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Endocrinology Vol. 142, No. 10 4363-4370
Copyright © 2001 by The Endocrine Society

ARTICLES

Spot 14 Gene Deletion Increases Hepatic de Novo Lipogenesis

Qihong Zhu, Ami Mariash, Mark R. Margosian, Sunil Gopinath, Mohammod T. Fareed, Grant W. Anderson 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 S.E., University of Minnesota, Minneapolis, Minnesota 55455. E-mail: cary{at}lenti.med.umn.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previous studies have investigated the relationship between the Spot 14 gene and hepatic lipogenesis. Those studies found that the Spot 14 protein was induced when lipogenesis was induced and suggested that induction of the Spot 14 protein was required for induction of hepatic lipogenesis by thyroid hormone and dietary carbohydrate. Analysis of those findings led us to hypothesize that the Spot 14 gene is required for induced hepatic de novo lipogenesis in vivo. To test this hypothesis, we created an in vivo deletion of the Spot 14 gene in mice using gene-targeting technology. Southern blot analysis showed that the Spot 14 gene was disrupted. Northern blot analysis showed that this disruption ablated expression of intact hepatic Spot 14 mRNA. In contrast to our hypothesis, acute thyroid hormone administration led to comparable induction of hepatic lipogenic enzyme mRNAs between the wild-type and knockout mice. Furthermore, long-term treatment with both thyroid hormone and a diet promoting lipogenesis led to enhanced lipogenic enzyme activity and a greater rate of hepatic de novo lipogenesis in the knockout, compared with the wild-type, mice. Although these data indicate that the Spot 14 protein is not required for induced hepatic de novo lipogenesis, they also suggest that Spot 14 plays some role in this process. It is possible that alternative pathways that complement the loss of the Spot 14 protein are present, and in the absence of Spot 14, these alternative pathways overcompensate to produce an enhanced rate of induced lipogenesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DE NOVO LIPOGENESIS is a process in which the cell makes fatty acids from nonfat materials (1). Because adipose fat is the only substantial storage form for surplus energy in mammals, it is inferred that the body must be able to transform surplus nonfat energy to fat. Although the contribution of de novo lipogenesis in humans remains controversial, it is generally agreed that in mammals it is the final common fate for surplus nonfat energy important in macronutrient energy economy (2). In most mammals, liver is the most important organ for de novo lipogenesis. However, de novo lipogenesis is also high in the mammary gland during lactation and in the adipose tissues under certain conditions (3).

The biochemical pathways of de novo lipogenesis were elucidated in the 1960s (4), but the regulation of these pathways has yet to be clarified. Among nutritional factors, fasting inhibits hepatic de novo lipogenesis. Although a diet rich in carbohydrates stimulates de novo lipogenesis, a diet rich in fat inhibits this process. The lipogenic pathways are also regulated by hormonal changes. Glucagon and catecholamines inhibit de novo lipogenesis, but insulin stimulates it (5, 6). Thyroid hormone stimulates de novo lipogenesis by primarily increasing the mRNA levels of lipogenic genes (7).

The Spot 14 protein was first discovered during the course of studying thyroid hormone action in the liver (8, 9, 10). The Spot 14 gene responds rapidly to T3 with an increase in nuclear precursor of Spot 14 mRNA 10 min after T3 injection (11, 12). Because of the rapid response indicating the S14 gene is a primary target gene for thyroid hormone, this gene has been widely used to study the mechanism of T3 action on gene expression.

Subsequent studies (13, 14, 15) suggested that the Spot 14 protein is involved in de novo lipogenesis. The evidence includes, firstly, Spot 14 is expressed only in lipogenic tissues such as liver, fat, and lactating mammary gland (13); secondly, the regulation of Spot 14 mimics the regulation of lipogenesis. For instance, the mRNA levels of Spot 14 are increased by thyroid hormone, carbohydrate feeding, and decreased by glucagon and catecholamine treatment (16). Similarly, the rate of de novo lipogenesis is increased by thyroid hormone, carbohydrate feeding, and decreased by glucagon and catecholamine treatment.

