©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Direct Evidence for a Role of the Spot 14 Protein in the Regulation of Lipid Synthesis (*)

William B. Kinlaw (1)(§), Jori L. Church (1), Jamie Harmon (2), Cary N. Mariash (2)

From the (1)Department of Medicine, Division of Endocrinology and Metabolism, Dartmouth Medical School, Lebanon, New Hampshire 03750, and the Department of Medicine, Division of Endocrinology and Metabolism and Department of Cell Biology and Neuroanatomy, (2)University of Minnesota Medical School, Minneapolis, Minnesota 55455

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

``Spot 14'' is a nuclear protein that is rapidly induced by thyroid hormone (T) and dietary carbohydrate in liver. We used an antisense oligonucleotide to inhibit induction of spot 14 protein by T and glucose in primary cultures of rat hepatocytes to test the hypothesis that the protein could function in the regulation of lipid synthesis. Spot 14 protein was undetectable in hepatocytes maintained in 5.5 mM glucose without T, and was induced within 4 h after addition of 27.5 mM glucose and 50 nM T to the culture medium, reaching a maximal level within 24 h. Accumulation of spot 14 protein was markedly inhibited in hepatocytes transfected with a spot 14 antisense oligonucleotide, but not in those treated with a control oligonucleotide. Transfection of the antisense, but not control, oligonucleotide also abrogated the increase in lipogenesis induced by T and glucose. Reduced triglyceride formation accounted for the diminished net lipid synthesis. In contrast to lipogenesis, glucose uptake was not significantly affected by the transfections. Antisense transfection inhibited the induction of both ATP-citrate lyase and fatty acid synthase immunoreactivities, as well as malic enzyme activity, indicating that the observed reduction in lipogenesis could be explained by diminished cellular content of lipogenic enzymes. Reduced malic enzyme activity in antisense-transfected hepatocytes was accompanied by lowered relative abundance of malic enzyme mRNA, suggesting that the antisense effects on lipogenic enzymes were mediated at the pretranslational level. The oligonucleotides did not significantly affect lipogenesis in a rat hepatoma cell line that does not express detectable spot 14 mRNA or protein. These data directly implicate the spot 14 protein in the transduction of hormonal and dietary signals for increased lipid metabolism in hepatocytes.


INTRODUCTION

Investigative attention focused on the ``spot 14'' gene because of its rapid and marked response to thyroid hormone (T)()in rat liver(1) . Subsequent observations established a strong correlation between the regulation of spot 14 gene expression and that of lipid formation. These included expression specific to metabolically responsive tissues that synthesize lipids for storage or export(2, 3) . In those tissues spot 14 mRNA expression was induced by lipogenic stimuli including T, dietary carbohydrate, or premature weening(4, 5, 6, 7, 8) , and it disappeared in catabolic circumstances such as fasting, experimental diabetes mellitus, or glucagon administration(9, 10, 11) . Hepatic content of the mRNA also exhibited a circadian variation that matched that of lipid synthesis(5, 12) . Moreover, we recently observed zonated distribution of induction of spot 14 protein by T and/or dietary carbohydrate in the liver identical to that of acetyl CoA-carboxylase, a rate-determining enzyme of long chain fatty acid synthesis(13) . Immunohistochemical analysis disclosed that the spot 14 protein was predominantly nuclear in location, suggesting the possibility that it could function in the modulation of gene expression (14). In the current studies we employed antisense transfection to directly examine the hypothesis that the spot 14 protein is involved in the transduction of signals initiated by T and glucose for increased lipid synthesis in hepatocytes.


MATERIALS AND METHODS

Rat Hepatocyte Culture

Collagenase perfusion of livers from male Sprague-Dawley rats (Charles River, Cambridge, MA) weighing approximately 150 g and maintained on a 12-h photoperiod (lights on at 0700 h) with ad libitum access to normal chow (Ralston Purina, St. Louis, MO) was as described previously(15) . Cells were plated in positively charged plastic dishes (143 10 cells/cm) in serum-free modified William's E medium containing penicillin, streptomycin, 5.5 mM glucose, and no linoleic acid (Life Technologies, Inc., Gaithersburg, MD).

