Endocrinology, doi:10.1210/en.2006-0473
Endocrinology Vol. 147, No. 9 4044-4047
Copyright © 2006 by The Endocrine Society
S14: Insights from Knockout Mice
Laura T. LaFave,
Lance B. Augustin and
Cary N. Mariash
Division of Endocrinology and Diabetes, Department of Medicine, University of Minnesota, Minneapolis, Minnesota 55455
Address all correspondence and requests for reprints to: Cary N. Mariash, M.D., MMC 101, 420 Delaware Street SE, Minneapolis, Minnesota 55455.
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Abstract
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Spot 14 (S14) is a protein whose mRNA is rapidly up-regulated by lipogenic stimuli including thyroid hormone and a high-carbohydrate diet. Previous investigation into the role of S14 suggested that it is involved in de novo lipogenesis. Knockout of the gene in mice has given further support to this hypothesis. The lack of S14 in different tissues resulted in varying phenotypic effects. In the lactating mammary gland, levels of lipogenesis, specifically the production of medium chain fatty acids, were decreased, whereas hepatic lipogenesis was not decreased. In fact, hepatic lipogenesis was increased, and the increase may be due to compensation by a paralog of S14 called S14-R. S14-R is expressed in the liver but not the mammary gland. Importantly, S14 knockout mice did not have reduced levels of lipogenic enzymes, implying that it does not affect the transcriptional rate of those enzymes. Instead, S14 may act in the cytoplasm to affect lipogenesis.
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Introduction
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THE STORY OF Spot 14 (S14) began more than 2 decades ago. Investigators used two-dimensional gel electrophoresis of in vitro-translated products to survey the effect of thyroid hormone on rat hepatic gene expression (1). Among the transcripts that responded to both thyroid hormone and a lipogenic (high carbohydrate) diet was the mRNA for S14, so named because it was the 14th spot on the gel noted to change in response to thyroid hormone status.
The S14 gene codes for a 17-kDa acidic protein that lacks any well-recognized functional motifs. Historically, the protein had not been known to share homology with any proteins of established function, making determination of its own function a challenge. Much of the work done on S14 has focused on regulation of its gene, although recent development of a knockout model has started to illuminate S14’s physiologic role (2, 3).
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Regulation of the S14 Gene
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Thyroid hormone
The most striking aspect of S14 regulation is its rapid and robust response to thyroid hormone (TH) and carbohydrate. To this end, the gene has been very useful in the study of TH action, even as the precise role of the protein itself has eluded discovery. Hypothyroid rats injected with T3 demonstrated induction of hepatic S14 mRNA within 20 min of treatment (4). Similar treatment induced a nuclear precursor of S14 mRNA within 10 min, suggesting that S14 is directly responsive to T3 at the nuclear level (5). Identification of several thyroid hormone response elements far upstream of the start site of transcription (6) confirmed the direct role of T3 in regulating the transcription of this gene.
Carbohydrate
Many hepatic enzymes induced by TH are also similarly induced by a lipogenic (high carbohydrate, fat free) diet (7). There is clearly a synergistic effect of TH and carbohydrate on S14 gene induction. Administration of sucrose to euthyroid rats caused a 25-fold increase in S14 mRNA by 4 h, whereas in hypothyroid rats there was only a 2- to 3-fold increase (8). Replacing the TH restored the rapid response to sucrose administration, implying that sucrose and TH act together to induce S14 mRNA. The S14 gene has also been useful in understanding how carbohydrate metabolism alters transcription of specific genes. Thus, a carbohydrate response element was identified in the S14 gene that contains a CACGTG motif (9). This motif is present in other carbohydrate-responsive genes, and the proteins that bind to and regulate transcription through this motif have recently been identified (2, 10).
Insulin
Levels of S14 are reduced in diabetic rats (11). Giving insulin to streptozotocin-induced diabetic rats restores levels of hepatic S14 mRNA to normal within 4 h, and the mechanism is thought to be principally transcriptional (11). Insulin also increased the ratio of mature to precursor S14 mRNA, suggesting that the hormone may act posttranscriptionally as well (12).
Fatty acids
Administration of long-chain polyunsaturated fatty acids (PUFAs) to rats down-regulated S14 mRNA. In one study this effect was mediated transcriptionally within the region of the S14 proximal promoter (13). A subsequent demonstrated that the inhibitory effect of PUFA was due to a peroxidative mechanism (14). They found that minimization of PUFA peroxidation abolishes the inhibition of lipogenic gene transcription by PUFAs.
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Evidence for the Role of S14 in Lipogenesis
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S14 mRNA has been detected exclusively in lipogenic tissues (15, 16), leading to speculation that the protein may be involved in lipogenesis. Levels of hepatic S14 mRNA are extremely low in fetal and newborn rats but increase at the time of weaning (17). The increase in S14 mRNA is accompanied by a similar rise in lipogenesis and lipogenic enzymes as well as a decrease in ß-oxidation. When rats are weaned prematurely, the rise is earlier and more marked, suggesting that S14 is involved in the synthesis of fatty acids in the neonate. Additionally, S14 and acetyl-coenzyme A-carboxylase, a rate-determining enzyme of fatty acid synthesis, are coexpressed as determined by immunohistochemistry in livers of euthyroid rats fed TH and a lipogenic diet (18). S14 involvement in lipogenesis is also indicated by abnormal fatty acid synthesis in a S14 knockout mouse model (see below).
