Dietary regulation of pancreatic amylase in transgenic mice mediated by a 126-base pair DNA fragment ROLAND Department

M. SCHMID

AND MIRIAM

H. MEISLER

of Human Genetics, University

of Michigan, Ann Arbor, Michigan

Schmid, Roland M., and Miriam H. Meisler. Dietary regulation of pancreatic amylasein transgenic mice mediatedby 262 a 126-base pair DNA fragment. Am. J. Physiol. (Gastrointest. Liver Physiol. 25): G971-G976, 1992.-Expression of the mouse pancreatic amylase gene Amy-2.2 is increased-lo-fold in responseto increasing the carbohydrate content of the diet from 9.6 to 74%. The DNA sequencemediating this responsehas been localized to the 5’ flanking region of the amylase geneby analysis of hybrid constructs in transgeniemice. The results define a 127base pair dietary response unit that includestwo previously describedregulatory elements, an insulin-responsiveelement and a pancreatic enhancer. Fragments containing these two elements alone fail to respond to diet, demonstrating a requirement for additional regulatory sequences.Another mouseamylasegene Amy-2.1 is only minimally responsiveto insulin and to diet. The data are consistent with the hypothesis that the insulin-responseelement is necessary but not sufficient for regulation of amylase by dietary carbohydrate. dietary carbohydrate; transcriptional regulation CELLS of the pancreas produce a variety of digestive enzymes that convert dietary macromolecules to low molecular weight absorbable products. The relative proportion of these digestive enzymes in the pancreatic secretion is influenced by the composition of the diet. Increased intake of starch results in increased synthesis of amylase, whereas a diet high in casein leads to greater synthesis of proteolytic enzymes (8, 11). Similar effects are seen when starch and casein are replaced by their digestion products, glucose or a casein hydrolysate (2, 8). The abundance of amylase messenger RNA in rat pancreas is increased eightfold by maintenance on a high carbohydrate diet (10). The molecular mechanisms responsible for dietary adaptation of the pancreatic enzymes are not well understood. Because of its role in regulating carbohydrate metabolism, insulin has been considered a potential mediator of amylase adaptation to a high carbohydrate diet. Insulin can be demonstrated to be a positive regulator of amylase expression in diabetic rodents. Chemical induction of diabetes results in a drastic reduction of amylase protein and mRNA, reductions that are reversed by administration of insulin (1, 3, 16,22, 27). The response to insulin in streptozotocin-treated diabetic mice appears to be a transcriptional effect, because it can be mediated by the proximal promoter region of the mouse pancreatic amylase gene Amy-2.2 (l&20, 21). The insulin-responsive element has been mapped to the region -158 to -137 of this gene (15). A second mouse gene Amy-2.1 is only slightly responsive to insulin (9, 12). In the present study, transgenic mice carrying constructs with the proximal promoter region of Amy-2.2 fused to the chloramphenicol acetyltransferase (CAT) reporter gene were maintained on various diets. The results demonstrate that response to diet can be mediTHE EXOCRINE

0193-1857/92

$2.00 Copyright

48109-0618

ated by a 127base pair (bp) fragment that includes the insulin responsive element of Amy-2.2. MATERIALSAND

