Dietary regulation of aldolase isozyme expression in rat intestinal mucosa

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 254, No. 1, April, pp. 116-123,1987

Dietary Regulation of Aldolase lsozyme Expression in Rat Intestinal Mucosa’ JUN-ICHI SAT0,2 KEN-ICHI TSUTSUMI, MAKOTO ISHIKAWA, AND KIICHI ISHIKAWA Lkpartment

of Biochenzistry,

Yamagata University

Sew

of Medicine,

Zaoiida,

Received May 23,1986, and in revised form September

Yamagata 990-25, Japan

3,1986

Changes of aldolase A and B protein levels and their mRNA levels due to starvation for 48 h in mucosae of the jejunum, ileum, and colon were determined by Western and Northern blot analyses. In fed rats, B protein and B mRNA were predominant in the jejunum. In the ileum, both A protein and A mRNA, as much as B protein and B mRNA, were present in significant amounts. In the colon, A protein and A mRNA were predominant. The enzyme activity levels in those segments of fed rat intestine were in parallel to total enzyme protein levels (A + B) and also to total mRNA levels (A + B), thus suggesting that aldolase isozyme expression in fed rat intestine is determined mainly at the level of transcription. Starvation for 48 h caused about 30% reduction of both B protein level and B mRNA level in the jejunum. In the ileum, both A and B mRNA levels were lowered 30-40% from those of fed rats, while A and B protein levels were reduced slightly (A, O%;B, 12%).In the colon, starvation caused about 50% increase of A mRNA level and about 10% reduction of A protein level. By measuring the synthetic rate of the enzyme proteins from in wivo [35S]methionine incorporation, the accumulation of A mRNA in this tissue was suggested to be due to the significant fall of the translation rate of A mRNA. The translational and post-translational controls of aldolase isozyme expressions in rat intestines are discussed. o 1987 Academic PWS, I~C. Three distinct isozymes (A, muscle type; B, liver type; C, brain type) of fructose-1,6bisphosphate aldolase (EC 4.1.2.13) are expressed tissue-specifically in mammals (1). The tissue-specific expressions of the isozymes change in response not only to some intracellular stimuli (e.g., development and carcinogenesis) but also to some extracellular stimuli (e.g., nutritional and hormonal conditions). It has been reported that dietary conditions influence the enzyme activities in digestive organs. For ex’ This work was supported in part by Grants-inAid 60470142 and 60770509 from the Ministry of Education, Science and Culture, Japan. e The Second Department of Internal Medicine, Yamagata University School of Medicine, Yamagata 99023, Japan. 0003-9861/87 $3.00 Copyright All rights

0 1987 by Academic Press, Inc. of reproduction in any form reserved.

116

ample, the enzyme activity in the liver is decreased by starvation, and can be induced again by a high fructose or glucose diet (2,3). Recently, this phenotypic change in the liver was suggested to involve selective inactivation of aldolase B (4-‘7), or regulation of B mRNA synthesis by some hormonal actions (8). Since digestive organ tissues show relatively rapid responses to such dietary conditions, we have been interested in investigating the multiplicity, if it exists, of control mechanisms of the aldolase expression in these tissues. For such investigations, we considered that the intestine is a preferable object, since the isozyme patterns are different in various parts of this organ and they change in response to dietary conditions. Using rat aldolase A and B cDNAs (9, 10) and specific antibodies, we have shown

