The Spot 14 Protein Is Required for de Novo Lipid Synthesis in the Lactating Mammary Gland
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内分泌学杂志 2005年第8期
Division of Endocrinology and Diabetes (Q.Z., G.W.A., G.T.M., J.K.M., C.N.M.), Department of Medicine, University of Minnesota, Minneapolis, Minnesota 55454; and Department of Food Science and Nutrition (E.J.P.), University of Minnesota, St. Paul, Minnesota 55108
Address all correspondence and requests for reprints to: Cary N. Mariash, M.D., Director, Division of Endocrinology and Diabetes, Department of Medicine, University of Minnesota, MMC 101, 420 Delaware Street SE, Minneapolis, Minnesota 55455. E-mail: mariasc@umn.edu.
Abstract
We generated a Spot 14 null mouse to assess the role of Spot 14 in de novo lipid synthesis and report the Spot 14 null mouse exhibits a phenotype in the lactating mammary gland. Spot 14 null pups nursed by Spot 14 null dams gain significantly less weight than wild-type pups nursed by wild-type dams. In contrast, Spot 14 null pups nursed by heterozygous dams show similar weight gain to wild-type littermates. We found the triglyceride content in Spot 14 null milk is significantly reduced. We demonstrate this reduction is the direct result of decreased de novo lipid synthesis in lactating mammary glands, corroborated by a marked reduction of medium-chain fatty acids in the triglyceride pool. Importantly, the reduced lipogenic rate is not associated with significant changes in the activities or mRNA of key lipogenic enzymes. Finally, we report the expression of a Spot 14-related gene in liver and adipose tissue, which is absent in the lactating mammary gland. We suggest that expression of both the Spot 14 and Spot 14-related proteins is required for maximum efficiency of de novo lipid synthesis in vivo and that these proteins impart a novel mechanism regulating de novo lipogenesis.
Introduction
REDUCED ENERGY INTAKE during early neonatal life has long-term effects on growth and adiposity in the adult mammal (1, 2, 3, 4). Thus, mammals have evolved to produce milk, a nutritionally controlled, high-energy neonatal food source. Triacylglycerols are major milk constituents and comprise a crucial energy source for the suckling neonate (5, 6, 7). The synthesis of triacylglycerols is a tightly controlled process in the lactating mammary gland. Triacylglycerols are synthesized in the mammary epithelial cell through the esterification of fatty acids. The fatty acids used in the synthesis of milk triacylglycerols are either exogenous fatty acids taken up from the plasma or are synthesized de novo in the mammary epithelial cell. De novo lipogenesis also occurs in other lipogenic tissues such as the liver and adipose tissue. The process of de novo lipogenesis in all these tissues includes the action of multiple enzymes including the rate-limiting enzyme acetyl coenzyme A (CoA) carboxylase (ACC) and the terminal enzyme fatty acid synthase (FAS) (8, 9). The lipogenic rate is closely correlated with lipogenic enzyme activities.
The Spot 14 gene has long been associated with de novo lipogenesis (10, 11). The Spot 14 mRNA was first identified while screening hepatic tissue for genes regulated by thyroid hormone (11). Spot 14 is expressed only in lipogenic tissues, and Spot 14 mRNA levels correlate well with the lipogenic rate associated with each tissue (12). Spot 14 is a small, acidic protein with no known functional motifs. The biochemical function of Spot 14 is currently unknown. Antisense experiments suggested that Spot 14 regulates the activation of lipogenic enzyme transcription by stimuli such as carbohydrate feeding and thyroid hormone administration (13). To test this hypothesis, we recently generated a Spot 14 null mouse (14). However, in contrast to the antisense studies, adult hepatic lipogenesis is not inhibited in the Spot 14 null animal (14). These data suggested two possibilities. First, the Spot 14 gene is not required for de novo lipogenesis in lipogenic tissues, or second, the adult Spot 14 null mouse liver possesses compensatory mechanisms to account for the absence of the Spot 14 protein.
We now report that the Spot 14 protein is required for de novo lipogenesis in a separate lipogenic tissue, the lactating mammary gland. We further show that the Spot 14 gene possesses a paralog. We have named the paralog the Spot 14-related (Spot 14-R) gene. Spot 14-R is expressed at high levels in the liver and white adipose tissue but is absent in the lactating mammary gland. The Spot 14-R gene is evolutionarily conserved and is present in the genome of vertebrates as divergent as Xenopus laevis and Homo sapiens. However, the Spot 14 gene is present only in the genome of mammalian species. Together these data suggest that the Spot 14 gene evolved to control milk energy content and that the Spot 14 gene family is an important regulator of de novo lipid synthesis in lipogenic tissues.
Materials and Methods
Animal use
All animal use and protocols were approved by the University of Minnesota Institutional Animal Care and Use Committee. Spot 14 knockout and wild-type mice were created as previously described (14). The mice were subsequently backcrossed to a C57BL6/J strain up to 11 times to assure the mice were studied with a homogeneous background. To create Spot 14 null mice, pups were bred by mating either Spot 14 null male and female mice (to create all Spot 14 null mice) or by mating Spot 14 heterozygous male and female mice (to create null, heterozygous, and wild-type littermates). Wild-type mice were obtained from breeding Spot 14 wild-type male and female mice generated from our heterozygous breeders. Lactating dams were fed a breeder chow diet containing 11% fat (Teklad chow 7004).
Lipid measurement
The rates of lactating mammary gland de novo fatty acid synthesis were measured after ip injection of 0.5 mCi 3H2O per gram body weight. Lactating dams were d 14 postpartum. The specific activity of labeled water was measured in whole blood at the time the animals were killed. Mammary glands and livers were rapidly dissected and frozen under liquid nitrogen until processed for lipid content as previously described (14).
The content of milk constituents was measured from milk curd obtained from nursing pups. Milk curd protein was measured by the method of Bradford (15) and triglyceride from a commercial kit (Infinity triglyceride reagent kit; Sigma Chemical Co., St. Louis, MO) (16, 17). Mammary gland triglycerides were measured by extraction of total lipid from mammary glands by the method of Stansbie et al. (18). The lipids were isolated by thin layer chromatography. The triglycerides were isolated, and a known quantity of pentadecanoic acid was added to the mixture to calculate recovery. The fatty acids were transesterified to fatty acid methyl esters. Methyl esters were then separated by gas chromatography, and fatty acid composition was determined by flame ionization and compared with external fatty acid standards as previously described (19).
ACC assay
Mammary gland samples were sliced into small pieces in a minimal volume of ice-cold saline and then homogenized in two volumes of cold 0.25 M sucrose for 30 sec (20, 21). The crude extract was centrifuged at 13,000 x g for 45 min. The top lipid layer was discarded, and the supernatant was removed and centrifuged at 100,000 x g for 1 h. After the final centrifugation, the supernatant was passed through a Sephadex G-50 fine-grade column, and the collected fractions were analyzed using a Bradford assay. A mixture containing 1 mg of the prepared tissue extract, 50 mM Tris-HCl (pH 7.5), 10 mM potassium citrate, 10 mM MgCl2, 3.75 mM reduced glutathione, and 0.75 mg BSA per ml was incubated at 37 C for 30 min. Another mixture containing 50 mM Tris-HCl (pH 7.5), 10 mM potassium citrate, 10 mM MgCl2, 3.75 mM reduced glutathione, 0.75 mg BSA per ml, 5 mM ATP, 0.125 mM acetyl-CoA, and 12.5 mM KH14CO3 was put into a scintillation vial. The vial also enclosed a microfuge tube without a cap containing 0.5 ml 10 M NaOH. After the enzyme incubation, the enzyme mixture was added to the scintillation vial and then sealed with a rubber stopper. The vial was further incubated at 37 C, and 0.5 ml 5 M HCl terminated the reaction and was added by inserting a 25-gauge needle through the rubber stopper. Samples were allowed to sit overnight to equilibrate. The next day, 200 μl of sample was counted in a scintillation counter to determine the amount of radioactivity remaining in the sample.
