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(Journal of Nutrition. 2000;130:2915-2921.)
© 2000 The American Society for Nutritional Sciences

Articles

Copper Deficiency Induces Hepatic Fatty Acid Synthase Gene Transcription in Rats by Increasing the Nuclear Content of Mature Sterol Regulatory Element Binding Protein 11

Zhongren Tang*, Daniela Gasperkova{dagger}, Jing Xu*, Rebecca Baillie*, Joo-Hee Lee* and Steven D. Clarke*2

* Graduate Program of Nutritional Sciences and the Institute of Cellular and Molecular Biology, The University of Texas, Austin, TX, 78712 and the {dagger} Diabetes and Nutrition Research Group, Institute of Experimental Endocrinology, Slovak Academy of Sciences, Vlarska 3, 833 06 Bratislava, Slovak Republic.

2To whom correspondence should be addressed.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Dietary copper (Cu) deficiency results in an accelerated rate of hepatic fatty acid synthase gene transcription and an enhanced rate of hepatic lipid synthesis. Because the nuclear transcription factor sterol regulatory element binding protein-1 (SREBP-1) is a strong enhancer of fatty acid synthase promoter activity, it was hypothesized that Cu deficiency induces fatty acid synthase gene transcription by increasing the nuclear localization of mature SREBP-1. Male weanling rats were pair-fed a Cu-adequate (6.0 mg/kg) or Cu-deficient (0.6 mg/kg) diet (AIN-93) for 28 d. DNase I hypersensitivity site mapping of the hepatic fatty acid synthase gene revealed the presence of four major hypersensitivity sites located at -8700 to -8600, -7300 to -6900, -600 to -400 and -100 to +50. Although Cu deficiency did not change the hypersensitivity site pattern or intensity, in vitro footprinting of the region between -100 and +50 indicated that Cu deficiency enhanced DNA protein interactions within this region. The sequence between -68 and -58 contains the DNA recognition sequence for SREBP-1 and upstream stimulatory element-1 (USF-1). Western blot analysis revealed that the dietary Cu deficiency increased the hepatic nuclear content of mature SREBP-1 by 150% (P < 0.05), and it concomitantly decreased the membrane content of precursor SREBP-1 by 45% (P < 0.05). Changes in the hepatic distribution of SREBP-1 associated with Cu deficiency were not accompanied by changes in SREBP-1 mRNA. The nuclear content of USF-1 was unaffected by dietary Cu status. The hepatic increase in mature SREBP-1 of Cu-deficient rats was accompanied by a 400% increase and an 80% decrease in the abundance of fatty acid synthase and cholesterol 7-{alpha} hydroxylase mRNA, respectively. hepatic These data indicate that a Cu deficiency stimulates hepatic lipogenic gene expression by increasing the hepatic translocation of mature SREBP-1.

KEY WORDS: • sterol regulatory element binding protein • copper • fatty acid synthase • rats


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Copper (Cu)3 deficiency is associated with a number of physiological changes, including anemia, neuronal degeneration, achromotrichia, cardiac hypertrophy, impaired immune function and impaired elastin cross-linking (Bala et al. 1991Citation , Prohaska 1990Citation , Rucker and Murray 1978Citation ). Many of these effects can be explained by the depletion of Cu from specific cuproenzymes (Bala et al. 1991Citation , Nelson et al. 1992Citation , Paynter et al. 1979Citation , Prohaska 1990Citation , Rucker and Murray 1978Citation ). For example, an inadequate supply of Cu leads to a reduction in the apo to holoenzyme conversion for cytochrome oxidase C, lysyloxidase and tyrosinase (Prohaska 1990Citation , Rucker and Murray 1978Citation ). The outcome is an increase in the fraction of inactive apo enzyme and a reduction in the flux of substrate through the respective metabolic pathway. On the other hand, several anomalies associated with a dietary Cu deficiency appear to be secondary responses to the Cu deficiency. One anomaly that is observed in Cu-deficient humans and experimental animals is an increase in hepatic triglyceride and cholesterol biosynthesis, which is accompanied by the development of hypertriglyceridemia and hypercholesterolemia (Allen and Klevay 1978aCitation and 1978bCitation , Al-Othman et al. 1992Citation , Klevay et al. 1984Citation ). This response to a dietary Cu-deficiency is surprising because none of the enzymes in lipid biosynthesis are known to be cuproenzymes. Moreover, if a unique cuproenzyme did exist in the lipid biosynthetic pathway, one would expect that Cu depletion would decrease, not increase, its activity. Recently, Fields and Lewis (1997)Citation reported that the 100–200% increase in hepatic lipid synthesis associated with a Cu deficiency may not be due to Cu depletion but rather iron accumulation. It is well recognized that a Cu deficiency increases the iron content of the liver approximately 100% (Fields and Lewis 1997Citation , Wilson et al. 1997Citation ). Moreover, overloading the liver with iron increases the expression of a number of genes, and it leads to hypercholesterolemia and hypertriglyceridemia that is comparable to that observed in Cu-deficient animals (Dabbagh et al. 1994Citation , Fields et al. 1994Citation , Fields et al. 1996Citation , Fields and Lewis 1997Citation ). Finally, when Cu-deficient rats were fed a low iron diet to prevent hepatic iron accumulation, the elevations in blood cholesterol and triglycerides characteristically observed in Cu deficiency were prevented (Fields and Lewis 1997Citation ).