DIETARY

CONTROL

OF INTESTINAL

ALDOLASE

GENE

EXPRESSION

117

Preparation of antibodies against akldase A and B. Aldolase A and B isozymes were purified from skeletal muscle and livers of Wistar rats by affinity chromatography on P-cellulose as described by Lebhertz (13). Purified aldolase A or B (4-5 mg) was mixed with Freund’s complete adjuvant and injected subcutaneously into the back of two rabbits. After 2 weeks, three booster injections were given intramuscularly each with about 1 mg antigen at e-week intervals. The antibodies were purified as described previously (11). Western blot analysis. Protein (100 pg) of the 105,OOOgsupernatant from each segment of fed and starved rats was applied to SDS-polyacrylamide gel electrophoresis (12.5%).The proteins separated were transferred electrophoretically to a nitrocellulose filter and incubated with anti-aldolase A or B antibodies. The filter was washed and then incubated with lzsIprotein A (14), and subjected to autoradiography. Densities of the bands at about 40 kDa were measured with Joyce-Loebl’s chromoscan 3. The amounts of aldolase A and B isozyme proteins in 100 c(g cytosolic proteins from each segment were quantitated by the MATERIALS AND METHODS standard curves prepared by using purified aldolase A or B proteins (0.5,1.0,1.5,2.0 pg proteins) as above. Materials. [cy-S2P$lCTP (3000 Ci/mmoI), [“S]RNA extraction and Northern blot hybridization methionine (1300 Ci/mmol), and ‘l-protein A (30 Mucosal cells from the jejunum, ileum, and colon were mCi/mg) were purchased from Amersham; the nitrohomogenized in guanidine thiocyanate as described cellulose filter was from Schleicher & Schtill (BA 85); by Harding et al (15). RNA was precipitated three agarose was from Sigma; oligo(dT)-cellulose was from times in ethanol in the presence of guanidine-HCl, Collaborative Research; fructose-1,6-bisphosphate and RNA containing polyadenylate segment (poly(A)’ tetrahydroxylammonium salt, glyceloaldehyde-3RNA) was isolated by oligo(dT)-cellulose column phosphate dehydrogenase, triose phosphate isomerchromatography (16). Poly(A)+ RNA (3 rg) was sepase, and NADH were from Boehringer Mannheim. All arated electrophoretically on agarose gel in a denaother reagents were of the highest grade available. tured condition and transferred to a nitrocellulose Animals and tissues. Male Wistar rats weighing 250paper. The paper was dried, baked, hybridized 300 g were used. One group of animals were fed a with ?-labeled nick-translated aldolase A cDNA commercial diet ad &turn and another group were (pAHA1352) or B cDNA (pRAB3031) with almost the starved for 48 h. Water was freely available. Rata were same specific activities as described previously (10, sacrificed by decapitation and intestines were excised. ll), and then subjected to autoradiography. Densities All subsequent steps were carried out at 4’C. Small of the bands at about 1600 nucleotides were measured intestine from just distal to Treiz ligament to ileocecal with Joyce-Loebl’s chromoscan 3. The relative junction was excised and divided into two halves, the amounts of A or B mRNAs from tissues to that of B jejunal segment and the ileal segment. Also the colon mRNA in 3 pg of poly(A)+ RNA from fed rat jejunum from the cecum to the rectum was excised. Each segwas determined. ment of the small intestine and the colon was cut lon1n viva protein &e&p. Fed and starved rats regitudinally and washed with ice-cold saline. Then, ceived each 600 &i of [“Slmethionine injection via mucosal cells were scraped off with a glass slide. tail vein, and after 20, 40, 90, and 180 min. mucosal Enzyme assay. Mucosal cells were homogenized in cells were scraped off from each segment of the in20 mM Tris-HCl buffer, pH 7.5, containing 80 mM KCl, testine and homogenized. After centrifugation at 5 mM MgClz, and 1 mM phenylmethanesulfonyl fluoride 105,OOOgfor 60 min, total aldolase protein in the suPMSF? The homogenate was centrifuged at 105,OOOg pernatant was purified by affinity chromatography on for 60 min and the resulting supernatant was used P-cellulose (13). The radioactivities of total cytosolic for the enzyme assay. Aldolase activity for fructoseprotein and purified aldolase protein were measured 1,6-bisphosphate was measured spectrophotometriwith a liquid scintillation counter of Packard Tri-Carb tally by the method of Blostein and Rutter (12). 4530. The counting efficiency of pS]methionine incorporated into the total and enzyme proteins was about 80%. Therefore, the radioactivity of 2288 cpm incorporated denotes 1 fmol of Cj5Slmethionine incorporated * Abbreviations used: PMSF, phenylmethanesul(1300 X 0.8 X 2.2 X 10” cpm/mmol = 2288 cpm/fmoI). fonyl fluoride; SDS, sodium dodecyl sulfate.

that the expression of aldolase isozyme genes in liver tissue is regulated primarily at the level of transcription (10,ll). However, when we examined similarly protein and mRNA levels of A and B isozymes in the intestines of fed and starved rats, we found that, in colon mucosal cells, A mRNA level increased while A protein level decreased due to starvation, suggesting the accumulation of A mRNA by translational control of aldolase A isozyme gene expression in this tissue. In the present paper, we describe changes due to starvation in the levels of aldolase A and B proteins and mRNAs in rat jejunum, ileum, and colon, and discuss control mechanisms of aldolase A and B gene expressions in rat intestines.

