dietary sucrose enhances processing of mrna-s14 nuclear precursor

THE JOURNAL OF BIOLOGICAL CHEMISTRY w 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Val. 266, No. 34, Issue of December 5, pp. 22905-22911,1991 Printed in U.S.A.

Dietary Sucrose Enhances Processing of mRNA-S 14 Nuclear Precursor*

(Received for publication, April 17, 1991)

Lynn A. Burmeister and Cary N. MariashS From the Diuision of Endocrinology and Metabolism, Department of Medicine and Department of Cell Biology and Neuroanatomy, University of Minnesota, Minneapolis, Minnesota 55455

The rapid response of rat hepatic mRNA-S14 to hormonal or dietary manipulation makes it an excellent model to study the control of lipogenic enzyme mRNA. The mechanism of regulation of this mRNA by triiodothyronine (T3) or sucrose remains controversial. Al- though initial studies suggested that Ts stabilized the nuclear precursor, subsequent studies suggest that Ts acts by increasing the transcriptional rate of this gene. More recently, the induction of mRNA-S14 by sucrose administration was shown to be associated with an increase in transcriptional “run-onn activity. Because Ts and carbohydrate feeding synergistically regulate this mRNA, we studied the response to short and long term high carbohydrate feeding in hypothyroid and euthyroid rats. We found the response to the lipogenic diet was rapid in hypothyroid rats, with maximal levels of mRNA-S14 attained by 4 h (2.2 f 0.6 chow fed versus 13.5 k 2.5 pg/pg RNA on lipogenic diet). The rapid induction by the lipogenic diet contrasts with the diminished response to sucrose by gastric gavage (4.6 f 1.2 pg/Mg RNA) over the same time interval. Despite the large increase in the mature mRNA induced by the lipogenic diet, the rise in the nuclear precursor was small and not different from that observed after sucrose gavage (0.14 f 0.01 chow, 0.26 f 0.03 sucrose gavage, 0.25 -C 0.04 pg/pg RNA lipogenic diet). The molar ratio of the mature to precursor mRNA-S14 showed progressive increases with the smallest level in the fasting rat, an intermediate level in the chow- fed and sucrose gavaged rats, and the highest level in the animals fed a lipogenic diet (2.1, 16.5, 16.3, 62.7, respectively). Based on the previously reported half- life for the mature mRNA-S14, these data show that feeding sucrose by gavage or by a lipogenic diet leads to enhanced fractional conversion of precursor to mature mRNA-S14 with a simultaneous stabilization of the precursor mRNA-$14.

The mechanisms of dietary regulation of hepatic gene expression are not well understood. Several enzymes involved in lipogenesis demonstrate increased activity after high carbohydrate feeding (1). Pretranslational regulation with increased mRNA levels caused by either enhanced transcrip-

Grant RO1-DK 32885 (to C. N. M.) and National Institutes of Health * This work was supported in part by National Institutes of Health

Training Grant T32-DK 07203 (to L. A. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Hospital, Minneapolis, MN 55455. Tel.: 612-624-5150; Fax: 612-626- $ To whom correspondence should be sent: Box 91, Mayo Memorial

4027.

tion, post-transcriptional mechanisms, or both have been proposed (2). We have employed rat hepatic mRNA-S14 as a model for the study of both thyroid hormone and carbohydrate regulation of lipogenic enzyme gene expression because it is rapidly responsive to triiodothyronine (TJ1 and a high carbohydrate or “lipogenic diet.” Specifically, the levels of nuclear precursor and cytosolic mature mRNA increase within 10 and 20 min, respectively, after administration of TB to hypothyroid rats (3, 4). Likewise, intragastric administration of sucrose to euthyroid rats results in increased mature mRNA-S14 levels within 30 min (5) and induction of the nuclear precursor signal by 15 min (6). In contrast, in hypothyroid rats sucrose gavage results in mature mRNA-S14 levels significantly lower than can be induced in starved euthyroid rats. A replacement dose of TR restores the diminished response (5). This suggests that T3 and a lipogenic diet interact to regulate this mRNA, as shown for other lipogenic enzymes ( 7 ) .