FAS assay
Mammary gland samples were homogenized in 0.25 M sucrose at a 1:10 dilution and centrifuged at 100,000 x g for 45 min (22). The clear cytoplasmic fraction was used for the enzyme assay. The cellular extract was diluted with an equal volume of 1 M potassium phosphate (pH 7.0) and 10 mM dithiothreitol. The assay contained 0.1 M potassium phosphate (pH 7.0), 0.05 mM acetyl-CoA, 0.2 mM reduced nicotinamide adenine dinucleotide phosphate (NADPH), and 1 mg/ml BSA in a volume of 0.9 ml. Fifty microliters of diluted cellular extract were added and mixed. The rate of NADPH oxidation was measured after 2 min at 340 nm. After the addition of 50 μl of the substrate malonyl-CoA, the rate of NADPH oxidation was calculated by measuring absorption after another 2-min incubation. The concentration of enzyme was adjusted to assure a linear reaction rate.
Other enzyme assays
Mammary gland samples were homogenized as previously described for liver (14). After centrifugation at 100,000 x g for 45 min, samples of the clarified supernatants were assayed for malic enzyme, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase as previously described for the liver (23). These enzymes are expressed in U/mg·min where 1 U is the amount of enzyme required to oxidize or reduce 1 nmol of NADP(H).
RNA extraction and real time RT-PCR
Total RNA was extracted from mouse tissue using a QIAGEN RNeasy Midikit (QIAGEN, Valencia, CA). Quantitative, real-time RT-PCR was performed using intron-spanning primer sequences targeting the 5' end of the mRNAs. The oligonucleotide sequences used to detect the Spot 14 and Spot 14-R mRNAs by PCR are as follows: Spot 14 forward, 5' TGA GAA CGA CGC TGC TGA AAC 3', and reverse, 5' AGG TGG GTA AGG ATG TGA TGG AG 3'; Spot 14-R forward, 5' GCA ACC ACA GTC GCC CTT ACT C 3' and reverse, 5' CCT TCT CCG CCC TCT CTA ACT TG 3'. The annealing temperatures were 56 C for Spot 14-specific oligonucleotides and 59 C for Spot 14-R-specific oligonucleotides.
RT-PCR were conducted using a Roche LightCycler (Roche Applied Science, Indianapolis, IN). Reagents used for the reactions were provided in the Roche SYBR Green I RNA amplification kit, and 100 ng total RNA was used for each reaction. RT-PCR was performed as follows: RT of template RNA for 30 min at 42 C, denaturation of the cDNA/RNA hybrid for 30 sec at 95 C, followed by 35 cycles of cDNA amplification consisting of a 15-sec denaturation at 95 C, primer annealing for 20 sec at 54 C, and product elongation for 15 sec at 72 C. The amplification process was monitored in real time via fluorescence data acquisition at the end of each amplification cycle at a temperature slightly lower than the temperature required to melt the PCR product (83 C). Threshold cycle (CT) values were determined in the log-linear amplification phase using LightCycler Software (version 3.5) and plotted vs. log RNA content.
In silico protein generation
The known sequence for the mouse Spot 14 protein was used to search the TIGR sequence databases (www.TIGR.org) using the tblastn parameter with the TIGR blast program (www.TIGRblast.TIGR.org). For each organism, potential homologous sequences were checked for the presence of multiple independent expressed sequence tags. The longest expressed sequence tag, or an identified contig, was used to translate a putative protein using the longest open reading frame that showed homology to the mouse Spot 14 protein.
Statistics
All values are expressed as the mean ± SD for single experiments and mean ± SEM when experiments were pooled. Differences between groups were determined by ANOVA using least-significant difference corrected for the number of comparisons performed. A probability of less than 0.05 is reported as significantly different between groups.
Results
The Spot 14 gene is required for the optimal growth of nursing mouse neonates
To fully characterize the Spot 14 null mouse phenotype, we assessed the Spot 14 null and wild-type neonatal growth rate. We first measured the weights of pups nursed on homozygous null or wild-type dams. No genotypic difference in birth weight was observed. However, null pups nursed on null dams were significantly reduced in weight compared with wild-type pups nursed on wild-type dams (Fig. 1). The pup weights began to diverge at postnatal d 7 and continued to diverge through weaning at postnatal d 22.
FIG. 1. Maternal Spot 14 expression is required for the maximal growth of suckling neonates. Homozygous Spot 14 null neonates nursing on Spot 14 null dams demonstrate reduced weight gain. Lactating dams were fed an 11% fat diet. Pups were allowed to nurse ad libitum and were individually weighed every 2 d after birth. These data represent first-generation backcross pups. Similar results were observed using fifth- and 11th-generation backcross progeny. The data are presented as the mean ± SEM of seven for each genotype. *, Statistical significance with a P value of <0.001 as determined by ANOVA. WT, Wild type.
We next assessed the weights of Spot 14 null and wild-type pups nursed on heterozygous dams. In contrast to the previous results, we observed no difference in neonatal weight gain between Spot 14 null and wild-type pups (data not shown). Furthermore, both genotypes grew at the same rate as the wild-type pups shown in Fig. 1. These data suggest that Spot 14 protein expression in the suckling neonate is not required for neonatal growth. Rather, optimal neonatal growth requires Spot 14 protein expression in the lactating dam.
Spot 14 is required for maximal de novo lipid synthesis in the lactating mammary gland
We next measured the triglyceride content in Spot 14 null and wild-type dam milk curd. Milk curd was obtained by allowing neonates to nurse ad libitum. The pups were then killed, and the milk curd was removed from their stomach. Measurement of Spot 14 null dam curd triglyceride levels revealed a 28% reduction when compared with wild type (P < 0.01) (Fig. 2A). No difference was observed in protein content.
FIG. 2. De novo lipid synthesis is significantly reduced in the Spot 14 null lactating mammary gland. Experiments were conducted on postpartum d 14 lactating dams and their nursing pups. A, Triglyceride content is significantly reduced in Spot 14 null dam milk. Milk was harvested from the stomachs of pups nursing lactating Spot 14 null and wild-type (WT) dams. Triglyceride and protein levels were subsequently assessed. The data are presented as the mean ± SEM of six for each genotype. *, Statistical significance with a P value of <0.001 as determined by ANOVA. B, Triglyceride and fatty acid levels are reduced in Spot 14 null dam lactating mammary gland. Mammary glands were harvested from lactating Spot 14 null and wild-type dams. Triglyceride and fatty acid levels were subsequently assessed. The data are presented as the mean ± SEM of four for each genotype. *, Statistical significance with a P value of <0.05 for the triglyceride levels and <0.001 for the fatty acid levels. C, Medium-chain fatty acid levels are reduced in the Spot 14 null lactating mammary gland. Triglycerides were extracted from lactating mammary glands, and the fatty acids were quantified by gas chromatography. The data are presented as the mean ± SEM of four for each genotype. P values were <0.05 for all fatty acid species. D, The rate of fatty acid synthesis is reduced in the Spot 14 null lactating mammary gland. Fatty acid synthesis was measured by following the incorporation of 3H into newly synthesized lipids. Each point represents an individual animal. Animals were killed at the indicated time (minutes) after 3H2O injection. Lipid synthesis was measured in an individual mammary gland.
