©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Function of the Human Insulin Promoter in Primary Cultured Islet Cells (*)

(Received for publication, April 26, 1995; and in revised form, October 10, 1995)

Hiroki Odagiri Juehu Wang Michael S. German (1)(§)

From the Hormone Research Institute and Department of Medicine, University of California at San Francisco, San Francisco, California 94143-0534

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Pancreatic islet beta cells regulate the rate of insulin gene transcription in response to a number of nutrients, the most potent of which is glucose. To test for its regulation by glucose, the promoter sequence was isolated from the human insulin gene. When linked to chloramphenicol acetyltransferase and transfected into primary islet cultures, the human insulin promoter is activated by glucose. In parallel islet transfections, glucose also activates the L-pyruvate kinase and islet amyloid polypeptide promoters and represses the branched chain ketoacid dehydrogenase E1a promoter, but it does not affect the beta cell glucose kinase promoter. Using deletion and substitution mutations of the proximal human insulin promoter, we mapped a metabolic response element to the E box, E1, at -100 base pairs relative to the transcription start site. Although the isolated E1 element responds to glucose, inclusion of either of two AT-rich sequences, A1 or A2/C1 on either side of E1, results in dramatic synergistic activation. Inclusion of A2/C1 also increases the response to glucose. The A2-E1-A1 region alone, however, does not explain all of the activity of the human insulin promoter in cultured islets, and other transcriptionally important elements likely contribute to the glucose response as well.


INTRODUCTION

The beta cells in the pancreatic islets of Langerhans play a central role in energy metabolism in vertebrates. The beta cells gauge the feeding state of the organism through hormonal and neural signals and by sensing the systemic levels of nutrients, especially glucose. Glucose is catabolized by the beta cells, and it is the changing levels of the products of glucose catabolism that signal the beta cells to respond to changes in glucose concentration. beta cell metabolism, therefore, is precisely regulated to respond proportionally to glucose levels in the physiologic range and to distinguish glucose from other nutrients. The beta cells then respond by modulating insulin synthesis and secretion.

Part of the synthetic response to glucose results from an increase in insulin gene transcription directed by the insulin gene promoter(1) . The insulin promoter consists of the untranscribed regulatory sequences immediately upstream of the transcription start site. In addition to responding to nutrients, the promoter also determines the correct transcription start site (2, 3) and limits expression of linked genes to the beta cell(2, 3, 4, 5, 6, 7, 8) . A series of distinct sequence elements along its length and the nuclear proteins that bind these elements interact to produce the characteristic functions of the insulin promoter. Studies in insulin-producing tumor cell lines have defined many of the sequence elements in the rat insulin I (rInsI)(9) , rat insulin II (rInsII)(7, 10) , and human insulin (hIns) (11) promoters. The positions of some of the well defined insulin promoter elements are shown in Fig. 1. The names of these elements have recently been simplified(12) .


Figure 1: Insulin promoter sequence elements. The names used in this manuscript are shown in boxes. Some of the older names used for the same elements in the human insulin, rat insulin I, and rat insulin II promoters are shown below the boxes.



The E elements are related to the E box sequences found in other genes (13) and bind several members of the basic helix-loop-helix class of transcription factors. In beta cell nuclei, the E elements bind a major protein complex formed by the ubiquitous E2A basic helix-loop-helix proteins, Pan1 and Pan2, and another protein of limited distribution (14, 15, 16, 17, 18) . The A elements generally contain an A/T-rich core sequence. Some of the A elements have been shown to bind members of the homeodomain class of transcription factors(19, 20, 21, 22, 23, 24) .

The functional elements that line the insulin promoter do not work in isolation; they synergistically interact to produce the full activity of the promoter. For example, the rInsI A3 and A4 elements synergize with the adjacent E2 element(25, 26) ; and in the rInsII promoter, the overlapping A2/C1 elements synergize with the E1 element(27) . This synergy reflects interactions between transcription factors such as is the case for lmx1 and Pan1, which bind to the A3/A4 and E2 sites, respectively, on the rInsI promoter(20) .

By transfecting insulin promoter constructs into primary cultured islet cells, we have shown previously that both the rInsI promoter and the rInsI E2A3/4 mini-enhancer can respond to glucose. In this system, the E2 site alone can respond to glucose, and binding of the Pan1- and Pan2-containing complex to E2 increases in response to glucose(28) . Interestingly, other investigators have mapped glucose responsiveness to other elements: the rInsI A3/4 elements(29) , the hIns A3 element (29, 30) , and the rInsII A2 element(31) . Probably several elements within the insulin promoter combine to give the full glucose response of the intact promoter(28) . In the present study we systematically test the hIns E and A elements for their ability to synergize and respond to glucose.


MATERIALS AND METHODS

Plasmid Constructions

Construction of pFOXCAT1 has been described previously(28) . The human insulin promoter constructs were derived from the HI1 genomic DNA(32) . The -1373D promoter was created by replacing the ILPR, (^1)-851 to -363 bp, with a synthetic XbaI site.