Previous reports on the molecular mechanism underlying the regulation of mRNA-S14 by TB and carbohydrate have been conflicting. The major controversy has centered on the relative contribution of transcriptional compared with post- transcriptional regulation. Initial reports favored nuclear precursor stabilization as the most significant site of mRNA-S14 regulation by both T3 and lipogenic diet (8,9). More recently, reports suggest that enhanced transcription plays a predomi- nant role combined with post-transcriptional regulation of mRNA-S14 by TB (10, 11). The specific post-transcriptional mechanism has not elucidated. Enhanced transcription has also been measured in response to sucrose gavage in euthyroid rats (6). In fact, Jump et al. (12) stated that post-transcriptional processes do not contribute significantly to the mRNA- S14 induction by sucrose gavage.

To clarify the role of sucrose and a lipogenic diet on mRNA- S14 regulation, we studied the time course of nuclear precursor and cytosolic mature mRNA-S14 response in hypothyroid and euthyroid rats to a lipogenic diet. We found that the hepatic nuclear precursor responded differently from the cytosolic mature mRNA-S14 to both sucrose gavage and a lipogenic diet. The difference in response of the precursor and mature mRNA was thyroid state independent. Specifically, the mature mRNA-S14 responded to a much greater extent than the precursor form of this mRNA to dietary changes. In addition, the response to a lipogenic diet was greater than to sucrose gavage. Besides enhanced transcription as reported previously, we propose that the lipogenic diet induces mRNA- S14 by a novel mechanism of decreasing precursor degradation and increasing the rate of processing to the mature form.

The abbreviation used is: T , triiodothyronine.

22905

22906 Sucrose Alters mRNA-S14 Processing

MATERIALS AND METHODS

Animal Treatment-Male Sprague-Dawley rats (BioLabs Inc., White Bear Lake, MN) weighing 150-175 g were rendered hypothyroid by the addition of 0.025% methimazole to their drinking water for 3 weeks. During the 3rd week hypothyroid rats showed the expected arrest of weight gain.

Dietary Manipulation-Hypothyroid and euthyroid rats, housed four/cage, were given either a standard lipogenic diet (58% sucrose, 20% casein, 20% nonabsorbable filler; fat-free test diet, ICN, Irvine, CA), maintained on regular rat chow (23% protein, 55% complex carbohydrate, 22% fat; Ralston Purina Co, St. Louis, MO), or fasted for 24 h and killed 4 h after either gavage by 60% sucrose (2 m1/100 f: of body weight) or saline. Diets were changed to allow the rats to be killed at the same time. All rats were killed a t 8 a.m. or noon by exsanguination via the abdominal aorta under light ether anesthesia. Livers were frozen in liquid nitrogen and stored a t -80 "C.

RNA Analysis-Total hepatic RNA was extracted by the guanidine t.hiocyanate method (13). Both mature and precursor mRNA-,914 were quantitated by RNAse protection assay (14) using a cRNA probe to either an 800-base nonrepetitive region of the single intervening sequence or a 400-base region in the 3"untranslated region of the S14 mRNA (15). Hybridization was carried out in 0.6 M NaCI, 22 mM Tris (pH 7.5), 5 mM EDTA, 0.15% sodium dodecyl sulfate, and 2.5% ethanol at 80 "C for 12-48 h. The cRNA was prepared with T7 polymerase as suggested by the manufacturer (U. S. Biochemical Corp.) from the respective cDNA subcloned into the plasmid PTZl8R and linearized with the appropriate restriction enzyme. A Mini-Spin Column (Cappell Life Science, Malvern, PA) was used to separate unincorporated nucleotides from the radiolabeled cRNA. Unlabeled mRNA standard was prepared by an identical technique with a plasmid in which the cDNA was cloned in the opposite orientation. Quantitative assays were done by comparing the hybridization signal from unknowns with a simultaneously run set of standards. The mass of precursor mRNA-S14 was corrected for the probe length to compare directly the molecules of precursor and mature mRNA-S14. The quantitiy of mature mRNA was also corrected for the amount of precursor mRNA present in the sample. Results from selected sam- ples were confirmed by Northern gel analysis utilizing a nick-translated ["'PP]cDNA probe (6). Corrections for RNA loading (mass of 28 S RNA present) and x-ray exposure were made prior to comparisons. For the actinomycin D studies we used used digoxigenin labeled random-primed cDNA with chemiluminescence detection (Genius Kit, Boeringer Mannheim) of Northern gels.