Although the induction of hepatic lipid synthesis and secretion accompanying a Cu deficiency may be secondary to hepatic iron enrichment, this still does not explain the cellular and molecular mechanisms responsible for the increase in hepatic lipid synthesis. In part the answer to this question resides in our observation that a dietary Cu deficiency induces transcription of lipogenic genes, that is, fatty acid synthase, and that this in turn increases fatty acid synthase mRNA abundance and finally elevates fatty acid synthase enzymatic activity (Wilson et al. 1997Citation ). How Cu deficiency and possibly iron overload coordinately induce the hepatic expression of genes encoding enzymes involved in fatty acid and cholesterol biosynthesis remains unclear. We hypothesized that Cu deficiency alters the protein/DNA interactions of transcription factors that are essential for fatty acid synthase gene transcription and that these changes in protein/DNA interactions alter chromatin structure such that fatty acid synthase promoter activity is increased.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals and diets.

The basal diet was purchased from Dyets (Bethlehem, PA) and was formulated according to the AIN-93-G rodent diet (Reeves et al. 1993Citation ) recommendations. The composition of the diet was specifically modified by using sucrose instead of dextrose and by the omission of Cu from the mineral mix. The basal diet, which contained 0.6 mg Cu/kg, served as the Cu-deficient diet, whereas the Cu-adequate diet was prepared by supplementing the basal diet with CuSO4 · 5H2O to a concentration of 6.0 mg/kg.

Weanling male Sprague-Dawley rats (Harlan Sprague-Dawley, Indianapolis, IN) weighing 40–55 g were housed individually in stainless steel mesh cages in an animal facility maintained at 24°C and 12/12-hour reversed light-dark cycle. Distilled water was provided in polyethylene bottles. During a 7-d adaptation period, food intake and body weight changes were determined daily. After this adaptation period, rats were paired according to food intake and weight gain. Each pair of rats was pair meal fed (8 h/d, 1000–1800 h) the Cu-deficient or Cu-adequate diet for 28 d. Food was allotted to Cu-adequate rats by giving an amount of Cu-adequate diet equal to that consumed on the previous day by its Cu-deficient pair. Body weights were measured once a week. Rats were killed and livers were collected for analysis 16 h after their last meal.

DNase I hypersensitivity site mapping.