METHODS

Animals. Mice from inbred strain YBR/Ki and transgenic mice were maintained under controlled temperature and light. Transgenic lines were generated by microinjection of (C57BL/6J x C3H)FB fertilized eggs(15, 21), followed by backcrossing with C57BL/6J mice. Transgenic individuals were identified by polymerase chain reaction (PCR) using primers complementary to CAT, asdescribedpreviously (15). Mice had accessto diet and water ad libitum. Diets. Diets were prepared by Purina Mills (St. Louis, MO) according to the compositions described by Giorgi et al. (10; personal communication). The high carbohydrate diet contained (in %) 71.7 dextrin, 15 casein,0.1 DL-methionine, and 4 peanut oil. The intermediate carbohydrate diet, which washigh in protein, contained (in %) 16.2 dextrin, 70 casein,0.6 DL-methionine, and 4 peanut oil. The low carbohydrate diet, which was high in fat, contained (in %) 11 dextrin, 25 casein, 0.15 DL-methionine, and 58 peanut oil. The composition of other components was the following (in %): 2 cu-cellulose,2 vitamin mix, 5 mineral mix no. 10, and 0.2 choline chloride. The calculated composition of the diets, obtained from the supplier, is presentedin Table 1. Mice weighing between 20 and 29 g (3-6 mo of age) were maintained on one of these diets for 14 days. Northern blot analysis. Total cellular RNA was prepared by the method of Chirgwin et al. (5). The integrity of the RNA was evaluated from the appearanceof the 28s and 18s rRNAs on nondenaturing agarosegels.Electrophoresis and transfer were carried out as described (20). Northern blots were probed with ~104, an amylase cDNA clone hybridizing with Amy-2.1 and Amy-2.2 transcripts (12). Probeswere radiolabeledby the random oligomer primed method to a specific activity >lOg counts per minute (cpm)/pg (20). Amylase assay and isozyme analysis. Amylase activity was assayedby a modification of the 3,5-dinitrosalicylate method (7, 13). Half of each pancreaswas homogenizedin 0.5 ml of 44 mM NaPO,, pH 6.9. Ten-microliter aliquots were incubated with 2% potato starch (Sigma no. S 2630) at 30°C for 2 min. The reaction was terminated by the addition of 1 ml of dinitrosalicylate reagent. After heating to 100°C for 10 min, absorbancewas measuredat 530 nm. Duplicate assaysdiffered by ~5%. Amylase isozymeswere separatedby electrophoresison 7.5% polyacrylamide gelsat pH 8.1 as previously described(25). Gels were stained by incubation with 2% starch, followed by staining with KI/12 to detect amylase activity. PCR assay of Amy-2.1 and Amy-2.2 transcripts. Five microgramsof total RNA in 15 ~1were heatedto 70°C and allowedto hybridize with 1 pg of oligo-dTls while cooling to room temperature. Synthesis of cDNA was carried out at 42°C for 60 min with 34 U of avian myeloblastosis virus reverse transcriptase (Seikagaku). Two microliters of this hybridization mixture was subjectedto PCR amplification with primers corresponding to sequencesthat are identical in Amy-2.1 and Amy-2.2 (12) [primer 1 (+1190 to +1219): 5’-CAATGACTGG GTCTGTGAAC ATAGATGGCG; and primer 2 (+1381 to +1410): 5’-TGACATCACA GTATGTGCCA GCAGGAAGAC]. The amplified product from both genesis 221 bp in length.

0 1992 the American

Physiological

Society

G971

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G972

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(Tris), pH 7.8, with a Polytron homogenizer and stored at -70°C. On the day of assay, extracts were heated at 65°C for 10 min and centrifuged. Protein concentration of the supernate was measured with the Bio-Rad reagent (14). CAT activity was assayed in the presence of 8 ~1 of [14C]chloramphenicol (0.1 &i/pl; 60 mCi/mmol) and 0.2 mg of n-butyrl coenzyme A in 188 mM Tris, pH 7.8, and 33 mM EDTA. After incubation at 37°C for 3 h, the aqueous phase was extracted with a 2:l mixture of tetramethylpentadecane:xylene and backextracted once in 0.01 M Tris and 1 mM EDTA. The radioactive product in the organic phase was counted in a scintillation counter. Data were obtained from the linear range of the CAT assay, which extended up to 17,000 cpm or 50% substrate conversion. Background values from substrate alone (20-40 cpm) were subtracted.

Table 1. Calculated dietary composition Diet High

%Carbohydrate %Protein %Fat Preparation of the carbohydrate.

carb

74 4 4 diets

Intermediate

carb

18 64 4 is described in

Low

cab

9.6 23 58 Carb,

METHODS.

OF AMYLASE

Amplification was carried out for 30 cycles with denaturation for 1 min at 96°C and annealing and polymerization for 2 min at 72°C. A 5-~1 aliquot was subjected to a final extension reaction at 72°C in a total volume of 50 ~1 in the presence of primer 2, which had been end labeled with [y-32P]deoxyadenosine 5’-triphosphate (5 x lo6 cpm/reaction) using T4 kinase. The PCR reactions were performed in a programmable thermal controller (PTC-100, MJ Research). The amplified products were digested with 20 U of HiltfI for 2 h at 37°C. Twenty-microliter aliquots were analyzed by electrophoresis on 12% nondenaturing polyacrylamide gels and visualized by direct autoradiography of the dried gels. CAT assay. CAT activity was assayed by a modification of the method described by Seed and Sheen (23). Pancreas was homogenized in 0.25 M tris(hydroxymethyl)aminomethane