S14 in humans
The human gene has been cloned and shares 78–81% homology with the rat gene (19, 20). In situ hybridization experiments demonstrated S14 expression in human liver (19). Regulation of human S14 gene expression by TH and carbohydrate is similar to that in rats (20). Comparison of S14 levels in obese and nonobese humans demonstrated abnormal regulation of adipose S14 in the obese subjects (21). After a 48-h fast, obese patients showed minimal down-regulation of S14 mRNA in adipose tissue obtained by biopsy, compared with nonobese patients. These data suggest a role for S14 in the development or maintenance of obesity in humans. Recent studies demonstrate that increased S14 levels in human breast cancer cells correlate with increased cell growth and predict patient survival (22, 23).
Oligonucleotide models
To better understand the role of S14 in lipogenesis, hepatocytes were transfected with an S14 antisense oligonucleotide (24). Western blotting confirmed that expression of the S14 protein was minimal in transfected cells. The transfected cells had lower levels of lipogenesis, compared with controls, and this was associated with a reduction in lipogenic enzymes. Specifically, there was diminished immunoreactivity of ATP-citrate lyase and fatty acid synthase and reduced induction of malic enzyme by T3 and carbohydrate. In addition, malic enzyme mRNA was decreased. A subsequent study supported this finding and was extended to other lipogenic enzymes including liver-type pyruvate kinase (25). Furthermore, an experiment using short inhibitory RNA to knock down S14 expression in breast cancer cells showed that S14 is required for lipogenesis (23). Although the oligonucleotide models are consistent with previous work showing immunohistochemical nuclear localization of S14 (26), it remains possible that S14 acts in the cytoplasm as well. The oligonucleotide experiments were done in cultured cells and not in an in vivo setting. Despite the use of multiple controls, antisense oligonucleotide and short inhibitory RNA experiments may also carry the potential weakness of nonspecificity.
Knockout models
Because of the limitations of the hepatocyte antisense model, investigators have created S14 knockout models to try to establish its role in vivo. Two separate models have demonstrated very disparate results.
The Dartmouth group deleted the proximal promoter and entire coding sequence of the gene (Kinlaw, W. B., personal communication). The homozygous mutation of this model was found to be early embryonic lethal.
Our group’s knockout construct deleted all but the first 21 amino acids of the S14 gene (3). In contrast to the Dartmouth model, the distribution of heterozygous and knockout offspring was normal. Northern analysis using a rat S14 probe showed a lack of both basal and induced S14 mRNA in the knockout liver. This was confirmed using a mouse cDNA probe. Western blotting with a monoclonal S14 antibody revealed no protein in the knockout mammary gland, compared with strong bands in the wild-type tissues (Fig. 1
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FIG. 1. S14 immunoblot. Liver and lactating mammary glands were homogenized in Tris-sodium dodecyl sulfate buffer, and 20 µg of proteins were separated by electrophoresis in a 15% polyacrylamide gel. After transfer to a polyvinyl difluoride membrane, they were visualized using an S14 monoclonal antibody (BD Biosciences, San Jose, CA). The location of the 18-kDa protein marker is indicated by the line. Tissues used were wild-type liver (lane A), wild-type mammary gland (lane B), and knockout mammary gland (lane C).
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The stark difference between the two knockout models is difficult to explain. One proposed explanation is that the 21 amino acids spared in the Minnesota model may code for a small peptide that supplies the same essential function as S14 in the embryo. It is also possible that the knockout of more than the entire coding sequence in the Dartmouth construct could have had adverse effects on other genes. Specifically, removal of the sequence containing the S14 promoter could have altered expression of other genes in this area of the chromosome essential to development that may not be expressed in the mature animal. Strain differences in mice may also account for the differences in phenotype if there were interaction between S14 and other strain-specific genes.
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Phenotype of the S14 Knockout Mouse
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Increased hepatic de novo lipogenesis in adult knockout mice
Given what was known about S14 at the time, including the temporal and spatial ability to act as a transcription factor as well as the results of antisense experiments, we expected to see reduced hepatic de novo lipogenesis in the knockout mouse. Instead, under TH and carbohydrate stimulation, the level of lipogenesis was increased in the knockout animal (3). Lipogenic enzymes were not reduced in the knockout animals, and in fact, when induced with T3 and a lipogenic diet, levels of several lipogenic enzymes such as fatty acid synthase (FAS) and glucose-6-phosphate dehydrogenase (G6PD) were higher in the knockout than the wild-type mouse. The surprisingly normal level of lipogenesis in the S14 null mouse suggested that another mRNA was present that could compensate for the lack of S14. To test this possibility, we examined several gene databases and found a paralog of S14. We named this paralog S14-R (2), but it is also referred to as MIG12 in a recent publication (27). Therefore, one possibility for the high level of hepatic lipogenesis in the knockout animal is the presence in the liver of S14-R, which may compensate for the lack of S14.