Rat liver nuclei were prepared as previously described (Rongnoparut et al. 1991Citation ). Briefly, 5 g liver was homogenized in 30 mL homogenization buffer [10 mmol HEPES/L (pH 7.6), 25 mmol KCl/L, 1 mmol sodium EDTA/L, 2 mol sucrose/L, 10% (v/v) glycerol, 0.15 mmol spermine/L and 2 mmol spermidine/L]. The homogenate was centrifuged through a 10-mL 2. 0 mol sucrose/L cushion [10 mmol HEPES/L (pH 7.6), 25 mmol KCl/L, 1 mmol sodium EDTA/L, 2 mol sucrose/L, 10% (v/v) glycerol, 0.15 mmol spermine/L and 2 mmol spermidine/L] at 75,000 x g for 1 h at 4°C. DNase I hypersensitivity site mapping of the 5'-flanking region of the fatty acid synthase gene was conducted as previously described (Jump et al. 1987Citation ). Briefly, 100 µL (500 µg) nuclei was digested in a buffer (0.25 mol sucrose/L, 15 mmol Tris-Cl/L [pH 8.0], 60 mmol KCl/L, 3 mmol MgCl2/L) that contained 0–20 U DNase I (Promega, Madison, WI). After 5 min of digestion at 30°C, the reaction was stopped by adding 10 µL 0.5 mol EDTA/L, and the nuclei were then incubated with RNase A (50 mg/L; Promega) for 1 h at 37°C. After RNase A digestion, 330 µL proteinase K digestion buffer [0.15% SDS, 100 mg proteinase K/L (Promega), 10 mmol/Tris–HCl (pH 7.5), 0.1 mol NaCl/L, 0.1 mmol EDTA/L] was added and incubated at 37°C overnight. DNA (15 µg) isolated from above nuclei was completely digested with BamHI (Promega). The digested fragments were size-fractionated by electrophoresis in 0.7% agarose gel. The DNA was electrotransferred onto Zeta–Probe GT blotting membrane (Bio–Rad, Hercules, CA). Blots were hybridized with 32P-labeled probes (NEN, Boston, MA) corresponding to the fatty acid synthase gene regions of (a) -5421 to 5050; (b) -4949 to -4651 and (c) -2711 to -2378. Hybridization was visualized by exposing the blots to X-OMAT AR film (Kodak, Rochester, NY) for 2 d at -80°C.

DNase I footprinting analysis.

Hepatic nuclear proteins were isolated as described previously (Rongnoparut et al. 1991Citation ). Briefly, 5 g liver was homogenized in 30-mL of homogenization buffer [10 mmol HEPES/L (pH 7.6), 25 mmol KCl/L, 1 mmol sodium EDTA/L, 2 mol sucrose/L, 10% (v/v) glycerol, 0.15 mmol spermine/L and 2 mmol spermidine/L]. The homogenate was layered over a 10-mL cushion of homogenization buffer and was centrifuged at 75,000 x g for 1 h at 4°C. The nuclear pellet was resuspended in 10 mL of lysis buffer [10 mmol HEPES/L (pH 7.6), 100 mmol KCl/L, 2 mmol MgCl2/L, 1 mmol EDTA/L, 1 mmol dithiothreitol/L and 10% (v/v) glycerol]. After the addition of 1 mL ammonium sulfate (4 mol/L, pH 7.9), the suspension was centrifuged at 75,000 x g for 45 min at 4°C. Solid ammonium sulfate (300 g/L) was added to the supernatant and was slowly dissolved for 15 min. After an additional 30 min on ice, it was centrifuged at 75,000 x g for 1 h at 4°C. The protein pellet was resuspended in dialysis buffer [10 mmol HEPES/L (pH 7.6), 40 mmol KCl/L, 0.1 mmol EDTA/L, 1 mmol dithiothreitol/L and 10% (v/v) glycerol] at 1 mL per 40 A260 U and subsequently dialyzed twice for 2 h against 250 mL of dialysis buffer.

DNase I footprinting was carried out by a modification of a previously described procedure (Rongnoparut et al. 1991Citation ). The -265/+65 fatty acid synthase fragment was labeled using klenow fragment (Clontech, San Francisco, CA) and [{gamma}-32P]–dATP (NEN) to fill in the Sau3AI cut site at the 3'-flanking end and then was purified using Spin column (Bio–Rad). Protein binding to the DNA fragment was conducted by incubating nuclear extracts with 75,000 dpm (0.5–1 ng) of probe in 50 µL of reaction buffer [50 mmol NaCl/L, 0.1 mmol EDTA/L, 20 mmol HEPES/L (pH 7.5), 0.5 mmol dithiothreitol/L and 10% glycerol]. After a 30-min incubation at room temperature, 6 µL of 0.1 mol MgCl2/L, 20 mmol CaCl2/L and 0.05 U of DNase I was added to the reaction. The reaction was stopped after 1 min by the addition of 20 µL of 0.1 mol EDTA/L (pH 7.6). The sample was extracted with phenol/chloroform and the DNA precipitated with ethanol. The DNA precipitate was resuspended in 95% formamide and resolved on a 5% polyacrylamide 8 mol/L urea sequence gel. The footprint was visualized by exposing the dried gel to X-OMAT AR film (Kodak) for 2 d at -80°C.

Western blot analysis.