RESULTS

Effect of dietary composition on amylase activity and mRNA. The three diets with carbohydrate content

between 9.6 and 74% (Table 1) were previously used in studies of rat pancreatic amylase (10). Mice of inbred strain YBR/Ki were maintained on each diet for a period of 2 wk. Measurement of total amylase in pancreas demonstrated a reduction from 69 + 5 U/mg protein on the high carbohydrate content to 14 + 2 on the low carbohy-

B

A

Diet:

100

I

1

2

3'4

5

6"7

8

9

'

l z l

l -

Hi

Int

Lo

Fig. 1. Effect of diet on pancreatic amylase activity and mRNA. Animals were maintained on various diets for a period of 2 wk. A: amylase activity of pancreatic homogenates was assayed as described in MATERIALS AND METHODS. Each point represents 1 animal. Mean for each group is indicated. B: total pancreatic RNA was prepared from 3 individuals on each diet. RNA was analyzed by Northern blotting and hybridization with an amylase cDNA probe. Each lane represents 1 individual. RNA concentrations are indicated by ethidium bromide stained gel at bottom. Lanes l-3, high carbohydrate diet; lanes 4-6, intermediate carbohydrate diet; lanes 7-9, low carbohydrate diet. Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on January 12, 2019.

DIETARY

REGULATION

drate diet (Fig. 1A). A Northern blot analysis of total pancreatic RNA demonstrated a similar effect on amylase mRNA (Fig. 1B). Both of these assays measure the combined expression of the two amylase genes in strain YBR, Amy-2.1 and Amy-2.2 (12). Differential effects of diet on Amy-2.1 and Amy-2.2. To distinguish the effects on the two amylase genes in YBR mice, the protein products of the two genes were separated by electrophoresis on nondenaturing polyacrylamide gels. On the high carbohydrate diet, the ratio of AMY-2.2: AMY-2.1 is -1.5 (Fig. 2A, lanes 1 and 2, and Ref. 4). After maintenance on the low carbohydrate diet, the ratio of the two isozymes is reversed. We estimate that the product of Amy-2.1 is reduced approximately twofold (cf. Fig. 2, lanes 1 and 2 with lanes 3 and 4 containing twice as much protein), and the product of Amy-2.2 is reduced approximately eightfold (cf. lanes 1 and 2 vs. 5 and 6). The overall fivefold reduction in amylase activity on the low carbohydrate diet (Fig. 1) can thus be accounted for by a twofold reduction in Amy-2.1 and an eightfold reduction of Amy-2.2. The response of Amy-2.2 to diet is comparable to that described for rat amylase (10). To determine whether the transcripts of Amy-2.1 and Amy-2.2 are differentially affected by diet, we developed a gene-specific assay based on the HinfI site, which is present in exon i of Amy-2.1 but not in the corresponding exon of Amy-2.2 (12). Both amylase transcripts were

G973

OF AMYLASE

amplified from total pancreatic RNA with primers complementary to sequences that are identical in the two genes. The amplified PCR products were subjected to a final extension cycle in the presence of an end-labeled primer and were then digested with HinfI. The resulting end-labeled fragments were separated by electrophoresis on polyacrylamide gels. The final Amy-2.2 product is 221 bp in length, and the Amy-2.1 product is 82 bp in length (Fig. 2, lanes 11 and 12). In mice maintained on the high carbohydrate diet, the majority of transcripts are derived from Amy-2.2 (lanes 7 and 8). On the low carbohydrate diet, the Amy-2.1 transcript is more abundant (lanes 9 and 10). These changes are qualitatively consistent with isozyme changes noted above. Regulation of hybrid genes in transgenic mice. To determine whether dietary response is the result of transcriptional regulation, we analyzed the expression of an Amy-2.2/CAT hybrid gene containing the Amy-2.2 proximal promoter region (-208 through +19). Mice of transgenie line 6833, previously shown to express this construct from the normal transcription start site (21), were maintained on the high or low carbohydrate diet for 2 wk. At the end of this period, pancreatic CAT activity of the two groups differed by fourteenfold (Table 2). This result demonstrates that c&acting sequences in the amylase proximal promoter can regulate the response to diet. To further localize the regulatory sequences, we studied three subfragments of the 228-bp amylase sequence,

A Diet:

