-TKCAT activity. These data indicate that SREBP-1 functions as an activator of transcription in chick hepatocytes.



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  		FIGURE 2 Chicken SREBP-1 activates transcription directed by a SRE. Chick embryo hepatocytes were transiently transfected with SRE-TKCAT or TKCAT (1 µg/plate) in the presence of varying amounts of pSV-SPORT1-based expression plasmids (0–1000 ng/plate) that express the mature form of chicken SREBP-1 [cSREBP-1 (1–464)] and a dominant negative form of chicken SREBP-1 (cSREBP-1 {Delta}22). For each experiment, CAT activity of cells transfected with TKCAT in the absence of expression of cSREBP-1 (1–464) and cSREBP-1 {Delta}22 was set at 1, and the other activities were adjusted proportionately. The results are means ± SEM, n = 3. aDifferent from all other means (P < 0.05) except cells transfected with SRE-TKCAT in the presence of 20 ng pSV-SPORT1–cSREBP-1 (1–464) and 10 ng pSV-SPORT1–cSREBP-1 {Delta}22.

 
    Modulation of the abundance of SREBP-1 in chicken liver. Feeding previously starved chicks a high-carbohydrate, low-fat diet stimulates a 11-fold increase in transcription of the ACC{alpha} gene in liver (10). The chicken ACC{alpha} gene contains a SRE that modulates ACC{alpha} transcription in chick hepatocytes (11,12). To investigate the role of SREBP-1 in mediating alterations in hepatic ACC{alpha} transcription during starvation and feeding a high-carbohydrate, low-fat diet, the concentration of mature SREBP-1 and precursor SREBP-1 was measured in livers of chicks subjected to this nutritional manipulation. In hepatic nuclear extracts of chicks starved for 24 h, the concentration of mature SREBP-1 was low; feeding a high-carbohydrate, low-fat diet for 5 and 24 h increased the concentration of mature SREBP-1 by 14- and 6-fold, respectively (Fig. 3A). Feeding a high-carbohydrate, low-fat diet also increased the concentration of precursor SREBP-1 in the membrane fraction; however, the magnitude of this effect (1.4- to 3.2-fold) was substantially lower than that observed for mature SREBP-1, particularly at the 5-h time point. This observation suggests that the increase in abundance of mature SREBP-1 caused by consumption of a high-carbohydrate, low-fat diet is mediated primarily by an elevation in the proteolytic processing of precursor SREBP-1 to mature SREBP-1 and/or by a decrease in the turnover of mature SREBP-1.



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  		FIGURE 3 Starvation and feeding a high-carbohydrate, low-fat (HC/LF) diet modulate the abundance of SREBP-1 (A) but not the abundance of SREBP-2 (B) in chick liver. Nuclear extracts and membrane fractions were prepared from livers of 10- to 12-d-old chicks starved for 24 h and then fed a high-carbohydrate, low-fat diet for 0, 5, and 24 h. The abundance of mature SREBPs and precursor SREBPs were measured in nuclear extracts and membrane fractions, respectively, by Western analysis. Levels of precursor SREBPs and mature SREBPs in starved chickens were set at 1, and the other values were adjusted proportionately. Insets show representative signals for each dietary condition. Values are means ± SEM, n = 5. aDifferent from starved chickens, P < 0.05.

 
The abundance of SREBP-1 mRNA was also measured in livers of chicks by using a RNase protection assay. Feeding previously starved chicks a high-carbohydrate, low-fat diet stimulated a 1.4-fold (5 h) and 2.6-fold (24 h) increase in SREBP-1 mRNA abundance at the indicated time points (Fig. 4A). These data provide further evidence that alterations in the synthesis of precursor SREBP-1 account for a minor portion of the increase in mature SREBP-1 abundance caused by consumption of a high-carbohydrate, low-fat diet.



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  		FIGURE 4 SREBP-1 mRNA abundance is controlled by alterations in nutritional status and by tissue-specific factors. A, the abundance of SREBP-1 mRNA, SREBP-2 mRNA, and ß-actin mRNA was measured in 10- to 12-d-old chicks starved for 24 h and then fed a high-carbohydrate, low-fat diet (HC/LF) for 0, 5, and 24 h. The left panel contains data from a representative experiment. Signals for SREBP-1 mRNA and SREBP-2 mRNA are quantitated in the right panel. Levels of SREBP-1 mRNA and SREBP-2 mRNA in starved chickens were set at 1, and the other values were adjusted proportionately. Values are means ± SEM, n = 5. aDifferent from starved chickens (P < 0.05). B, the abundance of SREBP-1 mRNA and 18S rRNA in brain, heart, liver, kidney, and pectoral muscle of 10- to 12-d-old chicks fed high-carbohydrate, low-fat diet. For each experiment, levels of SREBP-1 mRNA in brain were set at 1. Values are means ± SEM, n = 5. bDifferent from any other tissue (P < 0.05).