Statistics-RNA results from separate experiments, expressed as pg/pg of RNA, were pooled. We used an internal standard derived from normal rat liver RNA to assure consistency of results. Where necessary, the data were log or square root transformed to achieve homogeneity of variances. Groups were compared by analysis of variance and multiple pairwise comparisons made using Tukey's Honestly Significant Difference procedure, or Student's t test when appropriate. After dietary changes, we considered a new steady state to be established when two subsequent time points were no longer significantly different. Regression curves were compared with a test of slope homogeneity by analysis of covariance. Data are expressed as mean k S.E. unless otherwise indicated.

RESULTS

In confirmation of earlier studies, 24-h fasted hypothyroid rats were found to have a 90% reduction in mature mRNA- S14 levels compared with chow fed rats (Table I) (16). The administration of 60% sucrose by gavage to the fasted rat led to significant increases in mature mRNA-S14 levels to a level 2.1-fold higher than in chow-fed rats. The response to 4 days of the lipogenic diet was even greater than that found 4 h after a maximal dose of sucrose (6). Therefore, despite the previous observation that T3 is required for maximal response t o sucrose (5) , these hypothyroid rats can respond significantly to the carbohydrate challenge.

Because the response to 4 days of the lipogenic diet was greater than that observed 4 h after sucrose gavage, we next studied the time course of the mature mRNA-S14 response to shorter intervals of administration of the lipogenic diet (Fig. 1). To avoid the complications of diurnal changes in mRNA-S14 content, all rats were killed at the same time of

TABLE I Mature and precursor mRNA-,914 content in hypothyroid rats

Each value is the mean t- S.E. pooled from two to five experiments. Those values with different superscripts are significantly different from each other ( p < 0.05).

Treatment group Mature mRNA-S14 2:?&fs Pg/@g RNA

Fasting 0.238" f 0.064 8 Chow fed 2.15b f 0.563 20 Sucrose gavage (4 h) 4.62' f 1.24 8 Lipogenic diet (4 days) 9.86d f 5.16 12

Treatment group Precursor mRNA-S14 ;:ifs Pg/@ RNA

Fasting 0.115" f 0.009 8 Chow fed 0.142" f 0.013 20 Sucrose gavage (4 h) 0.263b f 0.029 8 Lipogenic diet (4 h) 0.253b f 0.036 12 Lipogenic diet (4 days) 0.235b f 0.016 12

20'o T T 16.0

12.0

8.00

4.00

T

0 4 8 12 24 48 72 96

Time (hours)

FIG. 1. Time course of mature mRNA-S14 response to a lipogenic diet. Hypothyroid rats were maintained on a regular chow diet until 8 a.m., 8 p.m., midnight, or 4 a.m. when they were switched to a fat-free 58% sucrose-containing diet. The rats were killed a t 8 a.m. after being on the diet for the indicated times after the switch, and hepatic mRNA-S14 was measured by RNase protection. The results from four different experiments are pooled. Each point is the mean k S.E. from 4-16 rats. The hours on the lipogenic diet are indicated on the x axis. All time points were significantly ( p < 0.05) different from the 0 time base line by analysis of variance. No other time points were different from each other.

day. Surprisingly, mRNA-S14 levels rose near maximally after only 4 h of conversion to the lipogenic diet. The only significant difference observed after the zero time point was between the value at 1 day and 4 days (14.1 _+ 1.43 versus 9.86 1.49 pg/pg RNA after 1 versus 4 days, p = 0.05). Although there is a small decline in mRNA-S14 after 4 days on the lipogenic diet, the major observation is the rapidity of response to the dietary change. The response to the dietary change is as rapid as and is greater than that after sucrose gavage. The mRNA- S14 content attained after the dietary change (an increase of 11.4 pg/pg RNA, Fig. 1) compared with that reached after sucrose gavage (an increase of 4.4 pg/pg RNA, Table I) suggests that the dietary stimulus is even more potent than a maximally effective quantity of intragastric sucrose (6). In addtion, because there is no further significant increase in mRNA-S14 content after 4 h, a new steady state is reached by 4 h on the lipogenic diet.