Hepatocyte Treatments

Cells were placed in modified William's E medium without antibiotics 5 h after plating. Some media contained 8 µg/ml Lipofectin (Life Technologies, Inc.) and 4 µM phosphorothioate oligonucleotides (Oligos Etc, Wilsonville, OR). Oligonucleotides employed were ``S14'' (GCGTTTCGTTAGCACTTGC; an antisense sequence of which the 3` residue corresponds to the ``G'' of the ATG translational start codon of the rat spot 14 mRNA sequence(16) ) and ``PPI'' (GAAGCGCATCCACAGGGCC; an antisense sequence of which the 3` residue corresponds to the ``G'' of the ATG translational start codon of the rat preproinsulin I mRNA, which is not expressed in liver tissue). Neither oligonucleotide displayed sequence similarity to rat malic enzyme mRNA. Media were replaced the following morning and again 24 h later with either modified William's E medium or modified William's E medium containing 27.5 mM glucose and 50 nM T. Oligonucleotides (2 µM, without Lipofectin) were also added to transfected cultures. Cells were harvested 24 h later. Rat H4IIE hepatoma cells (ATCC, Rockville, MD) were grown to confluence in the same media and transfected with the oligonucleotides described above in a control experiment.

Western Analysis

Affinity-purified rabbit anti-glutathione S-transferase-spot 14 fusion protein IgG was employed as reported previously(14) , except that a protein A-alkaline phosphatase conjugate (Sigma) was used for detection (1:5000 dilution). Western blot detection of rat ATP-citrate lyase and fatty acid synthase was as described(13) , employing affinity-purified goat IgG preparations (kindly supplied by L. Witters, Dartmouth Medical School). Band intensities were quantified by computer-assisted videodensitometry.

Analysis of Lipid Metabolism

Hepatocytes or hepatoma cells were placed in fresh media containing appropriate glucose, T, oligonucleotides, and either tritiated water (Sigma; 60 µCi/ml) or 1-[C]acetate (Sigma; 20.8 mCi/mmol, 4 µCi/ml) for an additional 60 or 180 min, respectively, prior to harvest. Preliminary studies showed that incorporation of radioactivity from these compounds was linear at those time points. Total lipids were extracted by the method of Bligh and Dyer(17) . Extracts were chromatographed on silica gel plates using petroleum ether:ethyl ether:acetic acid (80:19:1). Radioactive lipids were visualized by autoradiography, and identities were assigned to the labeled lipids by their comigration with nonradioactive standards. The protein concentration was determined on an aliquot of each sample prior to extraction using the method of Wadell(18) .

2-Deoxyglucose Uptake

Hepatocytes transfected as described above were incubated in the presence of 1-[C]2-deoxyglucose (Sigma; 57 mCi/mmol, 0.5 µCi/ml) for 30 min. Preliminary experiments showed linear uptake of this compound over a 60-min period. Cells were washed with phosphate-buffered saline and then suspended in 1.0 ml of phosphate-buffered saline. The same procedure used for extraction of lipids was then employed, except the aqueous phase was retained, dried in a stream of air, resuspended in 60 µl of water, and analyzed by liquid scintillation spectrophotometry.

Malic Enzyme Activity and mRNA

Malic enzyme activity was determined in total cellular protein by the method of Hsu and Lardy (19). Northern analysis of malic enzyme mRNA was as described previously(20) . Optical densities of autoradiographic bands were quantified by computer-assisted videodensitometry.

Statistical Analysis

Differences between means were assessed by analysis of variance (ANOVA(21) ).


RESULTS

Western analysis of the time course of induction of spot 14 protein after addition of 50 nM T and increased glucose (from 5.5 to 27.5 mM) to the hepatocyte culture medium is shown in Fig. 1. On blots loaded with 400 µg of total protein per lane, spot 14 protein was undetectable initially, became visible within 4 h, and was maximally induced within 1 day.


Figure 1: Time course of spot 14 protein induction by T and glucose in hepatocytes. A Western blot (400 µg of protein from individual culture dishes at each time point/lane) probed with antibodies directed against a spot 14 fusion protein is shown. T (50 nM) and increased glucose (from 5.5 to 27.5 mM) were added to the culture media at time 0, and duplicate plates were harvested at the indicated intervals.