Additional studies suggested that the Spot 14 protein regulates de novo lipogenesis by regulating the transcription of other lipogenic genes. Three lines of evidence support this hypothesis. First, immunohistochemistry showed that the Spot 14 protein is localized in the nucleus of liver cells, making it spatially possible for Spot 14 to act as a transcription factor (17). Second, induction kinetics after T3 injection indicated that the expression of Spot 14 protein largely preceded the expression of other lipogenic genes such as malic enzyme, making it temporally possible for Spot 14 to act as a transcription factor (18). Lastly, studies in primary hepatocytes transfected with a Spot 14 antisense oligonucleotide showed that the response of lipogenic enzyme activities and lipogenesis to glucose and T3 were abolished (19). These data led us to hypothesize that the Spot 14 protein is required for the induction of hepatic lipogenic genes upon stimulation with thyroid hormone and carbohydrates. To test this hypothesis in vivo, we deleted most of the Spot 14 gene using gene targeting technology and created a Spot 14 knockout mouse model. In this paper we report the hepatic phenotype of the Spot 14 knockout mouse and show that, contrary to our hypothesis, the Spot 14 gene is not required for induced de novo lipogenesis. In contrast, we find that the Spot 14 knockout mouse exhibits increased hepatic de novo lipogenesis when induced with thyroid hormone and carbohydrate feeding.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
The targeting construct and screening of embryonic stem (ES) cells
The murine Spot 14 gene and its flanking sequence were cloned by screening a 129/sv genomic library. The targeting vector, pPNT, was obtained from Dr. Beverly Kohler at the University of North Carolina, Chapel Hill, NC (20). This targeting vector uses the neomycin (neo) resistance gene as a positive selector and the thymidine kinase (TK) gene as a negative selector. A 6-kb XbaI-SstII insert spanning the promoter of the gene to the first 21 amino acids of the first exon was ligated to the NotI/XhoI site upstream of neomycin resistance cassette of the pPNT vector (Fig. 1Go). A 2.5-kb XbaI-Acc65 I fragment extending from part of the only intron and spanning all of the noncoding second exon was ligated to the XbaI/KpnI sites downstream of the neo gene but upstream of the TK gene of the pPNT vector. The vector was transfected into 129/sv ES cells by electroporation. A total of 116 ES clones were obtained from two electroporations. The ES clones were screened by Southern blot for appropriate homologous recombination. Eight correctly targeted ES clones were obtained and returned to Dr. Kohler’s laboratory for embryo injection and chimeric mice production. A total of 11 highly chimeric mice (9 males and 2 females) were obtained requiring two rounds of embryo injections. After breeding the chimeric males to B6D2/F1J females, germline transmission as assayed by Southern blot of the offspring was achieved in three of the males. The Spot 14 heterozygote founders were then backcrossed to C57B6/J mice to establish a line.



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  		Figure 1. The murine Spot 14 gene, the targeting construct and the mutant. The upper arm contains the promoter region and the first 21 amino acids of the coding region up to the SstII restriction enzyme site. The target region contains the rest of the coding region (amino acid 22 to amino acid 150) and most of the intron up to the XbaI restriction enzyme site. The entire coding region of this gene is located in the first exon. The lower arm contains the remainder of the intron and the second exon up to the Acc65 I restriction enzyme site. In the construct, the target region was replaced with a phospho-glycero-kinase (pgk)-neo cassette. Additionally, a pgk-TK cassette was inserted downstream of the lower arm as a negative selector. If successfully targeted, the mutant should have an intact upper and lower arm but its target region replaced with the neo cassette. Drawn to scale.

 
Mouse breeding and care
Mice were housed in a specific pathogen-free facility with a 14-h light and 10-h dark cycle (light turned on at 0600 h) and free access to food and water. Pups were weaned at postnatal day 21. The mice were fed with Teklad 7001 (4% fat by weight, 12% fat calories) diet (Harlan, Indianapolis, IN). Pregnant mothers were fed Teklad 7004 (Breeder chow, 11% fat by weight, 28% fat calories) starting 1 wk before birth to ensure good pup health. The mothers and pups were switched back to Teklad 7001 after the pups were weaned. More recently, mice were fed Teklad 8640 (5% fat by weight, 14% fat calories) owing to the discontinuation of Teklad 7001 by the manufacturer. Because lipogenic activity undergoes diurnal changes, mice were killed before noon. Mice treated with T3 were injected ip with 10 mg/ml T3 either one time 24 h before tissues were harvested (200 µg per 100 g of body weight) or daily starting 4 d before tissues were harvested (20 µg per 100 g of body weight). This amount of hormone has been previously shown to provide maximal stimulation of lipogenesis in rats (21). Some mice were fed a high-carbohydrate diet (ICN catalog no. 901683, 60% sucrose by weight) for either 24 h or 4 d before tissues were harvested.