In keeping with these data, triglyceride levels were reduced by 33% in the Spot 14 null lactating mammary gland (Fig. 2B). Assessment of the fatty acid composition of these mammary gland triglycerides revealed a marked reduction in associated medium-chain fatty acids (Fig. 2C). The 10:0, 12:0, and 14:0 fatty acids were all reduced by greater than 80% in the Spot 14 null lactating mammary gland. The 16:0 fatty acids were reduced by 59%. Reductions in longer-chain saturated and unsaturated associated fatty acids were less marked. Interestingly, fatty acids synthesized in the lactating mammary gland are predominantly medium chain in length (6). This is because the mammary gland FAS enzyme associates with a unique thioesterase enzyme (24). Mammary gland thioesterase II prematurely cleaves the elongated carbon chain from the FAS molecule to produce fatty acids that contain less than 16 carbons. As a consequence, inhibition of de novo lipid synthesis in the mammary gland will lead to marked reductions in medium-chain fatty acid levels while having a minimal effect on long-chain fatty acids.
We next assessed de novo lipid synthesis in Spot 14 null and wild-type postpartum d 14 lactating mammary glands in vivo. To assess lipid synthesis in the mammary gland, we injected lactating mice with 3H2O and harvested the mammary glands at various times after injection. The tritium is incorporated into newly synthesized lipid (18). Lipids were extracted from the harvested glands, and incorporation of tritium into the lipid was subsequently measured.
Figure 2D demonstrates the accumulation of radiolabeled lipids in Spot 14 null and wild-type lactating mammary glands. The rate of lipid synthesis is modeled by S = (/t)(1 – e–t) where S is the lipid synthesis rate, t is the time after injection of 3H2O, and is the fractional rate of degradation. Each point represents the accumulation of labeled lipids from an individual animal. This graph demonstrates that the lipid synthesis rate (S) in the null mammary gland is reduced by 62% compared with the wild type. However, the fractional degradation () rate is not different between the two genotypes. In contrast to the lactating mammary gland, however, the de novo lipid synthesis rate was not reduced in the lactating Spot 14 null liver (78 ± 9 μmol 3H/g·h, mean ± SEM for six wild-type mice) and as previously reported in male liver (14).
Reduced de novo lipid synthesis in the Spot 14 null mammary gland is not due to reduced FAS and ACC enzyme activities
ACC and FAS are two rate-limiting enzymes in the pathway of de novo lipid synthesis (9, 25). Thus, we next measured the activities of these enzymes. Postpartum d 14 lactating mammary glands were harvested from killed dams and quick frozen. Enzymes were subsequently extracted from the tissues. In contrast to the observed effects of the Spot 14 genotype on the rate of lipid synthesis, ACC and FAS enzyme activities were not reduced in the null mammary gland (Fig. 3). Indeed, ACC enzyme activities (as measured at 10 mM citrate) were significantly increased by 71% in the null glands. Because lipogenesis requires the production of NADPH, we also measured the activities of those enzymes required for the synthesis of NADPH such as malic enzyme and the hexose monophosphate shunt enzymes, 6-phosphogluconate dehydrogenase and glucose 6-phosphate dehydrogenase. We found that these enzymes were not different between the genotypes (values are mean ± SD for wild type vs. null for each enzyme, respectively: malic enzyme, 84 ± 11 vs. 84 ± 14; 6-phosphogluconate dehydrogenase, 26 ± 10 vs. 29 ± 10; glucose-6-phosphate dehydrogenase 63 ± 23 vs. 40 ± 9).
FIG. 3. The Spot 14 null mutation does not reduce ACC and FAS enzyme activities in the lactating mammary gland. ACC and FAS enzyme activities were measured in postpartum d 14 lactating mammary glands. Measurement of ACC and FAS mRNA levels revealed no significant change between genotypes for either gene (data not shown). The data are presented as the mean ± SEM of four for each genotype. *, Statistical significance with a P value of <0.01 as determined by ANOVA.
The Spot 14 gene possesses a paralog
The Spot 14 gene has been found in the genome of multiple mammals, including the human, rat, mouse, and bovine genome (Fig. 4). Interestingly, an in silico search of the gene data banks revealed the existence of another gene related in sequence to Spot 14. The Spot 14-R gene is found in the human, rat, mouse, and bovine genome (Fig. 4). However, nonmammalian vertebrates such as the frog, chicken, and fish also carry copies of the Spot 14-R gene as revealed by the close clustering of these proteins with the mammalian paralog. Interestingly, only mammals show a close clustering of the Spot 14 gene (highlighted sequences). Analysis of sequence homologies between the genes reveals three highly conserved regions (Fig. 5).
FIG. 4. The Spot 14 gene possesses a paralog. Progressive sequence alignments were performed using the Clustal series of programs (37 ) on the putative protein sequences as described in Materials and Methods. The alignments were created with MacVector version 7.1.1 (Accelrys Software Corp., San Diego, CA) using the default parameters. The figure displays the phylogeny tree developed from the cluster analysis. The numbers above the lines connecting each protein represent the dissimilarity of the proteins (and the clusters). The proteins highlighted by the bold rectangle are the mammalian Spot 14 (S14) proteins. The previously reported chicken-related proteins are labeled Chicken and ? per that report (27 ). The other proteins are arbitrarily labeled S14R and numbered sequentially within a species if there were more than one found in that species. The following are the GenBank accession numbers (those beginning with TC are TIGR database accession numbers): cow S14:TC244585, human S14:BC031989.1, mouse S14:NM_009381, rat S14:NM_012703.1, Oryzias latipes S14R3:TC39101, trout S14R3:TC49563, catfish S14R:TC6615, zebrafish S14R1:CB358783, Xenopus S14R:TC243826, chicken S14R:TC100212, human S14R:HSA272057, cow S14R:BF600856, mouse S14R:BC052899, rat S14R:NM_206950, O. latipes S14R2:TC37836, trout S14R1:TC61933, trout S14R2:TC49250, O. latipes S14R1:TC33469, and zebrafish S14R2:TC259145.
FIG. 5. The Spot 14 and Spot 14-R genes are evolutionarily conserved. Progressive sequence alignments were performed using the Clustal series of programs (37 ) as described in Fig. 4. The three most homologous regions are presented in this figure. The numbers above the sequence data represent the numbers of the amino acids in best alignment. The amino acids at the bottom of each region represent the consensus sequence. The stick figure on the right shows the location of the three homologous regions with the amino acid sequence number above the figure. The length of each darkened rectangle represents the length of each region. Note the third region has been identified as the protein-protein interaction (leucine zipper) region (27 28 ).
In our final experiments, we assessed Spot 14 and Spot 14-R mRNA levels in the lactating mammary gland, liver, and white adipose tissue. As previously reported (12), we found that Spot 14 mRNA levels were approximately the same in the lactating mammary gland and liver and 2-fold increased in white adipose tissue (Fig. 6). We further found that Spot 14-R mRNA levels are also approximately 2-fold increased in white adipose tissue compared with the liver (Fig. 6). However, lactating mammary gland Spot14-R mRNA levels are reduced to near background compared with robust expression in the liver and white adipose tissue. Finally, the absence of Spot 14 message in the null animals does not lead to a compensatory increase in mammary gland Spot 14-R mRNA levels.
FIG. 6. The Spot 14-R gene is not expressed in the lactating mammary gland. Extracted total RNA was subjected to quantitative real-time RT-PCR using primers specific for the mouse Spot 14 and Spot 14-R mRNA. All RNA samples were compared with the same reference RNA sample. No statistically significant differences in Spot 14-R mRNA levels were observed between genotypes in any tissue. The data are presented as the mean ± SEM of four to six for each genotype.