Sucrose Alters mRNA-S14 Processing 22907

To localize the site of the differential regulation by these dietary manipulations we also measured the content of the nuclear precursor for mRNA-S14 by RNAse protection assay. Since the quantity of precursor mRNA-S14 is 1-2 orders of magnitude less than that of the mature message, we also examined the relative quantity of the precursor mRNA-S14 by Northern analysis using a nick-translated cDNA probe directed against the intervening sequence of the primary transcript. The content of the 4.5Kb precursor on Northern gels correlated well ( r = 0.7) with the content of the precursor measured by RNase protection.

Table I lists the quantitative measurements of mRNA-S14 precursor in hypothyroid rats under different dietary treatments. Although the level of the precursor tended to be the lowest in the fasting state, it was not significantly lower than that in the chow-fed animals. As a consequence, the amount of precursor mRNA-S14 accounted for a large fraction of total mRNA-S14 in the fasted state. At the other extreme, all three sucrose-treated groups had levels of precursor only about twice that of the chow-fed controls and not different from each other. The much smaller precursor response and lack of difference among different groups stand in contrast to the response of the mature mRNA.

The time course of precursor response to changing the diet is depicted in Fig. 2. Although the relative response is less, it is evident that the precursor and mature mRNAs respond with a similar time course. The precursor rapidly rises and remains elevated for the entire 4 days. Also as noted with the mature mRNA, there appears to be a small, but insignificant, decrease in the content of the precursor as the animals adapt to the diet. Also, as with the mature mRNA-S14, by analysis of variance there was no statistical difference at any time after initiation of the diet although the 0 time point was significantly less than all other time points. Thus, steady- state precursor content was also reached by 4 h.

The greater increase in mature mRNA compared with that of the precursor indicates processes other than transcription are in part responsible for the changes in mature mRNA-S14. Formal proof

0.m

0.100 :i 0.W

of this statement derives from the following

I I

1 0 4 8 12 24 48 72 96

Time (hours)

FIG. 2. Time course of precursor mRNA-S14 to lipogenic diet. Precursor mRNA-S14 was measured by RNase protection. The RNA from the rats treated in Fig. 1 was used. Each point is the mean -C S.E. from 4-16 rats. The hours on the lipogenic diet are indicated on the x axis. All time points were significantly ( p < 0.05) different from the 0 time base line, except the 72-h point. No other time points were different from each other.

considerations. The content of the precursor at any time, t , after changing the diet is

where A = rate of precursor synthesis (transcription), X, = fractional degradation rate of the precursor (including processing), and C is the baseline content of the precursor prior to the dietary change. Once steady state is reached, when t is long compared with X,, the content of the precursor is

precursor mRNA content = - A (2) x,

By analogy, the content of the mature mRNA at steady state is

mature mRNA content = - B Am

(3)

where B is the synthesis rate of the mature mRNA, and X, is the fractional degradation rate of the mature mRNA. Since the synthesis rate, B, of the mature mRNA at steady state is the content of the precursor times the fractional rate of processing

then, assuming X, only represents processing and substitution of Equation 4 into Equation 3, the content of mature mRNA is

- A A m

Consequently, by dividing Equation 5 by Equation 2, the ratio of mature to precursor mRNA at steady state is

ratio = - x, x m

Thus, by Equation 6, the ratios of the mature to precurosr at two different steady states is constant and independent of the transcription rate as long as there is no change in the fractional degradation rates of the precursor and product.

Because both the precursor and mature mRNA-S14 reach steady state levels by 4 h after dietary manipulation (Figs. 1 and 2), a comparison of these ratios at 4 h allows one to analyze the roles of altered synthesis and metabolism of the products. Fig. 3 presents the ratio of mature to precursor mRNA at steady state after the dietary manipulations. The lowest ratio of mature to precursor RNA occurs in the fasted state. Four h after the administration of sucrose by gavage, the ratio increased %fold from 2.1 to 16.3 ( p < 0.05). In an analogous manner, 4 h after switching from rat chow to a lipogenic diet the ratio increased from 16.5 to 62.7 ( p < 0.05) and remained equally elevated at subsequent times (54.0,42.3, and 56.2 at 12, 24, and 48 h, respectively). Thus, administration of sucrose either by gavage to a fasted rat or as part of a high carbohydrate diet leads to a much greater rise in the mature form of mRNA-S14 compared with the precursor. This finding indicates that the dietary change leads to an alteration of either X, or X,.