Microsomal and nuclear proteins were isolated from liver removed from rats as described previously (Sheng et al. 1995Citation ). Briefly, 5 g liver was homogenized in 30 mL of buffer A [10 mmol HEPES/L (pH 7.6), 25 mmol KCl/L, 1 mmol sodium EDTA/L, 2 mol sucrose/L, 10% (v/v) glycerol, 0.15 mmol spermine/L, 2 mmol spermidine/L, 50 mg/L N-acetylleucylnorleucinal (Roche, Indianapolis, IN), 20 mg pefabloc/L (Roche), 5 mg pepstain A/L (Roche), 10 mg leupeptin/L (Roche) and 2 mg aprotinin/L (Roche)]. The homogenate was layered over a 10-mL cushion of buffer A and was centrifuged at 75,000 x g for 1 h at 4°C. The resultant pellet (nuclei) was resuspended in 1 mL of buffer B [10 mmol HEPES/L (pH 7.6), 100 mmol KCl/L, 2 mmol MgCl2/L, 1 mmol EDTA/L, 1 mmol dithiothreitol/L, 10% (v/v) glycerol, 50 mg N-acetylleucylnorleucinal/L (Roche), 20 mg pefabloc/L (Roche), 5 mg pepstain A/L (Roche), 10 mg leupeptin/L (Roche) and 2 mg aprotinin/L (Roche)]. After the addition of 100 µL ammonium sulfate (4 mol/L, pH 7.9) the suspension was centrifuged at 257,000 x g for 45 min at 4°C. The resultant supernatant was collected as nuclear protein extract. To isolate microsomal membranes, 1 g liver was homogenized in 5 mL homogenization buffer [20 mmol/L Tris–HCl (pH 8.0), 150 mmol NaCl/L, 1 mmol CaCl2/L, 50 mg N-acetylleucylnorleucinal/L (Roche), 20 mg pefabloc/L (Roche), 5 mg pepstain A/L (Roche), 10 mg leupeptin/L (Roche) and 2 mg aprotinin/L (Roche)]. The homogenate was centrifuged at 800 x g for 10 min at 4°C. The resultant supernatant was used to isolate microsomal membranes by centrifuging the supernatant for 1 h, 100,000 x g at 4°C. The pellet was rinsed with homogenization buffer and subsequently resuspended in 1.5 mL 250 mmol Tris—HCl/L, pH 6.0, and 2 mmol CaCl2/L. The membrane proteins were extracted by adding an equal volume of 2 mmol CaCl2/L, 320 mmol NaCl/L, 2% Triton X-100, 20 mg pefabloc/L (Roche), 5 mg pepstain A/L (Roche), 10 mg leupeptin/L (Roche) and 2 mg aprotinin/L (Roche) to the membrane pellet, mixing, and subsequently centrifuging the suspension for 45 min, 100,000 x g at 4°C.

Western immunoblot analyses for SREBP-1 and USF-1 were carried out as previously described (Towbin et al. 1979Citation ). Aliquots (50 µg protein) of microsomal or nuclear extract were subjected to 8% SDS–PAGE gel, transferred to nitrocellulose (Bio-Rad), and incubated with either monoclonal anti–SREBP-1 (IgG-2A4) prepared from hybridoma cells (American Type Culture Collection, Rockville, MA) or 0.1 mg/L anti–USF-1 (Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h at room temperature. The immunoreactive SREBP-1 and USF-1 were visualized with the enhanced chemiluminescence Western Blotting Detection System kits (Amersham, Arlington Heights, IL). Bands were quantified for relative intensity using the Ambis visual imaging 4000 system (AMBIS, San Diego, CA).

Northern blot analysis.

Total RNA was prepared from rat liver using the phenol-guanidinium-isothiocyanate procedure (Chomczynski and Sacchi 1987Citation ). For Northern gel analysis, 30 µg of total RNA was denatured, subjected to electrophoresis in 1.2% agarose gel and transferred onto Zeta Probe GT membrane (Bio-Rad). The abundance of the respective transcript was determined by sequentially hybridizing the membrane with radiolabeled cDNA probes using the following: hamster SREBP-1 (American Type Culture Collection cDNA Clone No. 87012), rat fatty acid synthase (Wilson et al. 1997Citation ), mouse cytoplasmic hydroxymethyglutaryl-CoA synthase (Gene-Bank Accession No.W85267), mouse cholesterol 7-{alpha}-hydroxylase (Gene-Bank Accession No. AA 254999) and human glyceraldehyde-3-phosphate dehydrogenase (American Type Culture Collection cDNA Clone No. 2007254). Each cDNA was labeled with [{alpha}-32P]–dCTP (NEN) using random prime labeling kits (GIBCO-BRL, Grand Island, NE). Hybridization was conducted overnight at 40°C or 42°C and was exposed at -80°C to Kodak X-Omat ARTM film (Kodak) with intensifying screens for 12–72 h. Corrections for variation in RNA loading of each lane were made using glyceraldehyde-3-phosphate dehydrogenase mRNA as the reference transcript.