Discussion
These data suggest that Spot 14 protein family members are required for maximal de novo lipid synthesis in the mouse. In lipogenic tissues where neither the Spot 14 nor Spot 14-R genes are expressed, such as the lactating Spot 14 null mouse mammary gland (Fig. 6), the rate of de novo lipid synthesis is severely repressed (Fig. 2). In contrast, de novo lipogenesis is normal in the adult Spot 14 null mouse liver (14) where the Spot 14-R gene is expressed at high levels (Fig. 6). These data suggest that in the liver, the Spot 14-R protein compensates for ablated Spot 14 expression in the null mouse. However, the Spot 14-R protein cannot compensate for ablated Spot 14 expression in the mammary gland as Spot 14-R is expressed at low levels in this tissue. Thus, de novo lipogenesis is repressed in the Spot 14 null lactating mammary gland. Confirmation of the role of the Spot 14-R protein in hepatic lipogenesis, and its lack of a role in mammary lipogenesis, must await the development of antibodies to measure the protein content, rather than relying on the expression of the mRNA.
These data lead us to hypothesize that deletion of both the Spot 14 and Spot 14-R genes will significantly impair de novo lipid synthesis in all lipogenic tissues. Global repression of de novo lipid synthesis in such a model animal would likely produce a profound phenotype because de novo synthesized fatty acids are used as an energy source, deposited as triacylglycerol in adipose tissue, and used in the formation of cellular membranes. Conversely, impaired fatty acid synthesis in the double-null mouse may provide protection from pathologies associated with increased fatty acid levels, such as obesity, insulin resistance, and coronary heart disease (26).
Interestingly, a recent paper reported the discovery of polymorphisms in the chicken Spot 14 gene family that are associated with abdominal fat traits (27). The polymorphisms involve either an insertion or deletion in a conserved region of the Spot 14 gene implicated in homodimer formation (28) (highlighted in the homology map) and result in decreased abdominal fat weight. In support of a role for this gene in obesity, we recently reported on a correlation between body mass index and the regulation of Spot 14 mRNA levels in human white adipose tissue (29). We found that Spot 14 mRNA levels were markedly down-regulated by fasting in nonobese subjects. However, Spot 14 mRNA levels were only minimally down-regulated by fasting in obese subjects.
The Spot 14 gene has also been linked to a subset of aggressive breast cancers (10, 30). Chromosomal duplication of chromosomal position 11q13 is associated with approximately 20% of human breast cancers and confers a poor prognosis. The human Spot 14 gene is contained within this region and is expressed in mammary tumor cell lines containing the 11q13 chromosomal amplification (30). Increased lipid synthesis has been hypothesized to enhance mammary cancer cell survival (10). Together, these findings suggest that the Spot 14 gene family plays important roles in vertebrate physiology.
The Spot 14-R gene is expressed in many species of vertebrates (Figs. 4 and 5). The absence of identified Spot 14-R genes in many vertebrate species to date is likely the result of incomplete sequence data. Indeed, we propose that the Spot 14-R gene is encoded in the genome of all vertebrates. Importantly, closely homologous genes are not encoded in the completed genomes of nonvertebrate animals including Drosophila melanogaster and Caenorhabditis elegans, and the unicellular eukaryote Saccharomyces cerevisiae. Thus, these data suggest that the Spot 14-R gene arose after divergence of the vertebrate phylum. The duplication and conservation of the Spot 14 and Spot 14-R genes further suggests that these genes play critical roles in vertebrate physiology.
The Spot 14 gene is found only in mammalian species based on the close similarity of these proteins and the dissimilarity of the proteins that merge into the Spot 14 gene group (Fig. 4). Interestingly, the Spot 14, but not the Spot 14-R, gene is expressed in the lactating mammary gland (Fig. 6). The ability to synthesize lipids in the lactating mammary gland allows the dam to maintain high milk lipid levels when feeding on a low-fat diet. Such physiological flexibility provides a significant survival advantage to offspring reliant on a high-energy milk diet. The effect of the Spot 14 null mutation on milk lipid levels suggests that expression of this gene in the lactating mammary gland confers a survival advantage that has been selected for during mammalian evolution. Whether this protein is also secreted into the milk and also plays a role in lipid absorption is an intriguing possibility. It would be of further interest to assess the expression of the Spot 14 gene in mammals that produce milk lipids in varying quantities such as the rhinoceros (0%) and seal (50%) (5). Deletion of the Spot 14 gene may also provide a mechanism for genetically manipulating the milk fat content of dairy animals. Reduction of dairy milk fat content may provide economic benefit to dairy producers.
The importance of the Spot 14 gene family begs the question of gene product function. Published data suggest that the Spot 14 protein functions in the nucleus as a transcription factor necessary for the induction of lipogenic enzyme gene expression (13, 31). This hypothesis relies on the results of experiments where the Spot 14 mRNA was targeted by antisense oligonucleotides in primary rat hepatocyte cultures (13, 32). These experiments demonstrated that Spot 14 antisense oligonucleotides repressed the mRNA levels of enzymes involved in de novo lipogenesis, including FAS. Additionally, de novo lipogenesis was also repressed in the cultured hepatocytes (13). In support of the transcriptional hypothesis, recent data suggest that the Spot 14 protein interacts with the orphan nuclear receptor chicken ovalbumin upstream promoter transcription factor on the L-type pyruvate kinase promoter (31). Immunohistochemical experiments suggest that the Spot 14 protein is localized in the nucleus (33). The protein is, however, also found in the cytoplasm (33). The data presented here do not support the hypothesis that Spot 14 regulates lipogenic enzyme gene mRNA levels. We find that lipogenic enzyme mRNA levels are not different in the Spot 14 null vs. wild-type lactating mammary gland (data not shown), and the enzyme activities are normal or elevated (Fig. 3). However, we cannot rule out the possibility that the Spot 14 gene regulates the mRNA levels of other genes.
What is the biochemical function of the Spot 14 gene product if it does not function to regulate lipogenic enzyme mRNA levels? The rate of liver lipogenesis is tightly correlated with lipogenic enzyme levels and activities (34, 35). However, whereas the mRNA levels and lipogenic enzyme activities are not different between the Spot 14 null and wild-type mouse lactating mammary gland (Fig. 3), the rate of de novo lipogenesis is significantly altered (Fig. 2D). We hypothesize that one of the rate-limiting enzyme’s activity is allosterically inhibited in the absence of Spot 14 protein. We further hypothesize that Spot 14 protein functions to relieve this allosteric inhibition. A similar observation in the lactating mammary gland has been reported previously (36). In that study, dietary administration of a specific polyunsaturated fatty acid markedly inhibited de novo lipogenesis without altering lipogenic enzyme levels. Administration of other polyunsaturated fatty acids did not affect lipogenesis. An alternative hypothesis is that the availability of substrate to FAS is reduced in the null mouse mammary gland.
In summary, our data demonstrate that the Spot 14 protein plays a unique role in mammary gland lipogenesis. The absence of Spot 14 protein in the lactating mammary gland leads to a reduced lipid synthesis rate, a corresponding reduction in medium-chain fatty acids, and inadequate pup nutrition leading to a reduced growth rate. We speculate that Spot 14 may function to relieve allosteric inhibition of key lipogenic enzymes. The existence and evolutionary conservation of the Spot 14-R gene suggests the importance of the Spot 14 gene family in vertebrate physiology. The unique expression of Spot 14 in the lactating mammary gland suggests the Spot 14 protein has evolved to enhance mammalian neonate survival. The Spot 14 null mouse model provides a unique opportunity to understand the biochemical mechanisms that regulate lipogenesis in vivo.