A decrease in X,, with a consequent increase in the half- life, cannot account for the change in ratios. Previously we have shown that the half-life of the mature mRNA-S14 is approximately 90 min on a regular chow diet. Because the new steady state on the lipogenic diet is attained by 4 h, the half-life of the mature mRNA after switching diets cannot be any longer than 90 min. This is consistent with our previous observation of similar half-lives of the mature mRNA-S14 on both a regular chow diet and after sucrose administration


T

Dietary Treatment

FIG. 3. Ratio of mature to precursor mRNA-S14 under different dietary regimens in hypothyroid rats. Hypothyroid rats were treated as indicated under “Materials and Methods.” Hepatic RNA was isolated and the content of the mature and precursor mRNA-S14 measured by RNase protection. The ratio of mature to precursor was calculated for each animal, and the mean & S.E. for each group presented. Each bar represents from 8 to 20 rats (two to five experiments). All groups are significantly different ( p < 0.05) from the fasting group, and both lipogenic diet groups are different ( p < 0.05) from the chow-fed and sucrose gavage groups.

10

5

2

1 l , , , , l I 1 I I l 1 I I 1 l 1 1 1 1

0 1 2 3 4 5

Time after Actinomycin-D (hours) FIG. 4. Disappearance of mature mRNA-S14 after actino-

mycin D administration. Euthyroid rats were switched to the lipogenic diet 24 h before administration of actinomycin D. At 8 a.m. actinomycin D (2 mg/kg) was given intravenously, and the rats were killed at the indicated times after administration of the drug. Three to four rats were used at each time point (1, 2, and 4 h). The content of mRNA-S14 is was measured by Northern gel analysis. The results are presented on a semilogarithm plot, and the line is fit by linear regression of the log-transformed data.

(17). To confirm that the half-life of the mature mRNA is 90 min on a lipogenic diet, we measured the disappearance of mature mRNA-S14 after actinomycin D administration. Fig. 4 demonstrates the half-life of the mRNA in rats on a lipogenic diet is also approximately 90 min. Since the dietary changes do not alter X, they must change X,.

Consideration of the determinants of X, yields the following model

such that x, = x, + x, (7)

where X, is the overall fractional turnover of the precursor, X, is the fractional rate of precursor degradation, and X, is the fractional rate of processing. By substitution of the appropriate fractional rate constants for X, into Equation 4, at steady state the rate of synthesis of mature mRNA,

The content of mature mRNA at steady state would be

mature mRNA content = A.X 1,. x, (9)

and, by dividing Equation 9 by Equation 2, the ratio of the mature to precursor content is XJX,. Therefore, if the fractional rate of processing of precursor to mature, X,, increased after the dietary change, the ratio of the mature to precursor would also increase. Moreover, if X, decreases to the same degree as X, increases, then X, will not change with the dietary manipulations. Consequently, the net rise in precursor mRNA would reflect only the increase in transcriptional rate whereas the rise in mature would reflect both the increase in transcriptional rate as well as the enhanced rate of processing of precursor to mature mRNA.

Fig. 5 presents further support for this conclusion. This figure shows that, except for the fasting state, in euthyroid rats the increase in mature to precursor ratio is nearly identical with that found in hypothyroid rats (Fig. 3). The re- markable similarity in mature to precursor ratios in the two thyroidal states emphasizes the thyroid-independent effect of dietary carbohydrate on precursor mRNA-S14 processing.

100 - 80 i T

Dietary Treatment

FIG. 5 . Ratio of mature to precursor mRNA-S14 under different dietary regimens in euthyroid rats. Euthyroid rats were treated and analyzed as in Fig. 3. Each bar represents from 4 to 16 rats (one to four experiments). All groups are significantly different ( p < 0.05) from the fasting group, and both lipogenic diet groups are different ( p < 0.05) from the chow-fed and sucrose gavage groups.


