Copyright 0 1986 by the Genetics Society of America

GLUCOSE REPRESSION OF AMYLASE GENE EXPRESSION IN DROSOPHILA MELANOGASTER

BERNHARD F. BENKEL AND DONAL A. HICKEY

Deflartment of Biology, University of Ottawa, Ottawa, Ontario, Canada K I N 6N5

Manuscript received June 7, 1985 Revised copy accepted May 23, 1986

ABSTRACT We have previously shown that dietary glucose can reduce amylase activity in

both adults and larvae of Drosophila; this reduction in enzyme activity reflects a reduction in the quantity of amylase protein, rather than an inhibition of enzyme activity. Here, we report that we have now defined conditions in which the repressive effect of glucose can be greater than 100-fold. Moreover, this repression is partially counteracted by the addition of exogenous cyclic AMP. We also show that there is a direct correlation between changes in amylase activity and changes in the amount of translatable mRNA as assayed in microin- jected Xenopus oocytes. This means that the glucose repression is occurring at a pretranslational stage.

ROSOPHILA amylases provide an excellent model system for the study D of many aspects of gene regulation in a higher eukaryote. Here, we focus on the regulatory effects of dietary glucose on amylase gene expression.

While there are some instances of glucose-mediated repression of transcrip- tion in lower eukaryotes (LEE and FREE 1984; BEIER and YOUNG 1982; FED- EROFF, ECCLESHALL and MARMUR 1983), in higher eukaryotes the modulation of gene expression by environmental parameters has only recently been dis- covered. Moreover, in the higher eukaryotic adaptive regulatory systems so far described, the magnitude of the response is usually much smaller than in typical prokaryote systems (NOGUCHI et al. 1983; GIORGI et al. 1984; GEER and LAURIE-AHLBERG 1984), and in addition, for some systems the physiological significance of the gene product is not known (LIN and LEE 1984). In contrast, D. melanogaster amylase activity is easily measured by a variety of methods; it represents an enzymatic function that has a specific, well-recognized physiolog- ical role and that is affected by the carbohydrate components of the diet (ABE 1958; DOANE 1969; HOSBACH, EGG and KUBLI 1972; HICKEY 1977; HOORN and SCHARLOO 1978; YAMAZAKI and MATSUO 1984). We and others have shown that the specific effect is one of glucose repression, rather than induc- tion by dietary starch (HICKEY and BENKEL 1982; ECHO and DOANE 1984).

In this report, we show that the degree of glucose repression can be several hundred fold in some instances. Moreover, we show that the “glucose effect” occurs at a pretranslational stage. Finally, this effect can be partially counter- Genetics 114 137-144 September, 1986

138 B. F. BENKEL AND D. A. HICKEY

acted by the addition of exogenous cyclic AMP. So far, we have not been able to identify any “inducer” of Drosophila amylase expression.

MATERIALS AND METHODS

To determine the sensitivity of D. melanogaster larvae to dietary glucose, we trans- ferred mature adults of two common wild-type strains, Oregon-R and Canton-S, to 8- dram shell vials containing 10 ml each of either of the two test diets for egg laying. The control diet consisted of 5% killed brewers’ yeast (w/v), 0.75% agar (w/v) and 0.8% propionic acid (v/v) in distilled water. The “glucose” diet consisted of 10% glucose (w/v), 5% killed brewers’ yeast (w/v), 0.75% agar (w/v) and 0.8% propionic acid (v/v) in distilled water. These two diets are identical except that one of them (“glucose medium”) contains 10% added dextrose, whereas the other (“sugar-free medium”) lacks this component. Larvae were allowed to grow to the late third instar stage and were harvested by washing them from the low agar foods with distilled water.

