(Received for publication, September 14, 1995)
From the
To study the molecular basis of tissue-specific and hormonally regulated expression of the fatty acid synthase (FAS) gene in vivo, we generated lines of transgenic mice carrying 2.1 kilobases of the 5`-flanking region (-2100 to +67) of the rat FAS gene fused to a chloramphenicol acetyltransferase (CAT) reporter gene. This reporter gene construct was strongly expressed in tissues that normally express high levels of FAS mRNA, which include liver and white adipose tissues. In contrast, CAT reporter activity was not detected in appreciable levels in lung, heart, kidney, and muscle tissues, which do not normally show significant levels of FAS activity. The relative levels of the CAT mRNA driven by the rat FAS promoter in various tissues of the transgenic animals approximated those of the endogenous mouse FAS mRNA. We also examined the hormonal and nutritional regulation of the FAS(2.1)-CAT reporter gene in transgenic mice. CAT activity was increased in both liver and white adipose tissue when fasted animals were refed a high carbohydrate, fat-free diet. These changes in CAT activity and CAT mRNA levels occurred in parallel to the changes in endogenous mouse FAS mRNA levels. On the other hand, fasting/refeeding did not change CAT activity appreciably in other tissues, such as muscle and brown adipose tissue. Administration of dibutyryl cAMP at the start of refeeding prevented an increase in CAT activity in liver. However, the cAMP effect was tissue-specific as cAMP treatment did not bring about change in CAT activity in adipose tissue. Next, to examine the effect of insulin, we made the transgenic mice insulin-deficient by streptozotocin treatment. Insulin treatment of the streptozotocin-diabetic mice increased both the CAT activity and CAT mRNA levels driven by the rat FAS promoter in liver and white adipose tissue. These changes in CAT expression by insulin paralleled those in endogenous FAS mRNA levels. Administration of glucocorticoids increased CAT activity in all tissues examined: liver, white and brown adipose tissues, lung, heart, and spleen. Overall, the first 2.1 kilobases of the 5`-flanking region of the rat FAS gene appear to contain sequence elements necessary to confer tissue-specific and hormonally regulated expression characteristic of the endogenous FAS gene.
Fatty acid synthase (FAS) ()plays a central role in de novo lipogenesis in mammals and birds by catalyzing all the
reactions in conversion of acetyl-CoA and malonyl-CoA to palmitate. FAS
activity is not known to be regulated by allosteric effectors or
covalent modification. However, FAS concentration in liver and adipose
tissue is highly sensitive to nutritional, hormonal, and developmental
states(1, 2, 3) . When rats are fasted for
1-2 days, the rate of synthesis of FAS declines, while refeeding
a high carbohydrate diet increases synthesis of FAS(4) . We
have previously reported that FAS mRNA was not detectable in livers of
fasted mice but dramatically increased upon refeeding a high
carbohydrate, fat-free diet(5) . This induction of FAS mRNA
resulted from increased transcription of the FAS gene(6) . Both
lipogenic and lipolytic hormones participate in regulating FAS
expression. Injection of cAMP prevented increase in FAS mRNA and
transcription during fasting/refeeding. FAS expression was very low in
diabetic mice, and insulin caused a marked and rapid increase in the
FAS mRNA levels and in the transcription rate of the FAS
gene(6) . Hormonal regulation of FAS expression in adipose
tissue, on the other hand, has not been studied extensively. We
observed that insulin increased and cAMP decreased FAS mRNA levels in
3T3-L1 adipocytes (5) . In these cells, thyroid hormone also
stimulated FAS gene transcription, indicating independent effects of
insulin, cAMP, and thyroid hormone on FAS gene expression(7) .
In addition to these various hormones and agents that regulate FAS
expression, dietary long-chain polyunsaturated fatty acids (PUFAs) have
been reported to have inhibitory effects on FAS expression, while
saturated and monounsaturated fatty acids show little to no inhibitory
effects(8) . The putative cis-acting elements responsible for
the hormonal/nutritional regulation of the FAS gene have not been
extensively studied. The only reported element is an insulin response
sequence at -67 to -52 base pairs of the FAS promoter that
we previously defined by transfection of various FAS
promoter/luciferase constructs into 3T3-L1
adipocytes(9, 10) .
Here, to investigate the molecular basis for the tissue-specific and nutritional/hormonal regulation of FAS gene expression in an appropriate in vivo physiological context, we generated transgenic mice carrying the 2.1-kb 5`-flanking promoter region of the FAS gene fused to a chloramphenicol acetyltransferase (CAT) reporter gene. Transient transfection into established cell lines is usually employed in defining promoter function. The relevance of cell culture studies to in vivo events, however, is not clear. Transgenic animal studies only will unequivocally demonstrate the presence of a true functional DNA region for tissue-specific expression and for hormonal regulation. In the present study, we show that the 2.1-kb region flanking the 5`-end of the rat FAS gene is sufficient to direct tissue specificity as well as hormonal regulation that mirrors the expression of the endogenous FAS gene.
