From the
``Spot 14'' is a nuclear protein that is rapidly
induced by thyroid hormone (T
Investigative attention focused on the ``spot 14''
gene because of its rapid and marked response to thyroid hormone
(T
Western analysis of the time course of induction of spot 14
protein after addition of 50 nM T
Thin layer chromatograpic separation of
The rat H4IIE hepatoma cell line does not express
detectable spot 14 mRNA or protein in response to T
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
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
Our major observation was the inhibition of glucose-
and T
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.
We thank Donald St. Germain, Jacqueline Sinclair,
Peter Sinclair, and Lee Witters for useful discussions, and Ami Mariash
for expert technical assistance.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
) 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.
)
(
)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.
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) ).
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.
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.
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).
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.
- and glucose-stimulated
hepatocytes.
-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) .
,
triiodothyronine.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.