Acknowledgments
We thank Mark Margosian and Mary Jo Stack for excellent technical support. The administrative and secretarial support of Lucy Mittag and Tanya Doble are gratefully appreciated.
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Address all correspondence and requests for reprints to: Cary N. Mariash, M.D., Director, Division of Endocrinology and Diabetes, Department of Medicine, University of Minnesota, MMC 101, 420 Delaware Street SE, Minneapolis, Minnesota 55455. E-mail: mariasc@umn.edu.
Abstract
We generated a Spot 14 null mouse to assess the role of Spot 14 in de novo lipid synthesis and report the Spot 14 null mouse exhibits a phenotype in the lactating mammary gland. Spot 14 null pups nursed by Spot 14 null dams gain significantly less weight than wild-type pups nursed by wild-type dams. In contrast, Spot 14 null pups nursed by heterozygous dams show similar weight gain to wild-type littermates. We found the triglyceride content in Spot 14 null milk is significantly reduced. We demonstrate this reduction is the direct result of decreased de novo lipid synthesis in lactating mammary glands, corroborated by a marked reduction of medium-chain fatty acids in the triglyceride pool. Importantly, the reduced lipogenic rate is not associated with significant changes in the activities or mRNA of key lipogenic enzymes. Finally, we report the expression of a Spot 14-related gene in liver and adipose tissue, which is absent in the lactating mammary gland. We suggest that expression of both the Spot 14 and Spot 14-related proteins is required for maximum efficiency of de novo lipid synthesis in vivo and that these proteins impart a novel mechanism regulating de novo lipogenesis.
Introduction
REDUCED ENERGY INTAKE during early neonatal life has long-term effects on growth and adiposity in the adult mammal (1, 2, 3, 4). Thus, mammals have evolved to produce milk, a nutritionally controlled, high-energy neonatal food source. Triacylglycerols are major milk constituents and comprise a crucial energy source for the suckling neonate (5, 6, 7). The synthesis of triacylglycerols is a tightly controlled process in the lactating mammary gland. Triacylglycerols are synthesized in the mammary epithelial cell through the esterification of fatty acids. The fatty acids used in the synthesis of milk triacylglycerols are either exogenous fatty acids taken up from the plasma or are synthesized de novo in the mammary epithelial cell. De novo lipogenesis also occurs in other lipogenic tissues such as the liver and adipose tissue. The process of de novo lipogenesis in all these tissues includes the action of multiple enzymes including the rate-limiting enzyme acetyl coenzyme A (CoA) carboxylase (ACC) and the terminal enzyme fatty acid synthase (FAS) (8, 9). The lipogenic rate is closely correlated with lipogenic enzyme activities.
The Spot 14 gene has long been associated with de novo lipogenesis (10, 11). The Spot 14 mRNA was first identified while screening hepatic tissue for genes regulated by thyroid hormone (11). Spot 14 is expressed only in lipogenic tissues, and Spot 14 mRNA levels correlate well with the lipogenic rate associated with each tissue (12). Spot 14 is a small, acidic protein with no known functional motifs. The biochemical function of Spot 14 is currently unknown. Antisense experiments suggested that Spot 14 regulates the activation of lipogenic enzyme transcription by stimuli such as carbohydrate feeding and thyroid hormone administration (13). To test this hypothesis, we recently generated a Spot 14 null mouse (14). However, in contrast to the antisense studies, adult hepatic lipogenesis is not inhibited in the Spot 14 null animal (14). These data suggested two possibilities. First, the Spot 14 gene is not required for de novo lipogenesis in lipogenic tissues, or second, the adult Spot 14 null mouse liver possesses compensatory mechanisms to account for the absence of the Spot 14 protein.
We now report that the Spot 14 protein is required for de novo lipogenesis in a separate lipogenic tissue, the lactating mammary gland. We further show that the Spot 14 gene possesses a paralog. We have named the paralog the Spot 14-related (Spot 14-R) gene. Spot 14-R is expressed at high levels in the liver and white adipose tissue but is absent in the lactating mammary gland. The Spot 14-R gene is evolutionarily conserved and is present in the genome of vertebrates as divergent as Xenopus laevis and Homo sapiens. However, the Spot 14 gene is present only in the genome of mammalian species. Together these data suggest that the Spot 14 gene evolved to control milk energy content and that the Spot 14 gene family is an important regulator of de novo lipid synthesis in lipogenic tissues.
Materials and Methods
Animal use
All animal use and protocols were approved by the University of Minnesota Institutional Animal Care and Use Committee. Spot 14 knockout and wild-type mice were created as previously described (14). The mice were subsequently backcrossed to a C57BL6/J strain up to 11 times to assure the mice were studied with a homogeneous background. To create Spot 14 null mice, pups were bred by mating either Spot 14 null male and female mice (to create all Spot 14 null mice) or by mating Spot 14 heterozygous male and female mice (to create null, heterozygous, and wild-type littermates). Wild-type mice were obtained from breeding Spot 14 wild-type male and female mice generated from our heterozygous breeders. Lactating dams were fed a breeder chow diet containing 11% fat (Teklad chow 7004).
Lipid measurement
The rates of lactating mammary gland de novo fatty acid synthesis were measured after ip injection of 0.5 mCi 3H2O per gram body weight. Lactating dams were d 14 postpartum. The specific activity of labeled water was measured in whole blood at the time the animals were killed. Mammary glands and livers were rapidly dissected and frozen under liquid nitrogen until processed for lipid content as previously described (14).
The content of milk constituents was measured from milk curd obtained from nursing pups. Milk curd protein was measured by the method of Bradford (15) and triglyceride from a commercial kit (Infinity triglyceride reagent kit; Sigma Chemical Co., St. Louis, MO) (16, 17). Mammary gland triglycerides were measured by extraction of total lipid from mammary glands by the method of Stansbie et al. (18). The lipids were isolated by thin layer chromatography. The triglycerides were isolated, and a known quantity of pentadecanoic acid was added to the mixture to calculate recovery. The fatty acids were transesterified to fatty acid methyl esters. Methyl esters were then separated by gas chromatography, and fatty acid composition was determined by flame ionization and compared with external fatty acid standards as previously described (19).
ACC assay
Mammary gland samples were sliced into small pieces in a minimal volume of ice-cold saline and then homogenized in two volumes of cold 0.25 M sucrose for 30 sec (20, 21). The crude extract was centrifuged at 13,000 x g for 45 min. The top lipid layer was discarded, and the supernatant was removed and centrifuged at 100,000 x g for 1 h. After the final centrifugation, the supernatant was passed through a Sephadex G-50 fine-grade column, and the collected fractions were analyzed using a Bradford assay. A mixture containing 1 mg of the prepared tissue extract, 50 mM Tris-HCl (pH 7.5), 10 mM potassium citrate, 10 mM MgCl2, 3.75 mM reduced glutathione, and 0.75 mg BSA per ml was incubated at 37 C for 30 min. Another mixture containing 50 mM Tris-HCl (pH 7.5), 10 mM potassium citrate, 10 mM MgCl2, 3.75 mM reduced glutathione, 0.75 mg BSA per ml, 5 mM ATP, 0.125 mM acetyl-CoA, and 12.5 mM KH14CO3 was put into a scintillation vial. The vial also enclosed a microfuge tube without a cap containing 0.5 ml 10 M NaOH. After the enzyme incubation, the enzyme mixture was added to the scintillation vial and then sealed with a rubber stopper. The vial was further incubated at 37 C, and 0.5 ml 5 M HCl terminated the reaction and was added by inserting a 25-gauge needle through the rubber stopper. Samples were allowed to sit overnight to equilibrate. The next day, 200 μl of sample was counted in a scintillation counter to determine the amount of radioactivity remaining in the sample.