Spot 14 is required for de novo lipogenesis in the lactating mammary gland
Interestingly, although adult knockout mice had normal hepatic lipogenesis, knockout pups nursed by knockout dams gained significantly less weight than wild-type pups (2). The milk of knockout dams contained 33% less triglyceride than wild-type dam milk. Analysis of the fatty acid makeup of the triglycerides revealed that the reduction was in medium-chain fatty acids. Because the mammary gland uniquely produces these medium-chain fatty acids, these data suggested that the reduction in milk triglyceride was due to a decrease in mammary gland lipogenesis. We verified this hypothesis by showing that lipogenesis was decreased (by 62%) in the S14 knockout mammary gland. In contrast to the liver, levels of S14-R are low in the mammary gland, and hence the absence of S14 was not compensated for by S14-R.
Despite the lower content of fatty acids and the decreased rate of lipogenesis, the activity and mRNA content of the rate limiting enzymes involved in lipogenesis (FAS, acetyl-coenzyme A-carboxylase) were not reduced. These data indicate that, whereas S14 does regulate lipogenesis, this regulation does not occur by altering the transcription of the rate-limiting lipogenic enzymes. We have recently shown that malonyl-CoA, the substrate of the FAS reaction, is actually increased in the mammary gland of the S14 knockout animal (29). The elevated level of malonyl-CoA suggests that the defect in lipogenesis occurs because of an in vivo decrease in FAS activity and is not due to limitation of substrate. Thus, in our S14 null knockout model, the S14 protein likely acts as an allosteric regulator of the in vivo activity of FAS.
The Spot 14 mouse is resistant to diet-induced obesity
In addition to the mammary gland observations, the S14 null mouse also shows a metabolic phenotype in the adult animal. On a mixed genetic background, we found the S14 null adult mouse gained significantly less weight than wild-type mice. The weight difference was significant by 4 months and continued through 7 months of age. The difference in weight could be accounted for completely by a difference in fat accumulation. When backcrossed to create a congenic strain on a C57BL/6J background, the differences in weight gain were abolished. However, the knockout mice continued to show marked increased insulin sensitivity. Whether these observations are related to the decreased growth rate of nursing pups or due to a direct effect of S14 deficiency in the adult will require further studies.
It is interesting to compare the difference in cellular response to the lack of S14 in breast cancer cells (30) and the response in normal mammary tissue. With depletion of S14 in cancer cells, there is an associated loss of lipogenic enzymes with subsequent cellular apoptosis. In contrast, the intact mammary gland does not seem to alter the level of the lipogenic enzymes with a loss of S14. Furthermore, we have not been able to document a significant difference in mammary gland development by whole mounts or histology in the S14 knockout mammary gland (Mucha, G., G. W. Anderson, and C. N. Mariash, unpublished results). These discrepant observations were recently supported by the finding that inhibition of lipid synthesis in neu-N transgenic mice decreased the growth of mammary cancers but had no effect on the morphology and development of normal mammary tissue in these mice (28).
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Speculation on S14 Function
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Our S14 null animals demonstrate that this protein is needed for normal in vivo lipogenesis in the mammary gland but is not required in the liver. Furthermore, we found that the liver, but not the mammary gland, contains a paralog of the S14 protein (S14-R). Interestingly, mRNA coding S14-R is readily detectable in breast cancer cells (23). It was recently shown that this paralog, also known as MIG12, associated with a protein (MID1) anchored to microtubules (27) and is involved in the stabilization of microtubules. Is it possible that S14 plays a related role in the mammary gland? Can it bind to the cytoskeleton and help to remove newly synthesized fatty acids from FAS to relieve end-product inhibition (Fig. 2)? We now have preliminary data to show that when S14 oligomerizes, it creates a very strong hydrophobic pocket that may be involved in the movement of fatty acids or function as a trafficking protein, physically linking different steps of lipogenesis.
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FIG. 2. Model of S14 and S14R involvement in lipogenesis. S14 and S14R oligomers associate with newly synthesized fatty acid (FA) and anchor the homodimeric FAS to the microtubule through interaction with MID1. They are also predicted to participate in substrate channeling of nascent fatty acids to unsaturases and other enzymes involved in the construction and trafficking of lipids.
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Acknowledgments
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We greatly appreciate the helpful discussions, support, and contributions of Grant W. Anderson to the work described in this review.
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Footnotes
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This work was supported by the Minnesota Obesity Center, National Institutes of Health Grant P30 DK-50456 and National Institutes of Health Training Grant T32-DK07203.
Disclosure summary: all authors have nothing to declare.
First Published Online June 29, 2006
Abbreviations: FAS, Fatty acid synthase; PUFA, polyunsaturated fatty acid; S14, Spot 14; TH, thyroid hormone.
Received April 13, 2006.
Accepted for publication June 2, 2006.
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References
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Abstract
Introduction