Mass homogenates of larvae grown on the two test media were prepared. The water- soluble fractions were electrophoresed, and the gels were stained for amylase activity as described by HICKEY (1981). Electrophoresis was carried out at 200 V for 2 hr. Following electrophoresis the gel was incubated in a 2% starch solution in 0.1 M Tris HCI, pH 7.4, 20 mM CaCI, for 1 hr at room temperature and was stained with iodine potassium iodide reagent. In order to visualize the faint glucose-treatment bands, gels were incubated in 0.1 M Tris HCI, pH 7.4, 20 mM CaCh without starch at 37”, following incubation in a starch solution, but before staining with the I-KI reagent. Homogenates were also tested for amylase activity in cuvettes using the DNSA (3,5- dinitrosalicylic acid) assay (BERNFELD 1955).

Sensitivity to CAMP was tested by growing larvae under repressing conditions, as already described, and adding 50 mg of CAMP in 250 PI of sterile water, per vial, 24 hr before harvesting the larvae. Controls were treated with either 50 mg of AMP in water or with water alone.

Total RNA was extracted from third instar larvae by a modification of the guanidine- HCI method of COX (1968). RNA was heated to 65” for 5 min in gel loading buffer containing SDS, then 6 pg per lane was loaded onto a 1.5% native agarose gel in 0.1 M Tris borate, pH 8.9, and was run for 2 hr at 80 V (SPARKS 1985). The ability of RNA from larvae to direct the synthesis of fly amylase was assayed by injecting total RNA into the vegetal hemispheres of XenoFpus laevis oocytes according to GURDON (1977). RNA was injected in sterile water at 10 mg/ml (50-100 nl per oocyte). Follow- ing injection, oocytes were incubated individually in microtiter plates containing sterile Barth’s medium for 3 days in the dark at 21 ’. Mass homogenates were prepared from 20-30 oocytes including incubation media and were brought to 0.1 M with Tris borate, pH 8.9, and 5% with sucrose, and five oocyte equivalents were loaded on a 5% poly- acrylamide gel in 0.1 M Tris borate, pH 8.9.

RESULTS

Oregon-R larvae show a dramatic response to dietary glucose [only trace activity on glucose medium (see Figure la, lane pairs 1 and 2)]. In order to quantify activity differences on electrophoretic gels, the high activity homog- enate was diluted until the band intensity of the diluted high activity homog- enate was about equal t o the band intensity of the low activity homogenate. Similar dilution series were used for the DNSA assays. In all cases, an Amy null strain was used to determine background activity values. In the gel assay, Oregon-R larvae show a difference of about 200X between the two dietary treatments, whereas for Canton-S the difference is approximately 5 0 X (Table

GLUCOSE REPRESSION OF AMYLASES 139

a

AMY'

I 2 3 4

AMY'

5 6 7 8 FIGURE 1 .-Effect of glucose and cAMP on amylase activity in D. mrlanogasttr larvae. a, Glucose

represses amylase activity in Oregon-R larvae: Lane pair 1, larvae grown on glucose medium; 2, larvae grown on no-sugar-added medium; S. IOX dilution of 2; 4, IOOX dilution of 2. Samples were equilibrated for protein content with the LOWRY assay (LOWRY. ROSEBROUGH and RANDALL 195 I) and Coomassic blue-stained polyacrylamide gels before activity comparisons. b, Dietary cAMP counteracts the glucose effect; Lane pair 5. larvae grown on glucose medium; 6. larvae grown on glucose and treated with cAMP as described in the text; 7, 4X dilution of 6; 8, 16X dilution of 6. Lane pairs 1 and 2 in (a) and 5 and 6 in (b) contain approximately two larval equivalents each from mass homogenates of several hundred late third instar larvae.

1). The DNSA assay consistently underestimates differences between the die- tary treatments, in comparison to optical, integrated densitometer scans of gel negatives, yielding difference estimates of 140X for Oregon-R and 33X for Canton-S. This is because a nonspecific activity that can be measured in Amy null larvae is comparable to the trace amounts of activity detected in glucose- treated Oregon-R larvae. Both assays show, however, that the repression effect on the Oregon-R strain is greater than 100-fold, and the effect on the Canton- S strain is about onequarter of this amount.