Statistical analysis.

The significance of the compared mean values was determined using two-tailed Student’s t test. Data are presented as mean ± SEM, and P < 0.05 is considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
DNA-protein interactions in the 5'-flanking region of fatty acid synthase gene.

The impact of the Cu-deficient diet on DNA-protein interactions within the 5'-flanking region of the hepatic fatty acid synthase gene was examined using DNase I hypersensitivity site mapping (Fig. 1Citation ) and in vitro footprinting (Fig. 2Citation ). DNase I hypersensitivity sites represent regions of local discontinuity in the DNA-protein interactions along the chromatin fiber (Gross and Garrard 1988Citation , Paranjape et al. 1994Citation ). Such sites typically identify regions within the genes that are actively involved in the regulation of gene transcription (Jump et al. 1987Citation ). Application of the DNase I hypersensitivity mapping technique to nuclei prepared from the livers of Cu-deficient and -adequate rats revealed that the 5'-flanking region of the fatty acid synthase gene contained four major hypersensitivity sites (HSS) within the region of -9700 to +658 (Fig. 1)Citation . The four sites mapped to the following regions: (a) -8700 to -8600 (HSS 1); (b) -7300 to -6900 (HSS 2); (c) -600 to -400 (HSS 3) and (d) -100 to +50 (HSS 4). The transcription factors that interact with HSS 1 and HSS 2 remain to be defined. HSS 3 reportedly contains cis-elements that enhance promoter activity in response to insulin (Roder et al. 1997Citation ). The sequences within HSS 4 instill insulin and sterol response to the fatty acid synthase promoter (Marisa and Osborne 1996Citation , Moustaid et al. 1994Citation ). Because Cu deficiency alters hepatic cholesterol homeostasis (Al-Othman et al. 1994Citation , Croswell and Lei 1985Citation , Lefevre et al. 1986Citation , Samman and Roberts 1985Citation ), the HSS 4 region was utilized in an in vitro footprint analysis to evaluate the impact of dietary Cu deficiency on protein-DNA interaction (Fig. 2)Citation . Hepatic nuclear proteins prepared from Cu-adequate rats weakly bound to the region of -88 to -78 and -68 to -58, which are the DNA recognition sites for Sp1 and SREBP-1, respectively (Fig. 2)Citation . On the other hand, DNA binding activity was much greater in nuclear protein extracts from Cu-deficient rats as can be seen by the very clear footprint in these respective regions, between -58 and -68 and between -78 and -88 (Fig. 2)Citation .



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  		Figure 1. Mapping DNase HSS in the fatty acid synthase gene in livers of Cu-adequate (CuA) and Cu-deficient (CuD) rats. Nuclei (500 µg) from CuD or CuA rats were digested with varying concentrations of DNase I. The hypersensitive sites were visualized after Southern blotting and hybridization with radiolabeled probes as described in the Materials and Methods section. The fragments at -9680 (A) and +658 (B) are the 5075- and 3383-bp BamHI fragments that are produced from the fatty acid synthase gene when the nuclei are not treated with DNase. The black boxes depict the four major HSS located within the fatty acid synthase gene region of -9700 to +658. The picture is representative of 3 rats/treatment.

 


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  		Figure 2. SREBP-1 binding to its DNA recognition sequence within the proximal promoter of the rat fatty acid synthase gene in livers of Cu-adequate (CuA) and Cu-deficient (CuD) rats. The region of -265 to +65 of the fatty acid synthase gene was subjected to DNase I footprinting using hepatic nuclear protein extracts (75 µg) prepared from CuA and CuD rats or no protein (NP). Boxes indicate areas of protein binding. The regions of -68 to -58 and -88 to -78 contain DNA recognition sites for SREBP-1 and Sp1, respectively. Binding was enhanced in the CuD rats. Comparable footprints were achieved with nuclear extracts from three different rats.