Our kinetic considerations suggest that X, may not change under various dietary treatments because X, decreases to the same degree as X, increases. To test this hypothesis further we studied the rate of disappearance of the precursor after inhibition of transcription by cycloheximide. We used this inhibitor because previous studies demonstrated that cycloheximide blocks transcription of mRNA-S14 and blocks the induction of this gene by T, or carbohydrate feeding (10, 18, 19). If our hypothesis is correct, the disappearance of the precursor in animals given sucrose would be the same as in controls. Fig. 6 is a semilogarithm plot of the precursor content after cycloheximide administration. There is no significant difference in the rate of loss of precursor mRNA-S14 after inhibition of transcription. The half-life of precursor disappearance in all three groups is 19 min. This value is similar to that reported previously using Northern gel anal- yses to measure the content of the precursor (10). If sucrose administration or the lipogenic diet led to only a decrease in X,, the half-life of the precursor would have been longer in the treated compared with the regular diet control. If sucrose or the lipogenic diet led only to an increase in X,, the half-life of the treated groups would have been shorter than the control. The similar half-lives in the treated and control groups argue for dual regulation of the precursor by sucrose and the lipogenic diet, namely, a decrease in X, and an increase in X, after sucrose or lipogenic diet administration.

Since the absolute mature mRNA-S14 response to sucrose

Regular Diel

J

50 30 40 i o 20

2 ' I I

10 20 30 50 40

z i t Sucrose Gavage

z I I

I I

I

10 20 30 40 50

Minutes Alter Cycloheximide

FIG. 6. Disappearance of precursor mRNA-S14 after inhibition of transcription. Euthyroid rats were either maintained on a regular chow diet (Regular Diet), placed on a lipogenic diet (Lipo- genic Diet) for 4 days or fasted for 24 h and then given a 60% sucrose solution (2 m1/100 g of body weight) by gastric gavage (Sucrose Guuuge). After administration of cycloheximide (1 mg/kg intraperi- toneally), shown previously to inhibit transcription of the S14 gene (19), four rats in each group were killed at the indicated times. The content of the precursor mRNA-S14 was measured by RNase protection. The natural logarithm of the precursor content is plotted against time after cycloheximide. The line represents the overall regression after correcting for differences in base line.

gavage was not identical with the response to the lipogenic diet (Table I), we examined the possibility that the different responses were caused by the differences in the time of administration of the stimulus. Although mRNA-14 displays a circadian variation, we found this was not responsible for the difference in response to the sucrose-containing diets. When we gave the lipogenic diet at 8 a.m. rather than 4 a.m., we found that the response was less (data not shown). However, the lower response at 8 a.m. could be fully accounted for by the diminished intake during this 4-h interval (0.39 at 4 a.m. versus 0.18 g/100 g rat/h at 8 a.m.). More importantly, sucrose gavage of hypothyroid rats at 4 a.m. or at 8 a.m. yielded similar precursor and mature mRNA-S14 content at the two times (Fig. 7). Therefore, the timing of administration of the lipogenic diet is unlikely to contribute to any significant difference in response.

Although the timing of food administration did not contribute to any difference in response, the caloric intake did. We found that the dietary intake was variable after switching rats to the lipogenic diet. In two separate experiments the hypothyroid rats consumed 0.32 and 0.46 g/100-g rat/h during the 4 h of exposure to the diet. Thus, during the second experi- ment the hypothyroid rats ate nearly 50% more. In two experiments with euthyroid animals we found that the rats ate 0.49 and 0.73 g/lOO-g rat/h, also showing 50% variability. In contrast, during a 24-h interval on regular rat chow we found that euthyroid rats consumed 0.54 and 0.42 g, and hypothyroid rats ate 0.17 and 0.19 g/lOO-g rat/h in separate studies. The variable food intake likely accounts for the variance in response observed on the lipogenic diet.

DISCUSSION

Although we and others have shown that enhanced transcription can account for part of the response of mRNA-S14 to sucrose (6, 12), the relative role of post-transcriptional processes has not been clarified. One approach to evaluating post-transcriptional regulation of mRNA-S14 is to measure the content of both the precursor and mature mRNA at steady state after various treatments. If only transcription events were responsible for alterations in mature mRNA, the relative

T

4 AM 8 AM

Time of Sucrose Gavage

FIG. 7. Effect of timing of sucrose gavage on mRNA-S14 precursor and mature mRNA content. Hypothyroid rats were fasted for 24 h. At the indicated hour each rat was given a 60% sucrose solution (2 m1/100 g of body weight) by gastric gavage and killed 4 h later. The content of hepatic precursor (shaded bar) and mature (hatched bar) mRNA-S14 was measured by RNase protection. Each point is the mean f S.E. of four (4 a.m.) or eight (8 a.m.) rats. There is no significant difference between the times of administration for either the precursor or mature content.


change in precursor and mature mRNA content should be the same. In the present study we found that sucrose produced a much greater increase in the mature mRNA compared with the response of the precursor from the fasted state. Therefore, transcription alone cannot account for our observations. The finding that the mature mRNA increased to a greater extent than the precursor is similar to that reported for malic enzyme mRNA (20). In that report the authors concluded that the greater response of mature message compared with precursor was caused by to stabilization of the mature mRNA for malic enzyme. Our data show that altered processing, rather than mature message stabilization, accounts for the change in mature:precursor ratios, at least for mRNA-S14.