140 B. F. BENKEL AND D. A. HICKEY

TABLE 1

Effect of dietary glucose and glucose + cAMP on amylase activity in D. melanogastet- larvae

Dietary treatment

Drosophila No sugar strain Assay method added Glucose Gluc + cAMP Gluc + AMP

Oregon-R DNSA assay 100 0.7 f 0.1 6.3 f 0.2 1.3 f 0.1 Gel scan 100 0.5 f 0.1 7.9 f 0.1 1.3 f 0.2

Canton-S DNSA assay 100 3.2 f 0.4 Gel scan 100 1.9 f 0.3 18.7 _+ 0.6 2.2 f 0.2

All values are shown as a percentage of no-sugar-added activity, mean +. SE of three replicates from mass homogenate for DNSA and mean f SE of two lanes from mass homogenate for gel scan.

We have previously shown that the modulation of amylase activity by dietary sugar in D. melanogaster involves a concomitant, parallel change in the amount of amylase protein (HICKEY and BENKEL 1982). In order to determine whether control of amylase enzyme abundance is post- or pretranslational, we extracted RNA from Oregon-R larvae grown under both dietary conditions and injected total Drosophila RNA into Xenopus oocytes. The oocytes provide a conven- ient, prepackaged translation system that is ideal for detecting low abundance messages (GURDON et a l . 1971) and has been used previously to monitor D. melanogaster RNA-directed amylase production (DOANE et al . 1983; BENKEL and HICKEY 1984). Total RNA from Oregon-R larvae grown on glucose me- dium did not direct the synthesis of any detectable D. melanogaster amylase (Figure 2b, lane 6). In contrast, a strong amylase electrophoretic band resulted from the injection of RNA from larvae fed the control, sugar-free medium (Figure 2b, lane 7). This activity comigrates with the Oregon-R Amy electro- phoretic band (Figure 2b, lane 4). The amylase standard in Figure 2b, lane 4, consists of a 50:50 mixture of Oregon-R (Amy’) and Belinga (Amy6) larval homogenates. The difference in translational activity was not due to degrada- tion of the RNA from glucose-treated larvae (see Figure 2a, lanes 2 and 3), nor was it due to batch-to-batch variation in the translational efficiency of oocytes (tested by co-injecting oocytes with RNA from an Amy6 strain that programs the synthesis of an electrophoretically distinguishable fly amylase band as an internal control, data not shown). Therefore, the observed differ- ence is due to the specific translational activities of the RNA samples, which demonstrates that modulation of amylase activity in Oregon-R larvae in re- sponse to dietary sugar involves regulation at a pretranslational level.

There are numerous examples of prokaryotic operons that show the “glucose effect.” A common feature of the regulation of these gene-enzyme systems is the interaction of cAMP (adenosine 3’:5‘-cyclic monophosphate) with genetic regulatory elements upstream of the structural genes (PASTAN and ADHYA 1976). Cyclic AMP has also been shown to increase transcriptional activity of several genes in higher eukaryotes (MURDOCH, ROSENFELD and EVANS 1982; HASHIMOTO, SCHMID and SHUETZ 1984; WILLIAMS, TSAND and MAHBUBANI

GLUCOSE REPRESSION OF AMYLASFS 141

a

23s- 16s-

b

A M Y ~

AMY'

I 2 3 4 5 6 7 FIGURE 2.-Pretranslational regulation of amylase activity in Oregon-R larvae. a. RNA integrity:

Lane 1. bacterial 23 S a n d 16 S rRNA standards; lane 2, total RNA from Oregon-R larvae grown o n glucose medium; lane 3. total RNA from Oregon-R larvae grown o n no-sugr-added medium. b. Translational activity o f Oregon-R RNAs: Lane 4. amylase standard (mixed homogenate o f Oregon-R. Amy', a n d Belinga. Amy); lane 5. oocytes injected with Barth's medium; lane 6, oocytes injected with total RNA from Oregon-R larvae grown o n glucose medium; lane 7, oocytes injected with total RNA from Oregon-R larvae grown o n no-sugar-added medium.