 
Enhanced nuclear localization of SREBP-1 in Cu deficiency.

The in vitro footprint of the HSS 4 region suggested that DNA sequence between -85 and -55 may play a role in the Cu-dependent regulation of fatty acid synthase gene transcription. This region of the proximal promoter of the fatty acid synthase gene contains DNA binding sites for USF-1 and SREBP-1 (Marisa and Osborne 1996Citation ). Western blot analysis revealed that the nuclear content of USF-1 was unaffected by a dietary Cu deficiency (Fig. 3Citation ). In contrast, the nuclear content of mature SREBP-1 was 150% greater in nuclei from Cu-deficient rats than from Cu-adequate rats (Fig. 4Citation ). This increase in nuclear concentration of mature SREBP-1 associated with a Cu deficiency was paralleled by a significant decrease in the hepatic content of membrane anchored precursor SREBP-1 (Fig. 5Citation ). However, the reduction in precursor SREBP-1 protein was not paralleled by a decrease in the hepatic abundance of SREBP-1 mRNA (Fig. 6Citation ). The 150% increase in nuclear content of mature SREBP-1 in the liver of Cu-deficient rats was accompanied by a threefold increase in hepatic fatty acid synthase mRNA abundance and an 80% decrease in the hepatic content of cholesterol 7-{alpha} hydroxylase mRNA. Surprisingly, the mRNA abundance for the cholesterologenic enzyme cytosolic HMG-CoA synthase was unaffected by dietary Cu status (Fig. 6)Citation .



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  		Figure 3. The hepatic nuclear content of USF-1 in livers of Cu-adequate (CuA) and Cu-deficient (CuD) rats. The nuclear abundance of USF-1 was determined by Western blot analysis using hepatic nuclear protein extracts (50 µg). (A) shows a representative Western blot and (B) summarizes data of n = 6 rats per diet, mean ± SEM.

 


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  		Figure 4. The hepatic abundance of mature nuclear SREBP-1 in livers of Cu-adequate (CuA) and Cu-deficient (CuD) rats. (A) shows a representative Western blot (50 µg/lane) depicting the hepatic abundance of SREBP-1. (B) depicts the nuclear abundance of SREBP-1 as quantified by image analysis scanning of the Western blots, n = 6/treatment. Data are expressed as mean ± SEM; *Significantly greater than the CuA group (P < 0.05).

 


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  		Figure 5. The hepatic abundance of membrane-bound precursor SREBP-1 in livers of Cu-adequate (CuA) and Cu-deficient (CuD) rats. (A) is a representative Western blot (50 µg/lane) depicting the hepatic abundance of precursor SREBP-1. (B) depicts the precursor abundance of SREBP-1 as quantified by image analysis scanning of the Western blots, n = 6/treatment. Data are expressed as mean + SEM; *Significantly greater than the CuD group (P < 0.05).

 