FAS assay
Mammary gland samples were homogenized in 0.25 M sucrose at a 1:10 dilution and centrifuged at 100,000 x g for 45 min (22). The clear cytoplasmic fraction was used for the enzyme assay. The cellular extract was diluted with an equal volume of 1 M potassium phosphate (pH 7.0) and 10 mM dithiothreitol. The assay contained 0.1 M potassium phosphate (pH 7.0), 0.05 mM acetyl-CoA, 0.2 mM reduced nicotinamide adenine dinucleotide phosphate (NADPH), and 1 mg/ml BSA in a volume of 0.9 ml. Fifty microliters of diluted cellular extract were added and mixed. The rate of NADPH oxidation was measured after 2 min at 340 nm. After the addition of 50 μl of the substrate malonyl-CoA, the rate of NADPH oxidation was calculated by measuring absorption after another 2-min incubation. The concentration of enzyme was adjusted to assure a linear reaction rate.
Other enzyme assays
Mammary gland samples were homogenized as previously described for liver (14). After centrifugation at 100,000 x g for 45 min, samples of the clarified supernatants were assayed for malic enzyme, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase as previously described for the liver (23). These enzymes are expressed in U/mg·min where 1 U is the amount of enzyme required to oxidize or reduce 1 nmol of NADP(H).
RNA extraction and real time RT-PCR
Total RNA was extracted from mouse tissue using a QIAGEN RNeasy Midikit (QIAGEN, Valencia, CA). Quantitative, real-time RT-PCR was performed using intron-spanning primer sequences targeting the 5' end of the mRNAs. The oligonucleotide sequences used to detect the Spot 14 and Spot 14-R mRNAs by PCR are as follows: Spot 14 forward, 5' TGA GAA CGA CGC TGC TGA AAC 3', and reverse, 5' AGG TGG GTA AGG ATG TGA TGG AG 3'; Spot 14-R forward, 5' GCA ACC ACA GTC GCC CTT ACT C 3' and reverse, 5' CCT TCT CCG CCC TCT CTA ACT TG 3'. The annealing temperatures were 56 C for Spot 14-specific oligonucleotides and 59 C for Spot 14-R-specific oligonucleotides.
RT-PCR were conducted using a Roche LightCycler (Roche Applied Science, Indianapolis, IN). Reagents used for the reactions were provided in the Roche SYBR Green I RNA amplification kit, and 100 ng total RNA was used for each reaction. RT-PCR was performed as follows: RT of template RNA for 30 min at 42 C, denaturation of the cDNA/RNA hybrid for 30 sec at 95 C, followed by 35 cycles of cDNA amplification consisting of a 15-sec denaturation at 95 C, primer annealing for 20 sec at 54 C, and product elongation for 15 sec at 72 C. The amplification process was monitored in real time via fluorescence data acquisition at the end of each amplification cycle at a temperature slightly lower than the temperature required to melt the PCR product (83 C). Threshold cycle (CT) values were determined in the log-linear amplification phase using LightCycler Software (version 3.5) and plotted vs. log RNA content.
In silico protein generation
The known sequence for the mouse Spot 14 protein was used to search the TIGR sequence databases (www.TIGR.org) using the tblastn parameter with the TIGR blast program (www.TIGRblast.TIGR.org). For each organism, potential homologous sequences were checked for the presence of multiple independent expressed sequence tags. The longest expressed sequence tag, or an identified contig, was used to translate a putative protein using the longest open reading frame that showed homology to the mouse Spot 14 protein.
Statistics
All values are expressed as the mean ± SD for single experiments and mean ± SEM when experiments were pooled. Differences between groups were determined by ANOVA using least-significant difference corrected for the number of comparisons performed. A probability of less than 0.05 is reported as significantly different between groups.
Results
The Spot 14 gene is required for the optimal growth of nursing mouse neonates
To fully characterize the Spot 14 null mouse phenotype, we assessed the Spot 14 null and wild-type neonatal growth rate. We first measured the weights of pups nursed on homozygous null or wild-type dams. No genotypic difference in birth weight was observed. However, null pups nursed on null dams were significantly reduced in weight compared with wild-type pups nursed on wild-type dams (Fig. 1). The pup weights began to diverge at postnatal d 7 and continued to diverge through weaning at postnatal d 22.
FIG. 1. Maternal Spot 14 expression is required for the maximal growth of suckling neonates. Homozygous Spot 14 null neonates nursing on Spot 14 null dams demonstrate reduced weight gain. Lactating dams were fed an 11% fat diet. Pups were allowed to nurse ad libitum and were individually weighed every 2 d after birth. These data represent first-generation backcross pups. Similar results were observed using fifth- and 11th-generation backcross progeny. The data are presented as the mean ± SEM of seven for each genotype. *, Statistical significance with a P value of <0.001 as determined by ANOVA. WT, Wild type.
We next assessed the weights of Spot 14 null and wild-type pups nursed on heterozygous dams. In contrast to the previous results, we observed no difference in neonatal weight gain between Spot 14 null and wild-type pups (data not shown). Furthermore, both genotypes grew at the same rate as the wild-type pups shown in Fig. 1. These data suggest that Spot 14 protein expression in the suckling neonate is not required for neonatal growth. Rather, optimal neonatal growth requires Spot 14 protein expression in the lactating dam.
Spot 14 is required for maximal de novo lipid synthesis in the lactating mammary gland
We next measured the triglyceride content in Spot 14 null and wild-type dam milk curd. Milk curd was obtained by allowing neonates to nurse ad libitum. The pups were then killed, and the milk curd was removed from their stomach. Measurement of Spot 14 null dam curd triglyceride levels revealed a 28% reduction when compared with wild type (P < 0.01) (Fig. 2A). No difference was observed in protein content.
FIG. 2. De novo lipid synthesis is significantly reduced in the Spot 14 null lactating mammary gland. Experiments were conducted on postpartum d 14 lactating dams and their nursing pups. A, Triglyceride content is significantly reduced in Spot 14 null dam milk. Milk was harvested from the stomachs of pups nursing lactating Spot 14 null and wild-type (WT) dams. Triglyceride and protein levels were subsequently assessed. The data are presented as the mean ± SEM of six for each genotype. *, Statistical significance with a P value of <0.001 as determined by ANOVA. B, Triglyceride and fatty acid levels are reduced in Spot 14 null dam lactating mammary gland. Mammary glands were harvested from lactating Spot 14 null and wild-type dams. Triglyceride and fatty acid levels were subsequently assessed. The data are presented as the mean ± SEM of four for each genotype. *, Statistical significance with a P value of <0.05 for the triglyceride levels and <0.001 for the fatty acid levels. C, Medium-chain fatty acid levels are reduced in the Spot 14 null lactating mammary gland. Triglycerides were extracted from lactating mammary glands, and the fatty acids were quantified by gas chromatography. The data are presented as the mean ± SEM of four for each genotype. P values were <0.05 for all fatty acid species. D, The rate of fatty acid synthesis is reduced in the Spot 14 null lactating mammary gland. Fatty acid synthesis was measured by following the incorporation of 3H into newly synthesized lipids. Each point represents an individual animal. Animals were killed at the indicated time (minutes) after 3H2O injection. Lipid synthesis was measured in an individual mammary gland.