The validity of our conclusion requires that several prereq- uisites be met. First, to compare the precursor and mature mRNA ratios the rat must be at steady state after applying the inducing stimulus. If the inducing stimulus was short lived, then precursor content would fall rapidly, and one could not compare the mature:precursor RNA ratios. However, it is evident from Fig. 1 that the maximum response to the lipogenic diet was attained by 4 h and remained elevated at that level for more than 12 h. In addition, previous data showed that mRNA-S14 undergoes diurnal variation with a nadir in the late a.m. (21). Since all animals were killed at 8 a.m. (near the nadir), diurnal factors do not contribute to the value attained at that time. In fact, if anything diurnal factors would lead to an even greater increase in the content of the mature mRNA at later hours. Therefore, the necessary con- ditions for steady-state analysis were present.

Because a new steady state is reached by 4 h and it takes three half-lives to reach 88% of the final value, the turnover rate of the mature message can be no longer than 1.5-2 h. Several lines of evidence indicate that the half-life of this message on a regular chow diet (10,22) and in hepatocytes in culture (23) is approximately 90 min. Further, studies after glucagon administration indicate a similar half-life in chow- fed and sucrose-gavaged rats (17). If stabilization of the mature mRNA were responsible for the preferential increase in mature message, then a new steady state could not be reached in 4 h. In fact, if there were only a &fold stabilization, the new steady state would not be reached for 14 h. It is important to emphasize that several lines of evidence were used to demonstrate that stabilization of the mature mRNA could not account for our results. Statistical analysis of the data from Fig. 1 shows that steady state is reached by 4 h. As noted above, measurement of the half-life of the mature mRNA was found to be similar in chow-fed and sucrose- gavaged rats. Lastly, the mature to precursor ratio did not change after 4 h on the lipogenic diet. Therefore, the dietary change did not lead to stabilization of the mature message.

Since dietary changes do not stabilize the mature message, the processing of the nuclear precursor must be altered. I t had been proposed previously that thyroid hormone stabilized the nuclear precursor for mRNA-S14 (8). This hypothesis was proposed to account for the rise in precursor RNA without a concomitant rise in transcriptional activity. A similar phenomenon was observed with the developmental changes that occur with this mRNA (24). However, subsequent studies in which enhanced transcriptional rates were observed cast doubt on this hypothesis for the thyroidal and developmental changes of this gene (11, 25). Despite these considerations, our data are distinctly different from the other studies because we find that the rise in the nuclear precursor is not propor- tional to the rise in mature mRNA. Most importantly, the ratio of the mature to precursor RNA increases with a change to a high carbohydrate diet. Therefore, neither transcriptional

rate changes nor precursor stability alone can account for our observations. Consequently, we propose that the administration of sucrose by gavage or in the lipogenic diet leads to two simultaneous changes in the processing of the nuclear precursor for mRNA-S14. The first is a decrease in the rate of nuclear degradation of the precursor mRNA-S14, X,, and the second is an increase in the rate of conversion of the precursor to mature mRNA-S14, X,. Because we have made all our measurements on total hepatic RNA, we cannot analyze any potential effect of nuclear transport on these processes. We are aware of only one other example of a similar phenomenon in a recently reported abstract (26).

Our hypothesis of the dual regulation of the precursor mRNA by sucrose is supported by the cycloheximide data. These data demonstrate convincingly that the overall turnover of the precursor is unchanged by sucrose feeding. How- ever, as with any metabolic inhibitor, the conclusions are valid only if the inhibitor stopped transcription and had no effect on processing. The same caution must be applied to the actinomycin D study on the mature mRNA turnover.