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  		Figure 6. SREBP-1, fatty acid synthase, cholesterol 7-{alpha} hydroxylase and HMG-CoA synthase in livers of Cu-adequate (CuA) and Cu-deficient (CuD) rats. RNA abundance was determined by Northern blot analysis using total hepatic RNA (40 µg/lane). The abundance of each transcript was determined by quantifying the amount of radioactivity associated with each transcript using a radioimagizer. The fold change in abundance for each transcript represents the mean ratio ± SEM (n = 6/treatment) for the Cu-deficient (CuD) response to the Cu-adequate response (CuA) after correction for loading difference with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). *Significant effect of Cu deficiency, P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
A dietary Cu deficiency is associated with the development of hypertriglyceridemia and hypercholesterolemia in both animals and humans (Allen and Klevay 1978aCitation and 1978bCitation , Al-Othman et al. 1993Citation and 1994Citation , Croswell and Lei 1985Citation , Kim et al. 1992Citation , Lefevre et al. 1986Citation , Samman and Roberts 1985Citation , Shao and Lei 1980Citation ). These changes in blood lipids in part reflect acceleration in hepatic lipogenesis and cholesterologenesis (Al-Othman et al. 1993Citation , Shao and Lei 1980Citation ). Enzymatic activity measures revealed that a dietary Cu deficiency increased the hepatic activity of fatty acid synthase, HMG-CoA reductase and glucose-6-phosphate dehydrogenase (Kim et al. 1992Citation , Wilson et al. 1997Citation ). Our prior studies indicated that the increase in hepatic fatty acid synthase activity elicited by Cu deficiency is the product of a two- to threefold increase in fatty acid synthase mRNA abundance that results from the induction of fatty acid synthase gene transcription (Wilson et al. 1997Citation ). Our objective in the current work was to identify the nuclear mechanism by which a dietary Cu deficiency leads to an increase in hepatic fatty acid synthase gene transcription. Our first approach to this question was to identify changes in chromatin structure within the fatty acid synthase gene that may be affected by changes in dietary Cu status. DNase I HSS mapping identified four areas within the regions of -9700 to +658 of the fatty acid synthase gene that were susceptible to DNase (Fig. 1)Citation : HSS 1 between -8700 and -8600; HSS 2 between -7300 and -6900; HSS 3 between -400 and -600 and HSS 4 between -100 and +50. No DNase I site was found upstream of -9700 (data not shown). The HSS 1 and HSS 2 were specific for hepatic nuclei. These sites are required for optimum promoter activity in response to insulin and glucocorticoids, but the cis-elements and transcription factors interacting with the region remain to be determined (Rufo et al. 1999Citation ). HSS 3 reportedly is an enhancer region that may amplify insulin’s induction of the fatty acid synthase promoter (Roder et al. 1997Citation ). HSS 4 contains the insulin response element and recognition sequences for the transcription factors SREBP-1 and USF-1 (Marisa and Osborne 1996Citation , Wang and Sul 1997Citation ). Unfortunately, none of the DNase I sites appeared to be affected by the dietary Cu deficiency. However, because HSS 4 instills both insulin and sterol regulation to the fatty acid synthase promoter, this region was utilized to examine the impact of dietary Cu deficiency on protein-DNA interactions within the sequence. In vitro footprinting revealed that nuclear proteins interacted with the region of -88 to -58. More importantly, the footprint appeared to be more intense with hepatic nuclear protein extracts from Cu-deficient rats (Fig. 2)Citation . The region of -88 to -58 of the fatty acid synthase gene contains DNA recognition elements for SREBP-1, USF-1 and Sp1 (Marisa and Osborne 1996Citation , Roder et al. 1997Citation , Wang and Sul 1997Citation ). Western blot analysis revealed that Cu deficiency had no effect on the nuclear content of USF-1. In contrast, Cu deficiency increased the nuclear content of SREBP-1 1.5-fold, which would appear to explain why nuclear extracts from the livers of Cu-deficient rats yielded a stronger footprint in the region of -68 to -58 (Fig. 2)Citation . In addition, the increase in SREBP-1 binding may explain the stronger footprint in the region of -88 to -78 (Fig. 1)Citation . This region of the fatty acid synthase promoter contains the DNA recognition sequence for Sp1. Sp1 binding to the region is enhanced by the downstream binding of SREBP-1 (Bennett et al. 1995Citation , Yieh et al. 1995Citation ). Thus, the increase in nuclear content of SREBP-1 would induce fatty acid synthase promoter activity directly by binding to its DNA recognition site in -68 to -58 of the fatty acid synthase promoter and indirectly by enhancing the upstream binding of Sp1 to the region of -88 to -78. Consistent with SREBP-1’s role as a strong stimulator of lipogenic gene expression (Horton et al. 1998Citation , Shimano et al. 1996Citation , Xu et al. 1999Citation ), the hepatic abundance of fatty acid synthase mRNA was increased nearly fourfold (Fig. 6)Citation . Although SREBP-1 induces hepatic lipogenic genes, it reportedly suppresses the transcription of cholesterol 7-{alpha} hydroxylase (Horton et al. 1998Citation , Shimano et al. 1996Citation ). Consistent with this suppressive function of SREBP-1, we found that Cu deficiency reduced the hepatic abundance of cholesterol 7-{alpha} hydroxylase >80% (Fig. 6)Citation . Interestingly, the hepatic content of cytosolic HMG-CoA synthase, a rate-determining enzyme in cholesterol biosynthesis, was unaffected by Cu status. Although this observation appears to be inconsistent with the increase in hepatic cholesterol biosynthesis that reportedly occurs with Cu deficiency (Allen and Klevay 1978Citation , Shao and Lei 1980Citation ), it is possible that the translation of HMG-CoA synthase mRNA was enhanced by the Cu deficiency. However, this conclusion is only speculative.