118

SAT0 RESULTS

Change of the Enqpne Activity in the Intestine Due to Starvation When we examined the total aldolase activities in mucosae from three separate segments of the intestine, i.e., jejunum, ileum, and colon, of fed and starved rats, the levels of total enzyme activity were not equal in these segments. In fed rats, the enzyme activity was highest in the jejunum and lowest in the colon (Table I). By starvation for 48 h, the wet weight of mucosa of each segment decreased to about a half of that of fed rats, and the enzyme activity levels of each segment also decreased to 5863% of those of fed rats (Table I). To see what causes these changes in more details, we next examined the changes due to starvation of the A and B isozyme protein contents, their cognate mRNA contents, and the rates of their protein syntheses in these intestinal segments. Quanti$cation of Aldoluse A and B Isozyme Proteins b Western Blotting Aldolase A and B isozyme protein levels in three segments of fed and starved rat intestines were detected and quantificated by Western blot analysis using anti-aldolase A and B antibodies (Fig. 1). In fed rats, the A protein level was highest in the colon (0.55 + O.lO%;data are the percentages of aldolase proteins to the total proteins in

ET AL.

the cytosol, and are the mean values + SD of the four experiments) and lowest in the jejunum (0.07 + O.O6%).0n the other hand, B protein level was highest in the jejunum (1.10 1 O.O5%)and lowest in the colon (0.04 + 0.01%). These results may indicate the existence of reciprocal concentration gradients of two isozyme proteins along the intestines of fed rats: A protein content becomes higher from the proximal to distal segment, and B protein content from the distal to proximal segment. In the jejunum of fed rats, A protein level was very low (about 6% of B protein), and conversely, in the colon of fed rats, B protein level was as low as about 7% of A protein. By starvation, A protein level slightly increased in the jejunum (from 0.07 f 0.06 to 0.13 f O.lO%), remained almost unchanged in the ileum (from 0.29 k 0.09 to 0.29 k 0.02%), and slightly decreased in the colon (from 0.55 f 0.10 to 0.49 f 0.05%). On the other hand, B protein content decreased in the jejunum (from 1.10 f 0.05 to 0.81 2 0.08%) and in the ileum (from 0.52 + 0.18 to 0.46 +: 0.13%), and remained unchanged in the colon (from 0.04 f 0.01 to 0.04 k 0.01% ). Therefore, the changes of the total enzyme activity in the jejunum and ileum are determined mainly by the change of B protein levels. Similarly, the change in the colon is determined by the change of A protein level. The levels of the total enzyme proteins (A + B) in starved rat intestines were 80% (jejunum), 92% (ileum), and 90% (co-

TABLE

I

EFFECTOFSTARVATIONONALDOLASEACTIVITIESFORFRUCTOSE-~,~-BISPHOSPHATE INJEJUNUM,ILEUM, ANDCOLON Starved

Fed

Jejunum Ileum Colon

Activity” (Units/mg protein)

No. of rats assayed

Activity ratio (starved/fed)

Activity” (Units/mg protein)

No. of rats assayed

0.071+ 0.016

9

0.041 + 0.009*

7

0.58

0.032+ 0.007 0.022 +0.003

9 9

0.020 + 0.004* 0.014+ 0.0046

7 7

0.63 0.64

“The 105,000~ supernatant of mucosal homogenate from intestine was assayed, and activities were defined as micromoles of substrate cleaved per minute per milligram of protein at 3’7’C. Values are given as means IL SD. 6 Statistical comparison between the values of fed and starved rats were carried out by the Mann-Whitney Utest, P < 0.02.

DIETARY 12

3

CONTROL 4

OF INTESTINAL

56

Aldolase

1

23

4

A

56

Aldolase

B

W!MR-

FIG. 1. Western blot analyses of aldolase A and B isozyme proteins in rat intestine. One hundred micrograms of the 105,OOOgsupernatant protein of the mucosal homogenates from the jejunum, ileum, and colon of fed and starved rats was applied to SDSpolyacrylamide gel electrophoresis. Aldolase A and B isozyme proteins in the jejunum of fed rats (lane 1) and starved rats (lane 2), those in the ileum of fed rats (lane 3) and starved rats (lane 4), and those in the colon of fed rats (lane 5) and staved rats (lane 6) were detected as described under Materials and Methods.

lon) of those in fed rat intestines. However, as shown in Table I, the levels of the total enzyme activity in starved rat intestines decreased to 58-63% of fed rat intestines. The reason for this discrepancy is unknown. Possibly, some selective inactivation of aldolase proteins similar to that observed in the liver (4-7) may also be induced by starvation in the intestines.