The effect of antisense transfection on spot 14 protein expression is shown in Fig. 2. The protein was not detectable in hepatocytes maintained in 5.5 mM glucose without T after 72 h in culture (lanes 1 and 2). Accumulation of spot 14 protein was observed after 48 h exposure to T and 27.5 mM glucose (lanes 3-6). This was not inhibited by either mock transfection (lanes 7-10) or transfection of the preproinsulin I oligonucleotide (lanes 15-18). Treatment with the spot 14 antisense oligonucleotide, however, resulted in a nearly complete inhibition of induction of the protein (lanes 11-14). Neither the yield of protein/culture dish nor trypan blue exclusion were reduced in antisense-treated plates.


Figure 2: Antisense-mediated inhibition of spot 14 protein induction in hepatocytes. A Western blot of total cellular protein (100 µg/lane) is shown. Hepatocytes were incubated in serum-free medium with 5.5 mM glucose and no T overnight, and then maintained for 48 h in identical medium (lanes 1 and 2) or medium containing 27.7 mM glucose and 50 nM T (lanes 3-18). Induced plates were either not transfected (lanes 3-6), mock transfected (Lipofectin without any oligonucleotide; lanes 7-10), transfected with the spot 14 antisense oligonucleotide (lanes 11-14), or transfected with a control oligonucleotide corresponding to rat preproinsulin I mRNA (lanes 15-18). The results typify those of more than 10 separate experiments. Bands at the bottom of each lane are tracking dye (pyronin Y). The arrow indicates the position of the 19-kDa size marker on the blot.



We assessed the impact of antisense transfection on lipid metabolism in hepatocytes labeled with tritiated water. Transfection of the antisense oligonucleotide reduced the incorporation of this label to 57% of that observed in cells mock transfected with Lipofectin alone (p < 0.05), a value not statistically different from that observed in cells maintained in 5.5 mM glucose without T (Fig. 3a). Incorporation in cells treated with the preproinsulin I oligonucleotide yielded rates not significantly different (p > 0.05) from that observed in mock-transfected cells. As was the case in tritium-labeled hepatocytes, the T- and glucose-induced increment in lipid labeling from acetate was also abrogated by transfection of the spot 14 antisense sequence (p < 0.05, Fig. 3b).


Figure 3: Lipid synthesis and glucose uptake in transfected hepatocytes. Data (12 culture plates/group, mean ± S.D.) are radioactive incorporation or uptake per microgram of total cellular protein. Cells were plated overnight, and then exposed to the indicated media for 48 h before labeling for 3 h. Low, 5.5 mM glucose, no T; all others were treated with 27.5 mM glucose and 50 nM T either alone (High), or plus mock transfection (Lf), transfection of the spot 14 antisense oligonucleotide (AS), or transfection of the preproinsulin I antisense control oligonucleotide (PPI). Mean values of groups marked with asterisks were not significantly different from each other, but were significantly (p < 0.05) different from the unmarked groups. Panel a, lipids labeled with tritiated water; panel b, lipids labeled with [C]acetate, panel c, [C]2-deoxyglucose uptake.



Reduced transport of glucose from the culture medium into the hepatocytes could explain the reduced lipogenesis in antisense-transfected cells. We therefore assessed the impact of the treatments on uptake of [C]2-deoxyglucose by hepatocytes (Fig. 3c). In contrast to lipid synthesis, none of the transfected groups exhibited significant differences in the uptake of the glucose analog.

Thin layer chromatograpic separation of C-labeled lipids revealed that triglyceride was the predominant lipid synthesized by the hepatocytes (data not shown). Analysis of equal amounts of radioactive lipids showed no major difference in the relative incorporation of label into triglycerides, free fatty acids, or cholesterol in antisense, as opposed to control-transfected cells. Diminished formation of triglyceride therefore accounted for the bulk of the reduced incorporation of radioactivity into total lipids caused by the antisense oligonucleotide.