All studies in this manuscript were conducted under a protocol approved by the University of Minnesota Committee on Animal Care and Use and in accordance with the NIH Guide for the Care and Use of Laboratory Animals.

Southern blot and PCR
Both the ES cells and the founder mice were screened by Southern blot. Genomic DNA was prepared from ES cells or a 1-cm piece of mouse tail using a high-salt protocol (22). DNA was digested with either EcoRI or BspHI. The probe was a 1.8-kb fragment of genomic DNA that was directly downstream of the "lower arm" and outside any region in the targeting vector. The transfer membrane was a Zeta-probe nylon filter (Bio-Rad Laboratories, Inc., Hercules, CA) and the manufacturer’s recommended protocol was followed. The hybridization solution contained 7% SDS, 10 mM sodium phosphate, and 1 mM EDTA. Hybridization was performed at 65 C.

Mouse genotyping was performed by multiplexed PCR. Templates were prepared as described for the Southern blot. Three primers, PUP, PDN, and PNEO, were used in each reaction. PUP, with a sequence of CAG TCT TCT GCA CCA AGT AC, binds to the 3'-end of the target region. PDN, with a sequence of AGC AGC AGA GCT AAG AGA AG, binds to the 5'-end of the lower arm. PNEO, with a sequence of CTG GGA TTC ATC GAC TGT GG, binds to the 3'-end of the neocassette. The PDN primer is used by both genotypes, the PUP primer binds only to the wild-type gene, and the PNEO primer binds only to the mutant gene (Fig. 2bGo). A GeneAmp 2400 (Perkin-Elmer Corp., Foster City, CA) was used with the following cycling parameters: 95 C for 15 sec, 55 C for 30 sec, and 72 C for 2.5 min. Thirty-five cycles were used followed by a 7-min extension at 72 C. Products were separated on a 1% agarose gel and visualized by ethidium bromide staining.



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  		Figure 2. Screening strategies. A, Screening with Southern blot. Genomic DNA from wild-type and mutant founder mice were digested with EcoRI and probed with a fragment that is outside the construct and downstream of the lower arm. The wild-type DNA produces a band of 4.3 kb, and the mutant DNA produces a band of about 12 kb owing to the deletion of EcoRI site in the target region. Depiction of the two genes is as described in Fig. 1Go. B, Screening with PCR. Three primers, PUP, PDN, and PNEO, are used in each reaction. The wild-type allele produces a band of 600 bp with primers PUP and PDN. The mutant allele produces a band of 1100 bp with primers PNEO and PDN. Depiction of the two genes is as described in Fig. 1Go.

 
Northern blot RNA quantification and real-time RT-PCR
TRIzol reagent from Life Technologies, Inc. (Grand Island, NY) was used to isolate RNA. Twenty µg of total RNA from each sample was denatured and separated by formaldehyde-agarose gel electrophoresis. RNA was electrotransferred to a Zeta-probe nylon filter (Bio-Rad Laboratories, Inc.) using the manufacturer’s recommended protocol. The hybridization protocol was adapted from the protocol by Brown and Macky. The fatty acid synthase (FAS) probe was obtained from Dr. William Kinlaw (23). The glucose-6-phosphate dehydrogenase (G6PD) probe was a gift from Dr. Lisa Salati (24). The malic enzyme (ME) probe was originally from Dr. C. S. Rubin (25). Blots were washed and subsequently exposed to phosphor screens (Molecular Dynamics, Inc., Sunnyvale, CA) and scanned (PhosphorImager 445 SI, Molecular Dynamics, Inc.). The images were corrected for variations in loading by calculating the ethidium bromide staining for the 18S ribosomal RNA as described by Correa-Rotter et al. (26).