The nuclear content of mature SREBP-1 is dependent on the synthesis of SREBP-1 precursor and/or the proteolytic release of the mature SREBP-1 from its endoplasmic reticulum–anchored precursor (Brown and Goldstein 1999Citation ). SREBP-1 is synthesized as a 125-kDa precursor that contains two trans-membrane domains that allow the protein to be anchored in the endoplasmic reticulum (Brown and Goldstein 1999Citation ). The 480 amino acid N-terminal domain corresponds to the mature SREBP-1 transcription factor and is released from the endoplasmic reticulum membrane by a two-step proteolytic cascade (Brown and Goldstein 1999Citation ). The observation that the dietary Cu deficiency was associated with a 2.5-fold increase in the nuclear content of SREBP-1 and a concomitant 45% reduction in membrane content of SREBP-1 precursor indicates that the Cu deficiency enhanced the proteolytic release and nuclear translocation of mature SREBP-1. This conclusion is strengthened by the observation that the hepatic abundance of SREBP-1 mRNA was not affected by the dietary Cu deficiency (Fig. 6)Citation .

The mechanism by which Cu deficiency enhances the nuclear localization of SREBP-1 may be related to the observation that Cu deficiency reduces hepatic cholesterol levels by as much as 50%. The proteolytic release of mature SREBP-1 may be dependent on the amount of cholesterol that is located in the endoplasmic reticulum (Brown and Goldstein 1999Citation ). As cholesterol is depleted from the endoplasmic reticulum membrane, the sterol-regulated protease within the lumen of the endoplasmic reticulum is activated, which in turn leads to the severing of the peptide anchor and subsequent release of the mature form of the SREBP-1. Such sterol control of SERBP-1 release is consistent with the increase in nuclear content of mature SREBP-1 and the decrease in the membrane amount of SREBP-1 that we observed in the livers of Cu-deficient rats. Thus, it appears that dietary Cu status may upregulate genes encoding enzymes of lipogenesis by decreasing the hepatic content of cholesterol and thereby increasing the nuclear content of SREBP-1. However, one thing remains to be explained, how cholesterol is depleted from the liver in a dietary Cu deficiency when the expression of the rate-limiting enzyme in bile acid synthesis (that is, cholesterol 7-{alpha} hydroxylase) is markedly suppressed. Wu et al. (1997)Citation have argued that the enhanced expression of hepatic apolipoprotein A-I observed in a Cu deficiency results in diversion of cholesterol away from bile acid synthesis and into lipoprotein assembly and secretion. Such a diversion may explain the depletion of hepatic cholesterol that occurs in a Cu deficiency and offers one explanation for the increase in nuclear concentrations of SREBP-1. An alternative explanation for the increase in nuclear content of SREBP-1 may lie with the observations that the hepatic concentration of reduced glutathione is increased 50–80% in the liver of Cu-deficient rats (Kim et al. 1992Citation ) and that the hepatic expression of fatty acid synthase is induced by an elevation in hepatic reduced glutathione (Wilson et al. 1997Citation ). Reduced glutathione inhibits neutral sphingomyelinase (Liu and Hannun 1998Citation ). Moreover, activation of sphingomyelinase is associated with a decreased proteolytic release of SREBP (Scheek et al. 1997Citation ). Thus, the increase in hepatic reduced glutathione associated with Cu deficiency may suppress the activity of neutral sphingomyelinase and thereby lead to an enhance the proteolytic release of SREBP-1. The consequence would be to stimulate fatty acid synthase gene transcription while suppressing the expression of cholesterol 7-{alpha} hydroxylase. Although this mechanism is only a hypothesis, it may offer an explanation for how Cu deficiency coordinately regulates the gene-encoding proteins that are involved in lipid and cholesterol metabolism and transport.


   FOOTNOTES
 
1 Supported by Grant DK 52573 from the National Institutes of Health and by the sponsors of the M. M. Love Chair in Nutritional, Cellular and Molecular Sciences at The University of Texas (SDC). Back

3 Abbreviations used: CuD, copper deficiency; CuA, copper adequate; FAS, fatty acid synthase; HMG-CoA, hydroxymethylglutaryl-CoA; hypersensitivity site, HSS; SREBP, sterol regulatory element binding protein; USF-1, upstream stimulatory factor-1. Back

Manuscript received March 30, 2000. Initial review completed June 6, 2000. Revision accepted August 11, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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