In keeping with these data, triglyceride levels were reduced by 33% in the Spot 14 null lactating mammary gland (Fig. 2B). Assessment of the fatty acid composition of these mammary gland triglycerides revealed a marked reduction in associated medium-chain fatty acids (Fig. 2C). The 10:0, 12:0, and 14:0 fatty acids were all reduced by greater than 80% in the Spot 14 null lactating mammary gland. The 16:0 fatty acids were reduced by 59%. Reductions in longer-chain saturated and unsaturated associated fatty acids were less marked. Interestingly, fatty acids synthesized in the lactating mammary gland are predominantly medium chain in length (6). This is because the mammary gland FAS enzyme associates with a unique thioesterase enzyme (24). Mammary gland thioesterase II prematurely cleaves the elongated carbon chain from the FAS molecule to produce fatty acids that contain less than 16 carbons. As a consequence, inhibition of de novo lipid synthesis in the mammary gland will lead to marked reductions in medium-chain fatty acid levels while having a minimal effect on long-chain fatty acids.
We next assessed de novo lipid synthesis in Spot 14 null and wild-type postpartum d 14 lactating mammary glands in vivo. To assess lipid synthesis in the mammary gland, we injected lactating mice with 3H2O and harvested the mammary glands at various times after injection. The tritium is incorporated into newly synthesized lipid (18). Lipids were extracted from the harvested glands, and incorporation of tritium into the lipid was subsequently measured.
Figure 2D demonstrates the accumulation of radiolabeled lipids in Spot 14 null and wild-type lactating mammary glands. The rate of lipid synthesis is modeled by S = (/t)(1 – e–t) where S is the lipid synthesis rate, t is the time after injection of 3H2O, and is the fractional rate of degradation. Each point represents the accumulation of labeled lipids from an individual animal. This graph demonstrates that the lipid synthesis rate (S) in the null mammary gland is reduced by 62% compared with the wild type. However, the fractional degradation () rate is not different between the two genotypes. In contrast to the lactating mammary gland, however, the de novo lipid synthesis rate was not reduced in the lactating Spot 14 null liver (78 ± 9 μmol 3H/g·h, mean ± SEM for six wild-type mice) and as previously reported in male liver (14).
Reduced de novo lipid synthesis in the Spot 14 null mammary gland is not due to reduced FAS and ACC enzyme activities
ACC and FAS are two rate-limiting enzymes in the pathway of de novo lipid synthesis (9, 25). Thus, we next measured the activities of these enzymes. Postpartum d 14 lactating mammary glands were harvested from killed dams and quick frozen. Enzymes were subsequently extracted from the tissues. In contrast to the observed effects of the Spot 14 genotype on the rate of lipid synthesis, ACC and FAS enzyme activities were not reduced in the null mammary gland (Fig. 3). Indeed, ACC enzyme activities (as measured at 10 mM citrate) were significantly increased by 71% in the null glands. Because lipogenesis requires the production of NADPH, we also measured the activities of those enzymes required for the synthesis of NADPH such as malic enzyme and the hexose monophosphate shunt enzymes, 6-phosphogluconate dehydrogenase and glucose 6-phosphate dehydrogenase. We found that these enzymes were not different between the genotypes (values are mean ± SD for wild type vs. null for each enzyme, respectively: malic enzyme, 84 ± 11 vs. 84 ± 14; 6-phosphogluconate dehydrogenase, 26 ± 10 vs. 29 ± 10; glucose-6-phosphate dehydrogenase 63 ± 23 vs. 40 ± 9).
FIG. 3. The Spot 14 null mutation does not reduce ACC and FAS enzyme activities in the lactating mammary gland. ACC and FAS enzyme activities were measured in postpartum d 14 lactating mammary glands. Measurement of ACC and FAS mRNA levels revealed no significant change between genotypes for either gene (data not shown). The data are presented as the mean ± SEM of four for each genotype. *, Statistical significance with a P value of <0.01 as determined by ANOVA.
The Spot 14 gene possesses a paralog
The Spot 14 gene has been found in the genome of multiple mammals, including the human, rat, mouse, and bovine genome (Fig. 4). Interestingly, an in silico search of the gene data banks revealed the existence of another gene related in sequence to Spot 14. The Spot 14-R gene is found in the human, rat, mouse, and bovine genome (Fig. 4). However, nonmammalian vertebrates such as the frog, chicken, and fish also carry copies of the Spot 14-R gene as revealed by the close clustering of these proteins with the mammalian paralog. Interestingly, only mammals show a close clustering of the Spot 14 gene (highlighted sequences). Analysis of sequence homologies between the genes reveals three highly conserved regions (Fig. 5).
FIG. 4. The Spot 14 gene possesses a paralog. Progressive sequence alignments were performed using the Clustal series of programs (37 ) on the putative protein sequences as described in Materials and Methods. The alignments were created with MacVector version 7.1.1 (Accelrys Software Corp., San Diego, CA) using the default parameters. The figure displays the phylogeny tree developed from the cluster analysis. The numbers above the lines connecting each protein represent the dissimilarity of the proteins (and the clusters). The proteins highlighted by the bold rectangle are the mammalian Spot 14 (S14) proteins. The previously reported chicken-related proteins are labeled Chicken and ? per that report (27 ). The other proteins are arbitrarily labeled S14R and numbered sequentially within a species if there were more than one found in that species. The following are the GenBank accession numbers (those beginning with TC are TIGR database accession numbers): cow S14:TC244585, human S14:BC031989.1, mouse S14:NM_009381, rat S14:NM_012703.1, Oryzias latipes S14R3:TC39101, trout S14R3:TC49563, catfish S14R:TC6615, zebrafish S14R1:CB358783, Xenopus S14R:TC243826, chicken S14R:TC100212, human S14R:HSA272057, cow S14R:BF600856, mouse S14R:BC052899, rat S14R:NM_206950, O. latipes S14R2:TC37836, trout S14R1:TC61933, trout S14R2:TC49250, O. latipes S14R1:TC33469, and zebrafish S14R2:TC259145.
FIG. 5. The Spot 14 and Spot 14-R genes are evolutionarily conserved. Progressive sequence alignments were performed using the Clustal series of programs (37 ) as described in Fig. 4. The three most homologous regions are presented in this figure. The numbers above the sequence data represent the numbers of the amino acids in best alignment. The amino acids at the bottom of each region represent the consensus sequence. The stick figure on the right shows the location of the three homologous regions with the amino acid sequence number above the figure. The length of each darkened rectangle represents the length of each region. Note the third region has been identified as the protein-protein interaction (leucine zipper) region (27 28 ).
In our final experiments, we assessed Spot 14 and Spot 14-R mRNA levels in the lactating mammary gland, liver, and white adipose tissue. As previously reported (12), we found that Spot 14 mRNA levels were approximately the same in the lactating mammary gland and liver and 2-fold increased in white adipose tissue (Fig. 6). We further found that Spot 14-R mRNA levels are also approximately 2-fold increased in white adipose tissue compared with the liver (Fig. 6). However, lactating mammary gland Spot14-R mRNA levels are reduced to near background compared with robust expression in the liver and white adipose tissue. Finally, the absence of Spot 14 message in the null animals does not lead to a compensatory increase in mammary gland Spot 14-R mRNA levels.
FIG. 6. The Spot 14-R gene is not expressed in the lactating mammary gland. Extracted total RNA was subjected to quantitative real-time RT-PCR using primers specific for the mouse Spot 14 and Spot 14-R mRNA. All RNA samples were compared with the same reference RNA sample. No statistically significant differences in Spot 14-R mRNA levels were observed between genotypes in any tissue. The data are presented as the mean ± SEM of four to six for each genotype.