To assess the relative contribution of wild-type and disrupted S14 mRNA to hepatic mRNA, Northern blots were prepared as above. However, the labeled probes were prepared from PCR-generated products. We prepared two separate probes from genomic DNA using primers as described above for genotyping mouse DNA. However, the common 5'-primer began just downstream from the start site of S14 translation (5'-TGC TAA CGA AAC GCT ATC CC-3'). The wild-type primer was derived from part of the first exon deleted in the knockout mouse (5'-TTC TCA GCC TCG CTG GTT TC-3') to yield a product of 273 bases. The knockout primer was derived from the neo gene (5'-GCG TGC AAT CCA TCT TGT TC-3') to yield a product of 604 bases. These primers were also used in an RT-PCR assay as described by the manufacturer for quantitative real-time RT-PCR (Roche Diagnostics Corp., Indianapolis, IN).

Enzymatic activity assays
Hepatic samples were homogenized in 0.25 M sucrose at a 1:10 dilution and centrifuged at 100,000 x g for 45 min. The clear cytoplasmic fraction was used for all enzyme assays. The assays were performed at 37 C. The protocol for FAS assay was adapted from Kim et al. (27). For the FAS assay, the cellular extract was diluted with an equal volume of 1 M potassium phosphate, pH 7.0, 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 [NADP(H)], and 1 mg/ml BSA in a volume of 0.9 ml. Fifty microliters diluted cellular extract was added and mixed. The rate of NADP(H) oxidation was measured after 2 min at 340 nm and used as a blank. Following addition of 50 µl of the substrate malonyl-CoA, the rate of NADP(H) oxidation was calculated after another 2 min. The concentration of enzyme was adjusted to assure a linear reaction rate. The G6PD and 6-phosphogluconate dehydrogenase (6PGD) assay used the method of McKerns (28). The combined activity of G6PD and 6PGD was measured in a 1-ml reaction mixture containing 0.1 M Tris-HCl, 1 mM NADP, 10 mM MgCl2, 2 mM of glucose-6-phosphate, and 0.5 mM of 6-phosphogluconate. The 6PGD was measured using the reaction mixture above except without glucose-6-phosphate. The activity of G6PD was obtained by subtracting the activity of 6PGD from the combined activity. The protocol for the ME activity assay was adapted from the method of Hsu and Lardy (29). Each 1-ml reaction mixture contained 0.1 M Tris-HCl, 4 mM MnCl2, 0.2 mM NAPD, and 2.5 mM malic acid. All enzyme activities are expressed as Units/milligram protein where 1 Unit is the amount of enzyme required to reduce (or oxidize) 1 nmol of NADP(H) per min.

In vivo lipogenesis
Mice were injected ip with 2 mCi of tritiated water per 100 g body weight and were killed either by cervical dislocation (adults) or decapitation (pups) after 30–45 min. Blood was collected in a heparinized syringe either from cardiac puncture or the hepatic vein, and plasma was obtained by centrifugation. The plasma was diluted 1:1 with 20% trichloracetic acid. After centrifugation, 20 µl of the supernatant was counted by liquid scintillation to obtain the specific activity of body water.

Tissues were frozen in liquid nitrogen after dissection. The protocol for fatty acid extraction was adapted from Stansbie et al. (30). In brief, a 0.5-g portion of liver was solubilized in 3 ml of 30% KOH in a 50-ml polypropylene tube. The lipids were saponified at 70 C for 15 min, followed by the addition of 3 ml of 95% ethanol and an additional 2 h of saponification at 70 C. Lipids were extracted with 10 ml petroleum ether three times, and the pooled petroleum ether was backextracted with 10 ml acidified water three times to remove any contaminating water. The petroleum ether extract was concentrated to 2 ml and then transferred to a liquid scintillation vial. Following evaporation of the petroleum ether, the lipids were dissolved in 5 ml scintillation cocktail and subjected to liquid scintillation counting. The rate of lipogenesis was calculated as micromoles 3H incorporated into lipids/gram tissue per hour using a molecular weight of 3H2O of 22.

Statistics
All data were analyzed by ANOVA. Comparison among groups was made by posthoc testing with the Bonferroni procedure. Groups were considered significantly different from each other if the probability reached <0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of the knockout mouse
To determine whether the Spot 14 protein was essential for hepatic lipid synthesis, we elected to delete this gene in vivo using gene targeting technology. The murine Spot 14 gene has only two exons with the entire coding region contained in the first exon (31). In making the construct, a 6-kb region upstream of exon 1 (upper arm) was ligated to the 5'-end of the pgk-neomycin resistance gene, and a 2.5-kb region downstream of the coding region (lower arm) was ligated to the 3'-end of the neo gene (Fig. 1Go). In addition, the construct contains a TK gene as a negative selector (driven by the pgk promoter). If targeted correctly, the construct should replace all but the first 21 amino acids of the Spot 14 coding sequence.