Figure 1:
Tissue distribution of CAT reporter
activity driven by the rat FAS promoter, CAT mRNA, and endogenous FAS
mRNA in transgenic mice. A, CAT activity was determined in
tissue extracts from a heterozygous transgenic mouse bearing a 2.1-kb
FAS promoter-CAT gene as described under ``Experimental
Procedures.'' Steady-state CAT and endogenous mouse FAS mRNA
levels were determined by Northern blot analysis employing RNA prepared
from the same tissues used for CAT assays as described under
``Experimental Procedures.'' 300 µg of extracts for CAT
assay and 20 µg of total RNA for Northern blot prepared from liver
of nontransgenic (Nt liver) and transgenic mice, white adipose
tissue (Wat), kidney, muscle, lung, and heart were used. The
ethidium bromide staining of the agarose gel showing 28 and 18 S RNA is
included. The 1.1-kb CAT coding region and 3.7-kb 3`-mouse FAS cDNA
sequence were used as probes after labeling with P by
random-priming method. Liver RNA from nontransgenic mice was used as a
control. B, CAT activity was determined using various tissue
extracts prepared from a different founder than that shown in A.
Figure 5: Effect of glucocorticoids on CAT activity of various tissues from transgenic mice. Transgenic mice bearing a 2.1-kb FAS promoter-CAT gene were injected with dexamethasone as described under ``Experimental Procedures'' and were sacrificed 5 h after the injection. 300 µg of extracts from liver, white adipose tissue (Wat), brown adipose tissue (Bat), muscle, lung spleen, kidney, and heart were used for determination of CAT activity. The thin layer chromatography plates were exposed for 2 days for all tissues except for liver, which was exposed for 9 days.
Figure 2: CAT reporter activity in fasted (F) and refed (R) transgenic mice. Heterozygous transgenic mice were either fasted for 48 h or fasted for 48 h then refed with a high carbohydrate, fat-free diet for 24 h. A, hepatic CAT and endogenous FAS mRNA levels were determined by Northern blot analysis as described under ``Experimental Procedures'' and in Fig. 1. The ethidium bromide staining of the agarose gel showing 28 and 18 S RNA is also included. Lane 1, fasted transgenic; lane 2, fasted nontransgenic; lane 3, refed transgenic mouse of the same founder as in lane 1; lane 4, refed transgenic mouse of a different founder; lane 5, refed nontransgenic mouse. B, CAT activity was measured in 300 µg of tissue extracts of liver, white adipose tissue (Wat), brown adipose tissue (Bat), and muscle.
Figure 3: Effects of insulin on CAT activity and CAT and endogenous mouse FAS mRNA levels in streptozotocin-diabetic transgenic mice bearing a 2.1-kb FAS promoter-CAT fusion gene. A, CAT activity was measured with 300 µg of tissue extracts from streptozotocin-diabetic and streptozotocin-diabetic transgenic mice treated with insulin as described under ``Experimental Procedures.'' B, Northern blot analysis was performed as described in Fig. 1using 20 µg of RNA prepared from livers of the same streptozotocin-diabetic and insulin-treated, streptozotocin-diabetic mice as those used in CAT activity measurement. The ethidium bromide staining of the agarose gel is also shown. Wat, white adipose tissue; Bat, brown adipose tissue.
We next studied the effect of cAMP on FAS promoter/CAT gene expression. Fasted transgenic mice were refed with a high carbohydrate, fat-free diet for 9 h to induce FAS/CAT fusion gene expression. Dibutyryl cAMP was administered at the start of refeeding. As shown in Fig. 4, both lipogenic tissues, liver and white adipose tissue, showed high levels of CAT activity after 9 h of refeeding of a high carbohydrate diet. cAMP administration at the start of the refeeding prevented this increase in CAT activity in liver driven by the 2.1-kb 5`-flanking region of the FAS gene. The present result is in agreement with our previous observation that cAMP prevents increase in FAS gene transcription that accompanies refeeding in mice(6) . However, the effect of cAMP appears to be tissue specific. Unlike in liver, there was no change in CAT activity in adipose tissue. Our observation may explain a previous report that glucagon decreased FAS activity in liver but not in adipose tissue(27) . On the other hand, we previously reported that cAMP decreased FAS mRNA levels in 3T3-L1 adipocytes in culture. We do not know the reason for this discrepancy. It is possible that cAMP has a dominant negative effect during fasting/refeeding only in liver, and the hormonal and nutritional milieu of cultured 3T3-L1 adipocytes is different from that of adipose tissue in vivo. Further studies are necessary to elucidate cAMP effects on the FAS gene transcription in different tissues. Nevertheless, our result clearly indicates that the 2.1-kb 5`-flanking sequence of the FAS gene is sufficient for dominant suppressive effect of cAMP on FAS gene transcription in liver during fasting/refeeding.