ALDOLASE

Aldolase A and B mRNA contents in the three segments of fed and starved rat intestines were analyzed by Northern blot hybridization using aldolase A cDNA (pAHA1352) and B cDNA (pRAB3031) as probes (Fig. 2). Since it was difficult to quantify aldolase mRNAs, we assumed aldolase B mRNA level in poly(A)+ RNA from the jejunum as 1.00 and determined the comparative levels of A and B mRNAs in various parts of fed and starved rat intestines to it. In fed rat intestines, the levels of aldolase A and B mRNAs were al-

EXPRESSION

119

most in parallel with those of A and B proteins. A mRNA level was highest in the colon and lowest in the jejunum, and B mRNA level was highest in the jejunum and lowest in the colon. Due to starvation, A mRNA level of the jejunum increased slightly (from 0.06 + 0.01 to 0.14 f 0.02, data are the comparative values to B mRNA level in the jejunum, and are the mean values + SD of the three experiments). In the ileum, A mRNA level decreased to 60% of that of the fed rats (from 0.19 + 0.04 to 0.11 + 0.02). In the colon, however, A mRNA level increased 1.5-fold (from 0.42 + 0.03 to 0.65 f 0.04), despite the decrease of the A protein level in this tissue. The changes of A mRNA levels in the jejunum and ileum were almost in accordance with those of A protein levels, but that in the colon was not. B mRNA levels in the jejunum and ileum decreased to about 70% of those of the fed rats (from 1.00 to 0.70 & 0.04, and from 0.56 -+ 0.05 to 0.39 f 0.07, respectively). No significant change, however, was observed in the colon (from 0.030 + 0.005 to 0.026 + 0.002). These results showed that in the jejunum and ileum, the changes of

123456

A mRNA

1

Quant$cation of Aldolase A and B mRNAs by Northern Blotting

GENE

2

3

4

5

6

1)

B mRNA

FIG. 2. Northern blot hybridization analyses of aldolase A and B mRNAs in poly(A)+ RNA. Poly(A)+ RNAs were prepared from the jejunum, ileum, and colon of fed and starved rats. Each 3 pg of poly(A)+ RNA was applied to agarose gel electrophoresis. Aldolase A and B mRNAs in the jejunum of fed rats (lane 1) and starved rats (lane 2), those in the ileum of fed rats (lane 3) and starved rats (lane 4), and those in the colon of fed rats (lane 5) and starved rats (lane 6) were detected as described under Materials and Methods.

120

SAT0

ET AL.

Jejunum

0

1

2

Ileum

3

0

1

2

Time after

injection

3

0

1

2

3

(h)

FIG. 3. In wivo incorporation of [ssS]methionine into total proteins and aldolase protein of mucosal cells of the intestines of fed and starved rats. After the injection of [%S]methionine to rats via tail vein, incorporation of the radioactivities into total proteins and aldolase proteins in 105,OOOgsupernatants of mucosal homogenates from the jejunum, ileum, and colon of fed and starved rats were determined at 20, 40, 90, and 180 min. Specific activities were showed as femtomoles of [85S]methionine incorporated per milligram of protein. Specific activities of total proteins of fed rat intestines (0), those of starved rat intestines (A), specific activities of aldolase proteins of fed rat intestines (o), those of starved rat intestines (A).