Two strategies were devised to identify positive embryonic stem cell clones and genotype mice. In the Southern blot method (Fig. 2AGo), a 1.8-kb fragment downstream of the lower arm was used as a probe. If genomic DNA is digested with EcoRI and hybridized with this probe, the wild-type allele will generate a 4-kb band. The mutant allele generates a 12-kb band owing to the elimination of the proximal EcoRI site when the target region is replaced with the neo gene. Some embryonic stem cells were also screened with BspHI (data not shown) to confirm that they were correctly targeted. There is a BspHI site inside the neomycin resistance gene but not in the target region. When hybridized with same 1.8-kb probe as mentioned above, the wild-type allele produces a band of more than 20 kb, but the mutant allele produces a band of about 8 kb.

A PCR method was also designed to genotype large numbers of mice (Fig. 2BGo). Three primers were used in each reaction, with the PUP primer complementary to the first exon, the PDN primer complementary to the lower arm, and the PNEO primer complementary to the neo gene. The wild-type allele produces a band of 600 bp, and the mutant allele produces a band of 1100 bp.

Breeding of heterozygous mice yielded a normal distribution of heterozygous and knockout progeny (Table 1Go). For example, 17 heterozygous matings yielded 37 knockout, 40 wild-type, and 70 heterozygous progeny. This finding suggests that the S14 gene deletion we created does not result in embryonic lethality. The Spot 14 knockout neonates were viable and have not yet exhibited any decrement in life span. Homozygous S14 knockout animals have been kept as long as 10 months after birth. Finally, both male and female Spot 14 knockout mice are fertile.


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  		Table 1. Genotype and sex ratios of pups from 17 heterozygous breeding pairs

 
To verify deletion of the Spot 14 gene, euthyroid knockout and wild-type mice were fed either normal mouse chow (uninduced) or a high-carbohydrate, low-fat diet and injected daily with thyroid hormone (20 µg of T3 per 100 g body weight) for 4 d (induced). Hepatic total RNA was prepared and subjected to Northern analysis. The filter was hybridized with a cDNA probe that contains the coding sequence of rat Spot 14 gene. As expected, euthyroid wild-type animals fed normal chow expressed Spot 14 mRNA at detectable but low levels (Fig. 3AGo). Treatment of wild-type animals with T3 and carbohydrate dramatically upregulated Spot 14 mRNA levels. In contrast, neither uninduced nor induced Spot 14 knockout animals expressed any Spot 14 mRNA. These data support the gene-targeting strategy because there is loss of both basal and induced Spot 14 mRNA expression in the knockout mouse.



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  		Figure 3. The Spot 14 mRNA is abolished in null mutant mice. A, Northern blot analysis using a rat S14 probe. Euthyroid male wild-type and mutant mice were maintained on either a normal diet for 4 d (-) or fed a high-carbohydrate diet and injected with 20 µg T3 per 100 g of body weight for 4 d (+). The Spot 14 message is present in wild-type mice maintained on a normal diet and is upregulated in the induced wild-type mice. However, neither treatment group has any Spot 14 expression in null mutant mice. B, Northern blot analysis using a 273-bp mouse S14 probe. Mice were treated as described in Fig. 3AGo. Only the wild-type mice show an inducible band. The other bands present on this blot represent nonspecific signals.

 
To test the possibility that the induced knockout mice produced significant quantities of mRNA coding for the first 21 amino acids of the S14 protein, we also measured the content of basal and induced Spot 14 mRNA using alternative methods. We used real-time quantitative RT-PCR to measure the amount of this potential product in the induced knockout mice. However, we found no such mRNA present because we could not produce an appropriately sized product with these primers. Therefore, we generated by PCR a mouse-specific cDNA probe using the common S14 5'-primer (used in the quantitative RT-PCR reaction) and a primer within the deleted S14 exon to generate a 273-bp probe. Approximately one-fourth of this cDNA contained the nondisrupted 5'-end of the Spot 14 gene and would be expected to give an easily measurable signal by Northern analysis if the knockout mouse produced similar quantity of mRNA from the S14 promoter as the wild-type animal. However, as can be seen in Fig. 3BGo, only the wild-type mouse produces an inducible mRNA upon stimulation with thyroid hormone and carbohydrate feeding. Therefore, it is likely that the knockout animal is not able to synthesize even a truncated Spot 14 protein.