Figure 4: Effect of cAMP on CAT activity in refed transgenic mouse liver. Mice were fasted for 48 h and then refed with a high carbohydrate, fat-free diet. Dibutyryl cAMP was injected at the start of refeeding. Mice were sacrificed 9 h after injection. Tissue extracts were prepared from liver and adipose tissue of transgenic mice. CAT activity was measured using 300-µg extracts as described under ``Experimental Procedures.'' Wat, white adipose tissue; Bat, brown adipose tissue.
The role of glucocorticoids in regulating FAS activity or FAS gene expression in different tissues has not been studied. Moreover, contradictory results were reported on glucocorticoid effects on fatty acid synthesis(28, 29) . Therefore, we examined the regulation of the 2.1-kb FAS promoter/CAT reporter gene by glucocorticoids in transgenic mice. As shown in Fig. 5, the basal CAT activity of transgenic animal on a normal chow diet was highest in adipose tissue as discussed in Fig. 1. On the other hand, CAT activity was difficult to detect in other tissues from normal chow-fed transgenic animal. Administration of synthetic glucocorticoids, such as dexamethasone, caused an increase in CAT expression in all tissues examined such as liver, white and brown adipose tissues, lung, heart, and spleen. Therefore, we conclude that glucocorticoids increase FAS expression in all tissues. Our observation that glucocorticoids up-regulate FAS gene agrees with a recent report on glucocorticoid stimulation of FAS gene transcription in fetal lung(30) . On the other hand, a previous report showed that FAS activity was decreased in adipose tissue, and adrenalectomy produced an increase in FAS activity that correlated with changes in fatty acid synthesis(27) . These changes, however, were tissue specific in that neither the glucocorticoid injection nor the adrenalectomy affected hepatic FAS activity. In those studies, the animals were on a high carbohydrate, fat-free diet, and the effects observed may partly reflect glucocorticoid effect during FAS induction by refeeding. Regardless, our data indicate that 2.1 kb of the FAS 5`-flanking sequence is sufficient for the transcriptional activation of FAS gene by glucocorticoids.
It has been known that, although their molecular
basis is not clear, dietary PUFAs can suppress expression of several
genes involved in hepatic lipid metabolism, including FAS(8) .
It was also shown that PUFA suppression of FAS expression in liver was
at the transcription level(13) . In adipose tissue, however,
although both saturated and unsaturated fatty acids depressed de
novo fatty acid synthesis, dietary saturated and polyunsaturated
fat had only a slight suppressive effect on FAS mRNA
levels(8) . We, therefore, compared the effects of triolein
containing essentially oleic acid (C18:19) and menhaden oil
enriched in long chain n-3 fatty acids (e.g. eicosapentaenoic acid (C20:5
3) and docasahexaenoic acid
(C22:6
3)) on the regulation of the 2.1-kb FAS promoter/CAT gene
of these transgenic mice. First, we carried out Northern blot analysis
to determine the effect of these oils on the endogenous FAS mRNA
levels. In both liver and adipose tissue, FAS mRNA levels were
dramatically suppressed in both liver and adipose tissue of mice fed
the menhaden oil diet as compared to those fed the triolein diet (Fig. 6). However, CAT activity in these tissues of mice on the
menhaden oil diet was only 2-fold lower than that of the mice on the
triolein diet. It is possible that multiple elements are present for
suppression of the FAS gene transcription by PUFAs and that one such
element exists in the first 2.1 kb of the 5`-flanking region of the FAS
gene. Another possibility is that FAS may also be regulated by PUFAs at
the level of mRNA stability as well as at the transcriptional level.
Figure 6: Effects of menhaden oil and triolein diets on CAT activity and endogenous FAS mRNA levels in transgenic mice bearing a 2.1-kb FAS promoter-CAT gene. Transgenic mice were either maintained on the triolein diet or switched to the menhaden oil diet for 5 days. Mice were killed 24 h after food removal. 300 µg of extracts from liver and adipose tissue were used for CAT activity measurement. RNA from liver and adipose tissue were isolated as described under ``Experimental Procedures,'' and 10 µg of RNA were employed for Northern blot analysis for murine FAS mRNA. The Northern blot membrane stained with methylene blue showing 28 and 18 S RNA is also included. Wat, white adipose tissue; Bat, brown adipose tissue.
In summary, a 2.1-kb region flanking the 5`-end of the rat FAS gene appears sufficient to direct tissue-specific expression of a reporter gene in transgenic mice. In addition to being sufficient for conferring tissue-specific expression in transgenic animals, the 2.1-kb FAS promoter fragment also displays appropriate hormonal control characteristic of the endogenous mouse FAS mRNA. Most of these studies were performed in at least two transgenic founder lines and gave similar results, indicating that the site of chromosomal integration did not influence expression. Additional FAS promoter-reporter constructs expressed in transgenic mice will be necessary to further define DNA sequences responsible for tissue-specific and regulated expression of the FAS gene in vivo.