 
Assaf et al. (27) have recently identified the chicken homolog of SREBP-2 and have shown that this protein is expressed in a wide variety of tissues, including liver. We investigated whether starvation and feeding modulated the abundance of SREBP-2 in chick liver. Feeding previously starved chicks a high-carbohydrate, low-fat diet had no effect on the concentration of mature SREBP-2, precursor SREBP-2, and SREBP-2 mRNA (Figs. 3Band 4A). These data suggest that SREBP-2 is not involved in mediating diet-induced changes in lipogenic enzyme expression in chickens.

In addition to nutritional regulation, the transcription of lipogenic genes is controlled by tissue-specific factors. For example, transcription of the ACC{alpha} gene in chickens is high in liver and low in brain, heart, kidney, and pectoral muscle (10). These differences in ACC{alpha} transcription may be mediated by alterations in SREBP-1 expression. To investigate this possibility, the abundance of SREBP-1 mRNA was measured in different tissues of chicks fed a high-carbohydrate, low-fat diet. The abundance of SREBP-1 mRNA was substantially higher in liver relative to brain, heart, kidney, and pectoral muscle (Fig. 4B). These results provide support for a role for SREBP-1 in mediating tissue-dependent differences in ACC{alpha} transcription in chickens.

    Modulation of the binding of nuclear proteins to the ACC-{alpha} SRE. Starvation and feeding a high-carbohydrate, low-fat diet modulated the concentration of mature SREBP-1 in chick liver (Fig. 3). Gel mobility shift experiments were performed to determine whether starvation and feeding also modulated the binding of SREBP-1 to the ACC{alpha} gene. Incubation of a 32P-labeled oligonucleotide probe containing the ACC{alpha} SRE with hepatic nuclear extracts from chicks fed a high-carbohydrate, low-fat diet resulted in the formation of 4 sequence-specific protein-DNA complexes (Fig. 5). These complexes are designated a, b, c, and d, in order of increasing mobility. Preincubation of nuclear extracts with an antibody against SREBP-1 disrupted the formation of complexes a, b, and c, but had no effect on the formation of complex d. Preincubation of nuclear extracts with an antibody against SREBP-2 disrupted the formation of complex c but had no effect on complexes a, b, and d. Incubation of the ACC{alpha} SRE probe with in vitro synthesized mature SREBP-1 resulted in a SREBP-1 homodimeric complex whose migration was similar to that of complex a. Collectively, these data suggest that complex a is composed of SREBP-1 homodimers. Complex b also appears to contain SREBP-1 or another protein that is immunologically similar to SREBP-1. The increased mobility of complex b relative to complex a may be due to a decrease in size or a posttranslational modification of 1 or both proteins comprising complex b. Complex c appears to contain both SREBP-1 and SREBP-2, or proteins that are immunologically similar to SREBP-1 and SREBP-2. The mobility of complex c is greater than that observed for homodimeric complexes containing in vitro synthesized mature SREBP-1 or mature SREBP-2. This observation suggests that 1 or both proteins comprising complex c is smaller in size than mature SREBP-1 and mature SREBP-2, or has undergone a posttranslational modification. The identity of the proteins in complex d is presently unclear. Previous work has shown that the SRE of the low-density lipoprotein receptor gene binds multiple protein complexes in crude nuclear extracts and that the transcriptional activity of this regulatory element is correlated only with the binding of nuclear complexes containing SREBP (28,29). Thus, complexes a, b, and c likely account for the activity of the ACC{alpha} SRE in chick liver.



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  		FIGURE 5 The ACC{alpha} SRE-1 binds multiple SREBP-containing complexes in chick liver. Nuclear extracts were prepared from livers of chicks starved for 24 h and then fed a high-carbohydrate, low-fat diet for 0, 5, and 24 h. Gel mobility shift assays were performed by using nuclear extracts, in vitro synthesized mature SREBP-1 (amino acids 1 to 464), and/or in vitro synthesized mature SREBP-2 (amino acids 1 to 455) and an oligonucleotide probe containing the ACC{alpha} SRE. Positions of specific protein-DNA complexes (arrows) and nonspecific complexes (asterisk) are indicated. In some incubations, nuclear extract was incubated with antibodies against SREBP-1, SREBP-2, or MEIS1 prior to addition of the probe.

 
Starvation and feeding a high-carbohydrate, low-fat diet modulated the pattern of binding of SREBP-containing complexes to the ACC{alpha} SRE. In livers of starved chicks, the binding activity of complex a and complex b was barely detectable, whereas the binding activity of complex c was readily observed (Fig. 5). Feeding a high-carbohydrate, low-fat diet for 5 and 24 h increased the binding activity of complex a and complex b but had little or no effect on the binding activity of complex c. The extent of the increase in the binding of complex a and complex b was greater at 5 h of feeding than at 24 h of feeding. These diet-induced changes in the binding of complex a and complex b paralleled changes in the concentration of mature SREBP-1 (Fig. 3). Collectively, these observations support the proposal that consumption of a high-carbohydrate, low-fat diet increases the binding of mature SREBP-1 to the ACC{alpha} gene in liver.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Results of the present study demonstrate that feeding previously starved chickens a high-carbohydrate, low-fat diet increases the concentration and the binding of mature SREBP-1 to the ACC{alpha} gene in liver. These observations, in combination with previous work demonstrating that the ACC{alpha} SRE modulates ACC{alpha} transcription in chick hepatocyte cultures, provide strong evidence that alterations in the abundance of mature SREBP-1 play a role in mediating the effects of starvation and feeding a high-carbohydrate, low-fat diet on ACC{alpha} transcription in chick liver.