Hepatic expression of lipogenic enzyme genes after acute stimulation with T3
We next wished to test the hypothesis that the S14 protein is required for acute induction of hepatic lipogenic gene transcription. To induce hepatic lipogenic gene transcription, we treated adult knockout and wild-type mice with thyroid hormone (200 µg per 100 g of body weight). After 24 h, the mice were killed and hepatic mRNA levels of four lipogenic genes were measured by Northern gel analysis. FAS is a key enzyme in the de novo lipogenesis pathway from acetyl-CoA to fatty acyl-CoA. Reducing equivalents are supplied by G6PD, which catalyzes the rate-limiting step in the hexose monophosphate pathway. ME is a key enzyme in the citrate shuttle that transports acetyl-CoA from mitochondria to the cytoplasm where de novo lipogenesis takes place (4) as well as providing additional reducing equivalents. Pyruvate kinase (PK), although not a lipogenic gene, was also measured because it responds to hormone and dietary stimuli in a manner similar to that of the lipogenic genes. We found no significant reduction in T3-induced FAS, ME, G6PD, or PK mRNA levels in Spot 14 knockout mice, compared with wild-type mice (Fig. 4Go). Thus, the S14 gene is not required for the acute up-regulation of these lipogenic mRNAs by thyroid hormone in the liver.



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  		Figure 4. Hepatic mRNA levels of lipogenic genes are unchanged in the Spot 14 knockout mouse when induced. Male wild-type and mutant mice were injected with 200 µg/100 g body weight of T3. After 24 h, the mice were killed and total liver RNA was prepared. The mRNA levels of FAS, G6PD, ME, and PK were measured. The levels in the mutant were normalized to those in the wild-type (mean ± SD, n = 3–4).

 
Hepatic ME mRNA levels and enzyme activity induction after long-term stimulation with T3 and carbohydrate feeding
Because we did not find a significant difference in the lipogenic gene mRNA levels after acute injection of thyroid hormone, we next tested the possibility that long-term lipogenic enzyme stimulation would be diminished in the Spot 14 knockout mice. We focused our attention on the response of ME because previous studies suggested that stimulation of ME mRNA production and enzyme activity required prior induction of Spot 14 (18). Knockout and wild-type mice were either fasted overnight or fed a high-carbohydrate diet and injected with thyroid hormone (20 µg T3 per 100 g of body weight) for four consecutive days. The mRNA levels as well as enzymatic activities of ME were measured after the treatments. As shown in Fig. 5Go, A and B, we saw no significant genotypic differences in either ME mRNA levels or enzymatic activities. These data indicate that Spot 14 is not required for long-term induction of ME mRNA expression and enzyme activity.



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  		Figure 5. Hepatic ME mRNA and activity show normal response to stimulation in the Spot 14 knockout mouse. Male wild-type and mutant mice were either fasted overnight (fasting) or fed with a lipogenic diet and injected with 20 µg/100 g body weight (T3/CHO) of T3 for 4 d. A, mRNA levels expressed in arbitrary units. B, Enzymatic activity (U/mg protein) (mean ± SD, n = 4).

 
Hepatic lipogenic enzyme activities after long-term stimulation with T3 and carbohydrate feeding
Although the previous data indicated that Spot 14 is not required for long-term induction of ME activity, it still remained possible that Spot 14 is required for long-term induction of other lipogenic enzymes. To test this possibility, we measured the activities of four lipogenic enzymes: ME, FAS, G6PD, and 6PGD. The S14 null and wild-type mice were fed a high-carbohydrate diet and injected with 20 µg T3 per 100 g of body weight for 4 d. As shown in Fig. 6Go, the activities of the lipogenic enzymes were not lower in the knockout mouse. In contrast, two of the enzymes (FAS and G6PD) were significantly higher in the Spot 14 knockout mice. These data suggest that Spot 14 is not required for the induction of lipogenic enzyme activity in the liver and also that the absence of Spot 14 leads to an increase in specific lipogenic enzyme activities.