A and B protein levels due to starvation roughly coincided with those of the cognate mRNA levels, but that in the colon the change of A protein level (decrease to 90% of the fed rats) did not coincide with that of A mRNA (1.5-fold increase), suggesting some translational control in this tissue. To confirm that aldolase A gene expression in the colon is controlled at the level of translation, we next examined the in tivo efficiency of aldolase protein synthesis. In Viva Aldolase Protein Synthesis Figure 3 shows the time course of [35S]methionine incorporation into the proteins in viva. After injection of the labeled methionine, aldolase was purified from the tissues by P-cellulose chromatography (13). Therefore, in this experiment, aldolase preparations contain both A and B proteins, and also possibly C protein, if it exists in the same tissue. Incorporation of r5S]methionine into total and aldolase proteins of the three segments of fed and starved rat intestines reached almost the maximum levels within 1 h. When we compared the incorporation rates during the initial 20 min as the in-

dications of the protein synthetic rates, the rates of total and aldolase protein syntheses were highest in the jejunum and lowest in the colon of fed rats, indicating the existence of the gradients of the protein synthetic activities along the intestine (Table II). This situation was the same in starved rat intestines. These protein synthetic rates in all of the intestinal segments were decreased due to starvation to 68-91s of those of fed rat intestines. The response of the tissues to starvation, as determined by the ratios of the protein synthetic rate of starved rat intestines to that of fed rat intestines, was highest in the jejunum, and lowest in the colon (Table II). When we measured and compared the rates of total aldolase protein synthesis ([35S]methionine incorporation into the aldolase proteins per minute during the initial 20 min) per unit amount of total aldolase mRNA (A + B) between fed and starved rat intestines, the ratios were 0.95 (jejunum), 1.1 (ileum), and 0.61 (colon). These results suggest that in starved rat colon the translation speed of aldolase mRNAs was slowed down to about half that in fed rat colon, suggesting the exis-

DIETARY

CONTROL

OF INTESTINAL TABLE

ALDOLASE

GENE

121

EXPRESSION

II

EFFECT OF STARVATION ON THE SYNTHETIC RATES OF TOTAL PROTEIN AND ALDOLASE PROTEIN Aldolase

Total protein

Jejunum Ileum Colon

Fed

Starved

2.29 1.78 1.53

1.57 1.43 1.21

Note. Values are given as femtomoles the period of the initial 20 min.

Starved/fed 0.68 0.80 0.79 I%lmethionine _ .

tence of translational control in this tissue, while no translational control may exist in small intestines. DISCUSSION

Aldolase is an important enzyme of the glycolytic pathway. It has three isozyme genes and their expressions are tissue and cell specific. Aldolase A seems a prototype in mammals and exists ubiquitously in almost all tissues and organs. Aldolase B is responsible for metabolism of fructose and gluconeogenesis, and exists in the liver, intestine, and kidney. C isozyme is expressed in the brain. To understand the regulation mechanisms of aldolase gene expressions, we have determined the mRNA levels and protein levels of aldolase A and B in various tissues and cells using A and B cDNAs and specific antibodies, and found that the expression of aldolase genes is controlled at the level of transcription (10, 11,29). On the other hand, it has been reported that in the liver in which B isozyme is predominantly expressed, the change of enzyme activity due to starvation and aging, is caused by the selective inactivation of B isozyme protein as a result of cleavage of several amino acids at the C-terminus (47,25,26). This suggested that, besides the transcriptional control, the post-translational control of aldolase B gene expression exerted some roles during starvation in the carbohydrate metabolism of liver. Since both A and B isozymes are expressed and their activities are also changed by dietary conditions in the in-

Fed

Starved

1.33 1.02 0.69

0.98 0.87 0.63

incorporated

protein

per minute per milligram

Starved/fed 0.73 0.85 0.91 of protein

in

testines (8), we are interested in the multiplicity of a control mechanism, if it exists, of aldolase gene expressions in the intestine. The estimation of the enzyme protein levels was carried out by Western blot analysis, using specific antibodies against A and B proteins. It was possible to quantitate the amounts of each isozyme protein in 100 pg cytosolic proteins from each segment of the intestine by the standard curves made in the same experiment using purified A and B proteins. However, the absolute quantification of A and B mRNAs by Northern blot analysis was difficult because of the shortage of the use of the purified mRNAs as standards. Nevertheless, the comparative estimation of the mRNA concentration in 3 pg of poly(A)+ RNA from various tissues could be carried out with a good reliability (11). Starvation for 48 h caused the following changes in the rat intestines. Wet weight of mucosal cells of each intestinal segment decreased to about a half of fed rat intestines. Although the cytosolic protein concentrations of each segment of starved rat intestine were almost the same as those of fed rat intestine, decrease ((level of fed rat - level of starved rat)/level of fed rat) in the enzyme activity levels was about 40% (Table I). Decrease in A + B isozyme protein levels was about lo-20%, and did not accord with that of the enzyme activity levels. Presumably, this discrepancy was caused by an occurrence of inactive forms of aldolase B similar to those observed in starved rabbit livers (4-7) or in aged mouse livers (25,26).