1980). Since amylase activity in D. melanoguster also shows the "glucose effect" and activity regulation takes place at a pretranslational (possibly transcriptional) level, we tested larvae of both strains for their response to dietary cAMP (Table 1). According to the gel assay, cAMP treatment restores activity to approximately 20% of the derepressed level in Canton-S larvae. This repre- sents an increase of roughly 1OX over the repressed activity level (Table 1). In Oregon-R the counteractive effect of cAMP is more pronounced (15X); however, due to the sensitivity of this strain to glucose, the amylase activity level of cAMP treated larvae is still well below that of derepressed larvae (see Table 1 and Figure 1 b). As a control, AMP was also added to larval diets; this dietary supplement did not have the same stimulatory effect on amylase activity as did cyclic AMP (Table 1).

DISCUSSION

The results presented here show that amylase activity in D. melanogaster larvae can be dramatically repressed simply by adding glucose to their diet and that the magnitude of the response is both large and strain-specific. The sim- plest explanation for this pretranslational regulation that can be partially coun- teracted by dietary cAMP is regulation at the level of transcriptional activity at the amylase region. The significance of this result is that glucose repression, although well known in prokaryotic and lower eukaryotic systems, has not

142 B. F. BENKEL AND D. A. HICKEY

previously been described for a Drosophila gene. In fact, there are very few examples of negatively regulated higher eukaryote genes (REYNOLDS et al . 1984).

We have recently cloned an amylase cDNA, and we have used this clone as a molecular probe on Northern blots of RNA from repressed and derepressed larval homogenates. Our preliminary results for such hybridizations indicate that the glucose repression is at the level of mRNA abundance ( B . BENKEL and D. A. HICKEY, unpublished results). Genomic sequences for D. melanogaster amylase have also been cloned (GEMMILL, LEVY and DOANE 1985; LEVY, GEM- MILL and DOANE 1985). The use of these sequences in conjunction with an Amy null strain isolated in our laboratory (HAJ-AHMED and HICKEY 1982), and the powerful P-element-mediated transformation system of RUBIN and SPRA- DLING (1 982) should allow us to identify the genetic regulatory elements that are responsible for the “glucose effect” and to determine the differences, be- tween these elements, that account for the range of sensitivities that we see in nature. Upstream regulatory elements that are involved in the glucose repres- sion of a mammalian gene have recently been identified (ATTENELLO and LEE 1984). The amylase system in D. melanogaster is of special interest because it displays genetically controlled patterns of tissue-specific expression (ABRAHAM and DOANE 1978), in addition to the diet-induced effects described here.

As the molecular details of this regulatory system are dissected out, we shall not only learn more about gene regulation in higher eukaryotes but also shall be in a better position to evaluate the degree of evolutionary divergence be- tween physiologically comparable eukaryotic and prokaryotic gene regulatory systems. For instance, two recent reports (STRUHL 1985; SAROKIN and CARLSON 1985) indicate that glucose repression of gene expression in lower eukaryotes is effected by molecular mechanisms that are distinct from the well-known prokaryotic systems. It will be of interest to ask whether the molecular mech- anism in Drosophila is similar to the lower eukaryotic system, or whether it involves yet a third distinct mechanism of glucose repression.

We thank JEAN DAVID and the Mid-America Drosophila Stock Center for supplying Drosophila stocks. We are grateful to ART BUCHBERG for advice on the Xenopus oocyte injections. Technical assistance was provided by K. BENKEL, C. MAGOULAS and Y. GENET. This work was supported by an operating grant from the Natural Sciences and Engineering Research Council of Canada (to D.A.H.) and by an Ontario Graduate Scholarship (to B.F.B.).

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