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  		Figure 6. Activities of hepatic lipogenic enzymes are higher in the Spot 14 knockout mutant when induced. Male wild-type and mutant mice were fed with a lipogenic diet and injected with 20 µg/100 g body weight of T3 for 4 d. The activities of FAS, 6PGD, G6PD, and ME were measured. The levels in the mutant were normalized to those in the wild-type. The values for FAS and G6PD are significantly higher in mutant mice as denoted by asterisks(mean ± SD, n = 4, ANOVA with posthoc testing using the Bonferroni method, for FAS, P = 0.02; for G6PD, P = 0.04).

 
Hepatic-induced lipogenic rates
Although we did not observe a decrease in the mRNA level or enzyme activity of lipogenic genes, it still remained possible that the in vivo lipogenic rate decreased in the Spot 14 knockout mice. This would be especially true if the Spot 14 protein were involved in aspects of de novo lipogenesis not related to transcriptional or enzyme activity regulation. To test this possibility, wild-type and knockout mice were injected with T3 alone, fed with a lipogenic diet alone, or subjected to the combination of these two treatments for 24 h. The mice were then injected with 2 mCi of tritiated water per 100 g of body weight and assayed for in vivo lipogenic rate as described. Figure 7Go shows that there is no significant difference between genotypes when the mice were acutely induced with T3, carbohydrate, or T3 plus carbohydrate for 24 h. However, if the animals were maintained on thyroid hormone and high-carbohydrate feeding for 4 d, the treatment led to a significantly increased lipogenic response in the knockout, compared with wild-type, control mice (Fig. 7Go). These data are in agreement with our observation of increased lipogenic enzyme activity in the knockout mice treated for 4 d with T3 and carbohydrate (Fig. 6Go). Moreover, in contrast to our original hypothesis, these data emphasize that Spot 14 knockout mouse liver exhibits enhanced rather than reduced lipogenic rates when induced.



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  		Figure 7. In vivo hepatic lipogenic rates are higher in the Spot 14 knockout mouse when induced by T3 and carbohydrate for 4 d. Wild-type and mutant mice were injected with 200 µg/100 g body of T3 for 24 h (T3 alone, mean ± SD, n = 4); starved for 24 h and refed with a lipogenic diet for 24 h (CHO alone, mean ± SD, n = 6); or treated with the combination of the aforesaid two treatments for 24 h (CHO/T3 24 h, mean ± SD, n = 6), or for 4 d (CHO/T3 4 d, mean ± SE, n = 12, three repeat experiments of four mice each). The levels in the Spot 14 knockout were normalized to those in the wild-type. Asterisks indicate significantly different values (ANOVA, P < 0.05).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Twenty years after the discovery of the Spot 14 gene, the precise function of this protein remains elusive. Several factors contributed to the slow advancement in this area of study. First, the primary structure of the Spot 14 polypeptide shares little if any homology with other proteins of known function. A recent BLAST search shows homology to only two other proteins of unknown function. One is a gastrulation-specific protein from the zebrafish (32); the other is a protein of unknown function present in both the human and mouse genomes (STRAIT 11499 and RIKEN cDNA 3110038L01, respectively). An alignment analysis shows that the STRAIT and RIKEN proteins are highly homologous to the zebrafish gastrulation protein. The homology to the Spot 14 protein is present in the amino terminus. However, the greatest degree of homology to the Spot 14 protein exists in the region of the protein deleted from the knockout mouse. Although the STRAIT, RIKEN, and the zebrafish gastrulation protein are more similar to one another than to the S14 protein, it is interesting to speculate that RIKEN protein may be expressed in the liver and contribute to the lack of expected hepatic phenotype.

Second, other functional assays of Spot 14 have failed to provide substantial information in regard to Spot 14 cellular function. For instance, an attempt to clone proteins interacting with S14 by yeast two hybrid system revealed that Spot 14 interacts with itself as a homodimer and possibly one other protein of 36 kDa (33). Thus, only antisense strategies employed in cultured hepatocytes provided any direct clue to the function of the Spot 14 protein (19). Because of the paucity of information regarding the molecular function of the Spot 14 protein we created the Spot 14 knockout mouse.