Hi Lo 12”34”5

LO

- 221 bp (2.2) AmY

AmY

Protein:

IL-lid lx

. 2x

E

- 82 bp (2.1)

8x

Fig. 2. Differential regulation of Amy-2.1 and Amy-2.2. A: protein products of the 2 pancreatic amylase genes were separated by electrophoresis on nondenaturing polyacrylamide gels and visualized with an amylase activity stain. Lanes 1 and 2: 0.2 pg protein; lanes 3 and 4: 0.4 pg protein; lanes 5 and 6: 1.6 Kg protein. Relative amount of protein is indicated below gel. Each lane contains pancreatic protein from a different animal. B: relative abundance of transcripts of the 2 genes was determined by HinfZ digestion of end-labeled polymerase chain reaction amplification products (see METHODS). Lanes 7-10 contain amplified products from 4 different animals. Lanes 11 and 12 contain standards prepared by amplification of cloned Amy-2.1 and Amy-2.2 cDNAs (12). Hi, high carbohydrate diet; Lo, low carbohydrate diet. Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (129.081.226.078) on January 12, 2019.

G974

DIETARY

Table 2. Pancreatic

CAT activity

Trangenic Line

in transgenic

-208 -208

CAT High

to +19 to -82

-208

Amy

SZ

Diet

-172

CAT

#-

+

-I-

-

-110

3. 167

-138 [

4. -205

CAT

1

*

a22

Fig. 3. Hybrid constructs expressed in pancreas of transgenic mice. Proximal promoter sequences from Amy-2.2 (solid boxes) and from rat elastase 1 (open boxes) were ligated to the chloramphenicol acetyltransferase (CAT) gene (14). The rat elastase 1 promoter extends from -205 to +22 (15, 17). These constructs were previously shown to be expressed in pancreas of transgenic mice and to be regulated by insulin (15). Sz, streptozotocin.

which were ligated to the heterologous pancreas-specific elastase 1 promoter (Fig. 3). These constructs were previously shown to be expressed in pancreas of transgenic mice and to respond to streptozotocin independently of insertion site (15). The expression of the construct with the fragment -208/-82 differed by 20-fold on the two diets (Table 2). However, constructs with the smaller fragments -172/-110 and -167/-138 were expressed at comparable levels on the two diets. These results demonstrate that dietary regulation of pancreatic amylase can be mediated by transcriptional regulatory sequences located in the 5’ flanking region of the Amy-22 gene and that the essential sequences are located between nucleotides -208 and -82. DISCUSSION

Regulation of amylase both by diet and by insulin was demonstrated first in rat pancreas, which appears to express a single type of amylase gene. Total rat pancreatic amylase is reduced approximately eightfold on a low carbohydrate diet (10) and lOO-fold in diabetic animals (16). In contrast, the mouse genome contains two pancreatic amylase gene copies, Amy-2.1 and Amy-2.2, which differ in their regulation. We previously demonstrated that Amy-2.2 responds to insulin like the rat pancreatic amylase gene, whereas Amy-2.1 is only slightly responsive (9, 20). The present study demonstrates that Amy-2.2 responds to changes in the carbohydrate content of the diet in the same manner as the rat pancreatic amylase gene. An eightfold difference in Amy-2.2 protein and mRNA concentration was observed when the carbohy-

U/mg

n

760tl70 12Ok50 of the chloramphenicol

-82 1

Activity,

12,900+2,400 1,100+250

-172 to -110 -167 to -138 of mice. Structures

Elastase

on high or low carbohydrate

carbohydrate

1.

2.

OF AMYLASE

mice maintained

Amylase Sequence

Regulated constructs Tg6833 Tg6208 Nonregulated constructs Tg5428 Tg287 Values are means t SD; n = no.

REGULATION

diet

protein

Low

carbohydrate

Fold Increase on High Carbohydrate

n

6 5

890t170 55t6

6 5

7 4 acyltransferase

430tl20 lOOt40 (CAT)

7 5 constructs

xl4 x20

are represented

x2 xl in Fig. 3.