Although we have emphasized in this manuscript the role of altered processing in the regulation of mRNA-S14 by dietary factors, enhanced transcription does play a role in the regulation of this mRNA. Previous studies have reported that sucrose administration leads to a 3-5-fold increase in the transcriptional rate of this gene (6, 12). In addition, the 5'- flanking region of this gene is responsive to enhanced media glucose concentrations in hepatocyte cultures (27). In the present study the increased level of mRNA-S14 precursor observed after sucrose gavage is entirely consistent with a transcriptional effect. For example, Table I shows that sucrose gavage led to a 2.4-fold increase in the precursor content. However, it is also important to note that the increase in transcriptional rates after sucrose gavage to a fasted rat is not immediate. Both in our earlier study (6) and in those by Jump et al. (E'), maximal increases in transcription did not occur for 2-4 h after sucrose administration. Further, the increases in transcription follow the earlier increases in mature mRNA- S14 content, implying that a process other than transcription is active. Consequently, dietary-induced altered processing of this gene must occur before enhanced transcription is maximal.

Although the content of the mature and precursor mRNA- S14 was higher in the euthyroid than hypothyroid rats (data not shown), the ratios of mature to precursor mRNA were nearly identical in the two thyroidal states in each treatment group (Fig. 3 versus Fig. 5 ) . The exception is in the fasted state, in which this ratio is higher in the euthyroid rats. Therefore, it is most likely that the major effect of thyroid hormone on this gene is transcriptional but that sucrose administration enhances transcription and stabilizes the precursor at the same time as it increases the processing of the precursor to mature message.

We initially undertook these studies to clarify the mechanism of regulation of mRNA-S14 by dietary carbohydrate. Previous studies used two dietary techniques to induce this mRNA: changing from regular rat chow to a lipogenic diet (9, 15), or administration of 60% sucrose by gavage to a fasted rat (5 , 6). It had been assumed that the mRNA-S14 response to these two diets was identical because both contained sucrose. Therefore, we thought the discrepancy between the large response to the lipogenic diet in hypothyroid rats reported by us (15) and the small response to sucrose gavage reported subsequently ( 5 ) was a result of the longer administration of the diet in the former study. However, in the present study we found that sucrose solution (60%) by gastric gavage


gave a smaller response than the 58% sucrose-containing lipogenic diet when administered to rats for 4 h. There are many potential reasons for the lipogenic diet giving a greater response than an equivalent amount of sucrose alone. A likely possibility relates to the additional protein in the lipogenic diet. It has been shown previously that sufficient dietary protein is required to obtain the maximal lipogenic enzyme response to simple sugar administration (28). Dietary protein may enhance the process of protein synthesis required for mRNA-S14 induction and blocked by cycloheximide administration. In addition to the protein in the lipogenic diet, this diet contains many other constituents, some of which also may influence the response to sucrose. For example, a more recent study showed that nonabsorbable fiber (contained in the lipogenic diet) enhances the lipogenic enzyme response to sucrose (29). Lastly, the lipogenic diet and sucrose gavage may ellicit different hormonal or gastrointestinal peptide responses. It is possible that insulin and glucagon responses are different between the two treatments. Despite the differences in response to the two dietary regimens, sucrose is clearly a major inducer of hepatic mRNA-S14.

In summary, we would propose the following model for the regulation of mRNA-S14 by sucrose. This model also may be applicable to other genes. Sucrose administration leads to a 3-5-fold increase in transcription of this gene (6, 12). The enhanced transcriptional rate is not immediate and may be associated with the development of new hypersensitive sites in the 5”flanking region of the S14 gene (12). In addition, sucrose administration leads to specific alteration of the processing of the nuclear precursor for mRNA-S14. The alteration in processing likely precedes the changes in transcription and account for the early increases in mature message reported previously. We envision a processing complex in which the single intron of the S14 gene is appropriately spliced. Under the influence of sucrose the splicing and ligation of the mature message occur more rapidly. In the absence of the sucrose stimulus, splicing may not occur appropriately, or ligation may not occur, and the message degrades within the nucleus. Thus, sucrose administration leads to both stabilization and enhanced processing of the nuclear precursor for mRNA-S14.

Acknowledgments-We thank Dr. Michel 0. Goumaz for help in performing the initial assays. We are also indebted to Mary Ellen Domeier and Ami Mariash for technical assistance.

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