Our observed phenotype, enhanced lipogenesis in the knockout animal, was surprising in the face of our original hypothesis. However, this phenotype was not entirely unexpected. It is well known that in complex organisms such as mammals there can be multiple pathways for the same cellular function (34, 35). In the case of the Spot 14 knockout mouse, as in many other knockout mouse models, the most probable cause of this surprising phenotype is the existence of complementary pathways that compensate for the loss of the Spot 14 gene. Because the function of the Spot 14 protein remains unknown, it is difficult to speculate as to which molecules or pathways substitute for the lack of the Spot 14 protein. In the stimulated Spot 14 knockout mice, we found that some lipogenic enzyme activities increased, compared with wild-type (Fig. 6Go). This phenomenon is possibly caused by overcompensation of compensatory pathways.

To assay compensation, several strategies can be employed. Because the mutation is present at the time of conception, the compensation may be at the organism level (e.g., the thyroid gland in the knockout mouse may secrete more thyroid hormone to compensate the loss of Spot 14). If this were true, we should be able to abolish the enhanced complementation by studying the effects of thyroid hormone and carbohydrates in primary hepatocyte cultures derived from wild-type and knockout mice. Experiments analogous to the ones reported here should not show enhanced complementation in hepatocyte cultures. Similarly, the mutant mouse may adapt to the deletion of Spot 14 gene during development. Thus, targeted deletion of hepatic S14 by cre-lox technology may show a very different hepatic phenotype. If we could delete this gene in only the adult mouse, we may observe a different phenotype.

Another possibility for the unexpected phenotype is that, in making the targeting construct, we left the N-terminal 21 amino acids of the Spot 14 gene in the genome. It is possible that these 21 amino acids are translated into a small peptide and this small peptide has the entire or partial function of the intact Spot 14 protein. Because part of the intron and the noncoding exon 2 are still intact, it is also possible that alternative promoters in the undeleted part of spot 14 can produce functional transcripts. Examples like this have been reported in the TR knockout mice (36, 37, 38). In our case this possibility is remote for several reasons. First, we have tried to identify an inducible transcript containing the 63 bases of the S14 protein in the knockout mouse. We have been unable to identify such a transcript (Fig. 3BGo). Second, we have observations of significant phenotypes in other physiological aspects of the Spot 14 knockout mouse such as marked reduction of lipogenesis in lactating mammary glands (data not shown). Additionally, in preliminary studies the knockout mice show diminished weight gain throughout life on a 11% fat diet (data not shown), suggesting an enhanced metabolic rate. Another possibility for the lack of expected hepatic phenotype is that S14 does not have any role in lipogenesis. But in light of the increased enzyme activities and lipogenic rates in mice that have been induced for 4 d, this possibility is also remote. Indeed, the increase in enzyme activity and lipogenesis in the knockout animals strongly suggests a role for Spot 14 in lipogenesis.

In conclusion, we believe that the Spot 14 gene is not absolutely required for the induction of lipogenesis in the liver. There are other pathways inside the cell that can also confer lipogenic signals to the enzyme machinery, although it is possible that the pathway represented by the Spot 14 gene is the main pathway under normal conditions. The higher activities of lipogenic enzymes and slightly higher lipogenic rates in the stimulated knockout mouse may be owing to overcompensation of the complementary pathways.


   Acknowledgments
 
We would like to thank Dawn Jolson for excellent technical support and Jack H. Oppenheimer for helpful discussions. We would also like to thank Dr. Beverly Koller and Anne Latour for their work on the generation of the knockout animal.


   Footnotes
 
This work was supported by NIH R-01-DK-32885, the Minnesota Medical Foundation, and the Minnesota Obesity Center NIH P30 DK-50456.

Abbreviations: ES, Embryonic stem; FAS, fatty acid synthase; G6PD, glucose-6-phosphate dehydrogenase; KO, knockout; ME, malic enzyme; NADP, nicotinamide-adenine dinucleotide phosphate; NAPD(H), reduced NAPD; neo, neomycin; 6PGD, 6-phosphogluconate dehydrogenase; PK, pyruvate kinase; TK, thymidine kinase; WT, wild-type.

Received February 16, 2001.

Accepted for publication June 18, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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