122

SAT0

Aldolase B protein levels and mRNA levels in the three segments were lowered by starvation. This response of the intestine to starvation is the same as that of the liver (8). Aldolase A protein level and A mRNA level increased in the jujunum, and decreased in the ileum. Furthermore, in the colon, A protein level decreased while A mRNA level increased significantly. If we assume that, in the colon of starved rat, each A mRNA molecule turns over after a definite cycle of translation, and A protein turns over with a similar rate as those in other tissues, the accumulation of A mRNA should be the result of the reduction of A mRNA translation rate in this tissue. To confirm the existence of translational control in the colon mucosal cells of starved rats, aldolase protein synthetic rates of the three segments of fed and starved rat intestines were determined by measuring the incorporation rates of [?S]methionine into aldolase protein in tivo, together with those of total cytosolic proteins. As shown in Table II and Fig. 3, the jejunum was the most active in protein synthesis of both total cytosolic proteins and aldolase proteins, and the colon was the least active. Aldolase is synthesized with about a half rate of total cytosolic proteins. Starvation caused some reduction of the apparent protein synthetic rates in all of the tissues. If we assume that changes of methionine pool and protein degradation rate in all of the tissues of starved rats are similar, the jejunum is the most affected, and the colon is the least affected tissue by starvation. But, the protein synthetic rates of aldolase per unit amount of mRNAs (A + B) in the jejunum and ileum of fed and starved rats were the same. Different from this, that of starved rat colon was about a half of that of fed rat colon. If transcription of aldolase genes is accelerated 1.5-fold by starvation, and no translational control exists, the aldolase proteins synthesized should also be increased l.&fold, although the possibility that the change of methionine pool due to starvation affected these apparent protein synthetic rates in the colon cannot be denied. This suggests that translation of aldolase mRNA was slowed down by some

ET AL.

reason in the starved rat colon. These points remained to be clarified. In the intestine, epitherial cells differentiate in crypts, move up along villi, and are exfoliated in l-3 days. Starvation is known to slow down this replacement of epitherial cells (21-23). Jones et al. also reported that aldolase activity in rat intestine was reduced by 24-60 h of starvation, but in that period of time, the change of cell population was small (24). It seems that the translational control rapidly minimizes the protein turnover and slows down the replacement of epitherial cells. In conclusion, the following can be said about the control of aldolase gene expression. The principal control is at the level of transcription (10, 11). Even when the change of the expression occurred, such as that during fetal liver development (11) or during chemical hepatocarcinogenesis (29), it is controlled at the level of transcription. However, starvation seems to induce some additional and presumably transient control mechanism at the levels of translation and post-translation in digestive organs, in order to adapt the drastic extracellular stimuli. ACKNOWLEDGMENTS The authors thank Professor A. Endo, Yamagata University School of Medicine, for the critical discussion about the statistical treatment of the data. Thanks are also given to Mrs. M. Seki for typing the manuscript. REFERENCES 1. PENHOET, E., RAJKUMAR, T., AND RUTTER, W. J. (1966) Proc iVuti Acd Sti USA 56,1275-X82. 2. ADELMAN, R. C., SPOLTER, P. D., AND WEINHOUSE, S. (1966) J. Biol Ch 241,5467-54’72. 3. SILLERO, M. A. G., SKLERO, A., AND SOLS,A. (1969) Eur. J. Bioch 10,345-350. 4. PONTREMOLI, S., MELLONI, E., SALAMINO, F., SPARATORE, B., MICHETIT, M., AND HORECKER, B. L. (1979) Proc. Nati Ad Sci USA 76,63236325. 5. PONTREMOLI, S., MELLONI, E., SALAMINO, F., SPARATORE, B., MICHETTI, M., AND HORECKER, B. L. (1980) Arch Biochmn. Bioph~s. 203,390394. 6. PONTREMORI, S., MELLONI, E., MICHETTI, M., SALAMINO, F., SPARATORE, B., AND HORECKER,

DIETARY

7.

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16. 17.

CONTROL

OF INTESTINAL

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