drate content of the diet was varied from 9.6 to 74%. The expression of Amy-2.1 varied by less than twofold in the same animals. Transgenic mice with an amylase CAT construct driven by the amylase proximal promoter region (-208 to +19) produced fourteenfold more CAT when fed the high carbohydrate diet, demonstrating that transcriptional regulation rather than mRNA stabilization is responsible for the increased abundance of amylase mRNA after intake of a high carbohydrate diet. To localize the element responsible for this transcriptional effect, we tested three constructs in which amylase flanking sequences were placed upstream of the heterologous pancreatic elastase promoter. One of these constructs, with amylase sequences -208 to -82, retained the dietary response. The 127-bp amylase sequence in this construct may be considered a functional dietary response unit. Fragments with only 63 or 30 bp of the dietary response unit were not regulated, indicating that the size of the minimal dietary response unit is between 63 and 127 bp. The lack of regulation of the two smaller fragments also demonstrates that the elastase promoter itself does not respond to the difference in composition of these two diets. In previous experiments, we demonstrated that the expression of the transgenes in all of these lines was independent of insertion site (15). It is striking that the dietary response unit and the previously defined minimal insulin response element are both located within the same 126-bp fragment (Fig. 4). Both the 63-bp and the 3O-bp amylase fragments were capable of mediating the insulin response (15) but were not sufficient to mediate the diet response (this study). The close physical association of the two functional elements is consistent with a role for insulin in mediating dietary response. The dietary response unit also includes a binding site for the pancreatic nuclear protein PTFI (Fig. 4), which

PTFI

I

I

-200

? I -100

-+I

DRU iI -82

I -228 I -167

Fig. 4. Regulatory Amy-2.2. PTFl, response element

-138

elements upstream of mouse pancreatic amylase gene pancreatic transcription factor 1 (14); IRE, insulin (15); DRU, dietary response unit (this paper).

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DIETARY

REGULATION

binds a consensus element common to genes expressed in the acinar cells of the pancreas (6, 14). Because PTFl is thought to coordinately regulate amylase, ribonuclease, and the proteases, it is not likely to be directly responsible for the response to dietary carbohydrate, which is specific for the amylase gene. We have identified another protein that binds within the dietary response unit, but we have not detected any difference in binding activity of this protein in response to diet (H. Yu and M. Meisler, unpublished observations). The sequencesof Amy-2.1 and Amy-2.2 differ by 40% (52/127) within the dietary response unit (20). The Amy-Z.1 gene is known to be less responsive to insulin than the Amy-2.2 gene (9, 12, 20). In the present study, the Amy-2.1 gene was also found to be less responsive to changes in diet. This coincident loss of regulation by diet and by insulin is consistent with the possibility that the insulin element is a functional component of the dietary response unit. Definitive evidence of the role of insulin in dietary response will depend on identification of transacting factors, which mediate the two regulatory pathways. Three other genes, L-pyruvate kinase, phosphoenolpyruvate carboxykinase (PEPCK), and glyceraldehyde phosphate dehydrogenase have been studied by similar methods to localize diet response elements. In transgenic mice, 3 kb of the pyruvate kinase 5’ flanking region is sufficient for response to high carbohydrate diet, glucose, and insulin (26, 28). The carbohydrate response element of this gene was localized to the region -197 to -96 by transfection of cultured hepatocytes (24). In transgenic mice expressing a construct with 460 bp of 5’ flanking sequences from PEPCK, treatment with a high carbohydrate diet resulted in >lO-fold reduction of circulating bovine growth hormone (18). The abundance of a nuclear protein, which binds the glyceraldehyde dehydrogenase gene promoter was also reported to be influenced by diet (19). All three of these diet-responsive genes are also regulated by insulin. Furthermore, the insulin response of L-pyruvate kinase was localized to the same 101 bp as the carbohydrate response (24). Our results with Amy-2.2 also demonstrate a close physical association of elements mediating response to diet and to insulin. This striking pattern of association strongly suggests that insulin is responses to dietary involved in transcriptional carbohydrate. We thank Thomas M. Johnson for the protocol for assay of CAT activity in pancreatic homogenates, Linda C. Samuelson for helpful discussions, J. C. Dagorn for detailed description of the diets, and Jane Santoro for expert manuscript preparation. This work was supported by the National Institute of General Medical Sciences Grant GM-24872. R. M. Schmid was supported by DFG Fellowship 740/2- 1. Present address of R. M. Schmid: Howard Hughes Medical Institute, MSRB I, University of Michigan Medical Center, Ann Arbor, MI 48109. Address for reprint requests: M. H. Meisler, Dept. of Human Genetics, Univ. of Michigan, Ann Arbor, MI 48109-0618. Received

2 December

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