What are the signaling pathways that mediate the increase in abundance of mature SREBP-1 in livers of chicks fed a high-carbohydrate, low-fat diet? Two hormones whose concentrations are elevated by the consumption of a high-carbohydrate, low-fat diet are insulin and T3 (9). In chick hepatocyte cultures, addition of insulin or T3 to the culture medium increases the concentration of mature SREBP-1 (12). The effect of insulin on mature SREBP-1 concentration is rapid (i.e., within the first 6 h of hormone treatment) and is mediated by a posttranslational mechanism. In contrast, the effect of T3 on mature SREBP-1 concentration is delayed (i.e., between 6 and 24 h of hormone treatment) and is mediated by both pretranslational and posttranslational mechanisms. We postulate that insulin plays a role in mediating the increase in mature SREBP-1 concentration observed at 5 h of feeding a high-carbohydrate, low-fat diet and that both insulin and T3 are involved in mediating the increase in SREBP-1 concentration observed at 24 h of feeding. In support of this proposal, the effect of feeding a high-carbohydrate, low-fat diet on mature SREBP-1 concentration is mediated by a posttranslational mechanism at the 5-h time point, whereas at the 24-h time point, it is mediated by both pretranslational and posttranslational mechanisms.

In contrast to SREBP-1, feeding previously starved chicks a high-carbohydrate, low-fat diet had no effect on the concentration of mature SREBP-2 and the binding of SREBP-2 (i.e., complex c) to the ACC{alpha} gene in liver. These observations suggest that SREBP-2 does not play a role in mediating the effects of starvation and feeding a high-carbohydrate, low-fat diet on ACC{alpha} transcription. Nevertheless, SREBP-2 may have a physiological function during the starved state, because complex c is the predominant SREBP-containing complex that binds the ACC{alpha} SRE during this dietary condition. We postulate that during conditions when ACC{alpha} is not induced by nutritional factors, complex c plays a role in ensuring a basal level of ACC{alpha} expression for the synthesis of structural lipids in cell membranes.

In rodents, SREBP-1c has been shown to play a critical role in the nutritional and hormonal regulation of lipogenic enzyme expression in liver (6,26). As observed with SREBP-1 in chicken liver, consumption of a high-carbohydrate, low-fat diet causes a marked increase in the concentration of mature SREBP-1c in rodent liver (5,6,8). Insulin treatment also increases the concentration of mature SREBP-1c in rodent hepatocytes (26,30,31). The effects of insulin and feeding a high-carbohydrate, low-fat diet on mature SREBP-1c concentration in rodent hepatocytes are mediated primarily by changes in SREBP-1c mRNA abundance (26). In contrast, we have found that the effects of insulin and feeding a high-carbohydrate, low-fat diet on mature SREBP-1 concentration in chick hepatocytes are mediated primarily by a posttranslational mechanism. Thus, avians and rodents differ in the mechanisms by which nutrients and hormones modulate the concentration of mature SREBP-1 in liver. The reason for these differences is not clear. They may reflect subtle class-dependent differences in the role of SREBP-1 in the regulation of fatty acid synthesis and/or other metabolic processes.

In summary, consumption of a high-carbohydrate, low-fat diet increases the concentration of mature SREBP-1 in chick liver. This effect is mediated primarily by a posttranslational mechanism. Diet-induced changes in the abundance of mature SREBP-1 in chick liver are associated with alterations in the binding of SREBP-1 and the rate of transcription of the ACC{alpha} gene. SREBP-1 expression is substantially higher in liver relative to brain, heart, kidney, and pectoral muscle, and thus may play a role in mediating tissue-dependent differences in ACC{alpha} transcription.


   FOOTNOTES
 
1 Supported by a grant from the American Heart Association (0355193B) and by a grant from the Cooperative State Research Service/USDA (2001–35206-11133). Back

3 Abbreviations used: ACC{alpha}, acetyl-CoA carboxylase-{alpha}; bHLH-Zip, basic helix-loop-helix-leucine zipper; CAT, chloramphenicol acetyltransferase; mRNA, messenger RNA; 5' RACE, rapid amplification of 5'-cDNA ends; rRNA, ribosomal RNA; SRE, sterol regulatory element; SREBP, sterol regulatory element-binding protein; T3, triiodothyronine. Back

Manuscript received 11 May 2004. Initial review completed 20 May 2004. Revision accepted 7 June 2004.


   LITERATURE CITED
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 ABSTRACT
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 RESULTS
 DISCUSSION
 LITERATURE CITED
 

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