The rat H4IIE hepatoma cell line does not express detectable spot 14 mRNA or protein in response to T and 27.5 mM glucose. These cells also did not exhibit a significant increase in lipid labeling from [C]acetate 48 h after the transition from 5.5 mM glucose to 27.5 mM glucose plus 50 nM T (low, 9.8 ± 1.4; high, 11.0 ± 1.8 nmol incorporated/90 min/µg, mean ± S.D., n = 6 plates/group, p = 0.18). We measured lipid synthesis in H4IIE cells transfected with the spot 14 or preproinsulin I antisense oligonucleotides to further control for potential nonspecific metabolic effects of the treatments. No significant differences in mean rates of lipid labeling from [C]acetate among the groups were observed in these cells (data are mean ± S.D., nanamole/90 min/µg, 6 plates/group): 27.5 mM glucose and 50 nM T alone, 8.0 ± 0.6; plus mock transfection, 6.4 ± 1.8; plus transfection of the spot 14 antisense oligonucleotide, 6.4 ± 1.2; plus transfection of the preproinsulin I oligonucleotide 7.0 ± 1.2).

We wished to determine whether reduced lipid synthesis in antisense-treated hepatocytes resulted from diminished expression of lipogenic enzymes. Western analysis showed that the cellular content of ATP-citrate lyase was induced by treatment of hepatocytes with T and 27.5 mM glucose, and that this was significantly (p < 0.05) inhibited by transfection with the antisense, but not the control, oligonucleotide (Fig. 4). A similar result was observed for fatty acid synthase (Fig. 5). Induction of malic enzyme activity was also significantly (p < 0.05) inhibited by exposure to the antisense, but not the control, oligonucleotide (Fig. 6).


Figure 4: Induction of ATP-citrate lyase immunoreactivity is inhibited by a spot 14 antisense oligonucleotide. A Western blot of total cellular protein (50 µg/lane) from individual plates of hepatocytes treated as described in the legend to Fig. 2 was probed with an antibody directed against rat ATP-citrate lyase. Intensities of the ATP-citrate lyase bands were: low, 0.72 ± 0.02; high, 7.19 ± 2.32; Lipofectin, 13.78 ± 1.19; antisense, 2.87 ± 0.93; preproinsulin I, 10.25 ± 1.71 (mean ± S.D.; * indicates significantly different (p < 0.05) from other groups; Lipofectin was also significantly greater than the preproinsulin I group).




Figure 5: Induction of fatty acid synthase immunoreactivity is inhibited by a spot 14 antisense oligonucleotide. A Western blot of total cellular protein (50 µg/lane) from individual plates of hepatocytes treated as described in the legend to Fig. 2 was probed with an antibody directed against rat fatty acid synthase. The arrow indicates the position of the 265 kDa enzyme. Intensities of the fatty acid synthase bands were: low, 1.29 ± 1.41; high, 8.08 ± 3.45; Lipofectin only, 9.63 ± 1.78; antisense, 1.89 ± 0.81; preproinsulinI, 10.40 ± 1.63 (mean ± S.D.; * indicates different from other groups, p < 0.05).




Figure 6: Malic enzyme induction by T and glucose is inhibited by antisense transfection. Data (mean ± S.D., 8 culture plates/group) are malic enzyme activity corrected for protein content. Treatment groups are designated as in the legend to Fig. 2. The asterisks indicate groups that are not significantly different from each other, but are different (p < 0.05) from unmarked groups.



We performed Northern analysis of total RNA extracted from the hepatocytes using a rat malic enzyme cDNA to define the mechanism of the antisense inhibition of enzyme induction (Fig. 7). Densitometric quantitation of autoradiographs, normalized to the intensity of actin signals seen on reprobing of the blots, indicated that expression of malic enzyme mRNA was significantly reduced in the antisense, as opposed to preproinsulin I-transfected cells. Optical densities of the malic enzyme bands were 1.0 ± 0.03 in preproinsulin I, and 0.39 ± 0.21 in antisense-treated groups (mean S.D., n = 4/group, p < 0.05).


Figure 7: Northern analysis of malic enzyme mRNA in transfected hepatocytes. An autoradiograph prepared from total RNA (10 µg/lane) prepared from hepatocytes and probed with a rat malic enzyme cDNA is shown. Two individual culture plates are represented for each treatment group. Numbers below the treatment designations indicate the average optical density of the lower (21 S) autoradiographic bands in each group, corrected for that of the actin signal seen on reprobing the blots. The two bands observed in each lane result from the two potential polyadenylation sites in the rat malic enzyme gene.