Discussion
These data suggest that Spot 14 protein family members are required for maximal de novo lipid synthesis in the mouse. In lipogenic tissues where neither the Spot 14 nor Spot 14-R genes are expressed, such as the lactating Spot 14 null mouse mammary gland (Fig. 6), the rate of de novo lipid synthesis is severely repressed (Fig. 2). In contrast, de novo lipogenesis is normal in the adult Spot 14 null mouse liver (14) where the Spot 14-R gene is expressed at high levels (Fig. 6). These data suggest that in the liver, the Spot 14-R protein compensates for ablated Spot 14 expression in the null mouse. However, the Spot 14-R protein cannot compensate for ablated Spot 14 expression in the mammary gland as Spot 14-R is expressed at low levels in this tissue. Thus, de novo lipogenesis is repressed in the Spot 14 null lactating mammary gland. Confirmation of the role of the Spot 14-R protein in hepatic lipogenesis, and its lack of a role in mammary lipogenesis, must await the development of antibodies to measure the protein content, rather than relying on the expression of the mRNA.
These data lead us to hypothesize that deletion of both the Spot 14 and Spot 14-R genes will significantly impair de novo lipid synthesis in all lipogenic tissues. Global repression of de novo lipid synthesis in such a model animal would likely produce a profound phenotype because de novo synthesized fatty acids are used as an energy source, deposited as triacylglycerol in adipose tissue, and used in the formation of cellular membranes. Conversely, impaired fatty acid synthesis in the double-null mouse may provide protection from pathologies associated with increased fatty acid levels, such as obesity, insulin resistance, and coronary heart disease (26).
Interestingly, a recent paper reported the discovery of polymorphisms in the chicken Spot 14 gene family that are associated with abdominal fat traits (27). The polymorphisms involve either an insertion or deletion in a conserved region of the Spot 14 gene implicated in homodimer formation (28) (highlighted in the homology map) and result in decreased abdominal fat weight. In support of a role for this gene in obesity, we recently reported on a correlation between body mass index and the regulation of Spot 14 mRNA levels in human white adipose tissue (29). We found that Spot 14 mRNA levels were markedly down-regulated by fasting in nonobese subjects. However, Spot 14 mRNA levels were only minimally down-regulated by fasting in obese subjects.
The Spot 14 gene has also been linked to a subset of aggressive breast cancers (10, 30). Chromosomal duplication of chromosomal position 11q13 is associated with approximately 20% of human breast cancers and confers a poor prognosis. The human Spot 14 gene is contained within this region and is expressed in mammary tumor cell lines containing the 11q13 chromosomal amplification (30). Increased lipid synthesis has been hypothesized to enhance mammary cancer cell survival (10). Together, these findings suggest that the Spot 14 gene family plays important roles in vertebrate physiology.
The Spot 14-R gene is expressed in many species of vertebrates (Figs. 4 and 5). The absence of identified Spot 14-R genes in many vertebrate species to date is likely the result of incomplete sequence data. Indeed, we propose that the Spot 14-R gene is encoded in the genome of all vertebrates. Importantly, closely homologous genes are not encoded in the completed genomes of nonvertebrate animals including Drosophila melanogaster and Caenorhabditis elegans, and the unicellular eukaryote Saccharomyces cerevisiae. Thus, these data suggest that the Spot 14-R gene arose after divergence of the vertebrate phylum. The duplication and conservation of the Spot 14 and Spot 14-R genes further suggests that these genes play critical roles in vertebrate physiology.
The Spot 14 gene is found only in mammalian species based on the close similarity of these proteins and the dissimilarity of the proteins that merge into the Spot 14 gene group (Fig. 4). Interestingly, the Spot 14, but not the Spot 14-R, gene is expressed in the lactating mammary gland (Fig. 6). The ability to synthesize lipids in the lactating mammary gland allows the dam to maintain high milk lipid levels when feeding on a low-fat diet. Such physiological flexibility provides a significant survival advantage to offspring reliant on a high-energy milk diet. The effect of the Spot 14 null mutation on milk lipid levels suggests that expression of this gene in the lactating mammary gland confers a survival advantage that has been selected for during mammalian evolution. Whether this protein is also secreted into the milk and also plays a role in lipid absorption is an intriguing possibility. It would be of further interest to assess the expression of the Spot 14 gene in mammals that produce milk lipids in varying quantities such as the rhinoceros (0%) and seal (50%) (5). Deletion of the Spot 14 gene may also provide a mechanism for genetically manipulating the milk fat content of dairy animals. Reduction of dairy milk fat content may provide economic benefit to dairy producers.
The importance of the Spot 14 gene family begs the question of gene product function. Published data suggest that the Spot 14 protein functions in the nucleus as a transcription factor necessary for the induction of lipogenic enzyme gene expression (13, 31). This hypothesis relies on the results of experiments where the Spot 14 mRNA was targeted by antisense oligonucleotides in primary rat hepatocyte cultures (13, 32). These experiments demonstrated that Spot 14 antisense oligonucleotides repressed the mRNA levels of enzymes involved in de novo lipogenesis, including FAS. Additionally, de novo lipogenesis was also repressed in the cultured hepatocytes (13). In support of the transcriptional hypothesis, recent data suggest that the Spot 14 protein interacts with the orphan nuclear receptor chicken ovalbumin upstream promoter transcription factor on the L-type pyruvate kinase promoter (31). Immunohistochemical experiments suggest that the Spot 14 protein is localized in the nucleus (33). The protein is, however, also found in the cytoplasm (33). The data presented here do not support the hypothesis that Spot 14 regulates lipogenic enzyme gene mRNA levels. We find that lipogenic enzyme mRNA levels are not different in the Spot 14 null vs. wild-type lactating mammary gland (data not shown), and the enzyme activities are normal or elevated (Fig. 3). However, we cannot rule out the possibility that the Spot 14 gene regulates the mRNA levels of other genes.
What is the biochemical function of the Spot 14 gene product if it does not function to regulate lipogenic enzyme mRNA levels? The rate of liver lipogenesis is tightly correlated with lipogenic enzyme levels and activities (34, 35). However, whereas the mRNA levels and lipogenic enzyme activities are not different between the Spot 14 null and wild-type mouse lactating mammary gland (Fig. 3), the rate of de novo lipogenesis is significantly altered (Fig. 2D). We hypothesize that one of the rate-limiting enzyme’s activity is allosterically inhibited in the absence of Spot 14 protein. We further hypothesize that Spot 14 protein functions to relieve this allosteric inhibition. A similar observation in the lactating mammary gland has been reported previously (36). In that study, dietary administration of a specific polyunsaturated fatty acid markedly inhibited de novo lipogenesis without altering lipogenic enzyme levels. Administration of other polyunsaturated fatty acids did not affect lipogenesis. An alternative hypothesis is that the availability of substrate to FAS is reduced in the null mouse mammary gland.
In summary, our data demonstrate that the Spot 14 protein plays a unique role in mammary gland lipogenesis. The absence of Spot 14 protein in the lactating mammary gland leads to a reduced lipid synthesis rate, a corresponding reduction in medium-chain fatty acids, and inadequate pup nutrition leading to a reduced growth rate. We speculate that Spot 14 may function to relieve allosteric inhibition of key lipogenic enzymes. The existence and evolutionary conservation of the Spot 14-R gene suggests the importance of the Spot 14 gene family in vertebrate physiology. The unique expression of Spot 14 in the lactating mammary gland suggests the Spot 14 protein has evolved to enhance mammalian neonate survival. The Spot 14 null mouse model provides a unique opportunity to understand the biochemical mechanisms that regulate lipogenesis in vivo.
Acknowledgments
We thank Mark Margosian and Mary Jo Stack for excellent technical support. The administrative and secretarial support of Lucy Mittag and Tanya Doble are gratefully appreciated.
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