DISCUSSION

The correlative data cited in the introduction led to the association of spot 14 mRNA expression and the lipogenic activities of liver, white and brown adipose, and lactating mammary tissues. The association rested on multiple examples of concordant regulation of the mRNA and lipogenic rates in those tissues by various stimuli in both the intact animal and cultured cells. Our demonstration that the regulation of the spot 14 protein was as rapid and marked as that of the mRNA(22) , and that the protein co-localized with lipogenic enzymes within the hepatic lobule (13) extended the association to the level of the protein itself. Immunohistochemical detection of the protein in hepatic nuclei further prompted the hypothesis that the spot 14 protein could be involved in the regulation of the lipogenic pathway at the level of gene expression(14) . In the current studies transfection of a phosphorothioate antisense oligonucleotide allowed us to directly assess the metabolic consequences of inhibition of spot 14 protein induction in T- and glucose-stimulated hepatocytes.

Our major observation was the inhibition of glucose- and T-induced lipogenesis in antisense-transfected hepatocytes. In order to understand the inhibitory effect of antisense transfection on lipid formation, we used available antibodies to analyze the cellular content of both ATP-citrate lyase and fatty acid synthase. In both cases, inhibition of enzyme induction by the antisense, but not the control, oligonucleotide was observed. Others have shown that the expression of these enzymes is regulated at the pretranslational level(23, 24) , although the lyase also exhibits insulin-induced serine phosphorylation(25) . We observed a similar inhibition of induction of the activity of malic enzyme, which is also known to be regulated at the pretranslational level(26) . Although this enzyme is not believed to be rate-determining for de novo long chain fatty acid synthesis, its well characterized regulation by T and glucose rendered it attractive as a representative lipogenic enzyme. In view of our previous demonstration of immunostaining of hepatic nuclei with antibodies directed against a spot 14 fusion protein(14) , and our current observation of reduced malic enzyme mRNA expression in antisense-treated hepatocytes, we hypothesize that the metabolic effects of the spot 14 antisense knockout were mediated at the pretranslational level. Our previous comparison of the time course of accumulation of malic enzyme mRNA and spot 14 protein in rat liver following T injection was also consistent with this formulation(20) .

In contrast to lipogenesis, antisense transfection had no significant impact on glucose uptake. This observation had two major implications. The first was that the spot 14 antisense oligomer did not nonspecifically injure the cells with resultant inhibition of either the facilitated transport of glucose or its energy-dependent phosphorylation. The second was that diminished availability of glucose from the culture medium did not underlie the observed reduction in lipid formation in antisense-treated cells.

Fractional turnover of modified oligonucleotides such as those employed in this study is markedly reduced compared to that of unmodified single-stranded DNA in tissue culture(27) . The possibility of nonspecific effects requires consideration. The starkly contrasting effects of the antisense and control oligonucleotides on both the expression of spot 14 protein and on lipid formation, as assessed by incorporation of two different radioactive precursors, assured us of the specificity of the effect of the antisense oligomer on these variables. This conclusion was further supported by the observation that H4IIE cells, a line derived from the same organ and species as the hepatocytes that does not express detectable spot 14 mRNA or protein, did not exhibit any differential effects of the oligonucleotides on lipid synthesis. The metabolic impact of antisense transfection was therefore dependent on both the sequence of the oligonucleotide and the presence of the intended target sequence within the transfected cell.

Taken together, the data indicate that spot 14 protein functions in the transduction of hormone- and substrate-initiated signals for lipid metabolism in hepatocytes. Transcriptional activation of the spot 14 gene by thyroid hormone (28) and glucose metabolism(29, 30) , as well its inhibition by polyunsaturated fatty acids (31) and cyclic AMP (11) suggest the possibility that levels of the protein serve to integrate a variety of physiological stimuli that regulate lipogenesis in selected tissues.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant DK-43142 (to W. B. K.), the Hitchcock Foundation (to W. B. K.), and National Institutes of Health Grant DK-32855 (to C. N. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: 714 West Borwell Bldg., 1 Medical Center Dr., Lebanon, NH 03750.

The abbreviation used is: T, triiodothyronine.


ACKNOWLEDGEMENTS

We thank Donald St. Germain, Jacqueline Sinclair, Peter Sinclair, and Lee Witters for useful discussions, and Ami Mariash for expert technical assistance.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.