The pFOXCAT2 plasmid was derived from pFOXCAT1 by inserting the human beta-globin gene intron between the polylinker sequence and the 5`-end of the CAT gene. This results in a significant increase in CAT activity in transfected cells. The minienhancer multimers were constructed as described previously(26) . All of the minienhancers, except for RSV, contain five copies of the mini-enhancer inserted upstream of the -85-bp minimal promoter from rIns I in pFOXCAT2. The Rous sarcoma virus construct has only a single copy of the enhancer sequence from the Rous sarcoma virus long terminal repeat (28) inserted upstream of the -85-bp minimal promoter from rIns I in pFOXCAT2.

All of the other beta cell promoters were inserted into the polylinker in pFOXCAT1. The rat beta cell glucokinase -1000 promoter extends from -1000 to +14 bp and was a gift from M. Magnuson, Vanderbilt University (33) ; the rat L-pyruvate kinase promoters extend from the 5`-ends shown to +11 bp and were a gift of A. Kahn, INSERM, Paris, France(34) ; the human IAPP promoters extend from the 5`-ends shown to +1 bp on the 3`-end and were constructed from a genomic clone provided by D. Steiner, University of Chicago(35) , using available restriction sites; the rat branched chain ketoacid dehydrogenase A promoters extend from the 5`-ends shown to +1 bp and were obtained by polymerase chain reaction using rat genomic DNA and primers based on the published sequence(36) .

Sequences of all plasmid constructs were verified by the Sanger dideoxy sequencing method.

Islet Transfections

21-day gestation fetal Sprague-Dawley rat islets were isolated and transfected as described previously(1) . Each 10-cm plate of cultured islet cells was transfected with 25 µg of double cesium chloride-purified CAT plasmid DNA. The transfected cells were grown in RPMI 1640 media with the glucose concentration shown for approximately 36 h prior to harvesting and protein extraction. CAT enzyme assays were performed with 10 µg of protein for 2 h(3) .

Numbering for hIns Promoter

A sequencing error resulted in a two-base pair deletion at -87 bp in the proximal promoter in the original published sequence of HI1(32) . As a result, the positions reported for the 5`-ends of the hIns promoter in previous studies that used this clone (2, 11) are inaccurate. Compared with these previous studies(2, 11) , the promoter names used here are two base pairs longer.

The exact ends of the ILPR depend on how the repeats are defined, ACAGGGGTGTGGGG or GGGGTGTGGGGACA. We chose to include the final ACA on the 3`-end with the ILPR, and therefore defined the 3`-end as -363 bp.


RESULTS

5`-Promoter Deletions

We made a series of deletions from the 5`-end of the human insulin promoter (Fig. 2c) starting with the -1373 to +1 bp fragment from the original HI1 genomic clone (32) and linked these to the chloramphenicol acetyltransferase (CAT) gene in the pFOXCAT1 plasmid(28) . The CAT plasmids were transfected into primary cultured fetal rat islet cells by electroporation as described previously(1) . The cells were harvested 36 h later and assayed for CAT activity (Fig. 2a).


Figure 2: Function of the truncated human insulin promoters in primary cultured beta cells. A and B, cultured fetal rat islets were transfected with the truncated human insulin promoter-CAT plasmids shown and grown in 2 mM (solid bars) or 16 mM (hatched bars) glucose. CAT enzyme activity was assayed with equal amounts of protein 36 h after transfection. Each data point represents the mean of at least three independent transfections. CAT activity of cultures transfected with the promoterless pFOXCAT1 plasmid and grown in 2 mM glucose was arbitrarily set at 1.0. C, the positions of the 5` deletions and the construction of -1373D are shown.



The deletion series demonstrates several areas that contribute to the overall transcriptional activity of the promoter. Removal of the distal promoter from -1373 to -882 bp, the ILPR (see below) from -882 to -362 bp, and the short sequence from -362 to -341 bp all result in modest decreases in activity. The two largest percentage drops in activity result from removal of the sequences from -341 to -260 and -233 to -171 bp.

The sequence from -341 to -260 contains three previously characterized sequence elements: A5 (-323 to -314)(37) , the enhancer core element (-317 to -309)(9, 28, 38, 39) , and the negative regulatory element (-281 to -260)(11) . The sequence from -233 to -171 bp also contains several previously characterized elements: the A3 element, two potential cyclic AMP response elements(40) , and the CCAAT element. The remaining -171 to +1 promoter has only weak transcriptional activity, but the relative contribution of these proximal sequences are difficult to gauge without 3` deletions or selective mutation in the context of the intact promoter.

All of the truncated promoters retain the ability to respond to glucose (Table 1). The -171 deletion causes a marked reduction in the -fold stimulation by glucose, but the overall activity of this construct is very low; activity at 2 mM glucose is only 50% greater than the activity of the promoterless pFOXCAT1 vector. If the background activity of the promoterless vector is subtracted, the recalculated -fold stimulation of the -171 promoter by glucose is 4.0 ± 0.6, similar to the other promoters.



Distal Promoter Elements

The sequences immediately upstream from -362 bp comprise the ILPR. This region consists of tandem repeats of a G-rich 14-bp sequence(41) . The number of repeats, and therefore the length of the ILPR, varies considerably within the population. In the U. S. population, most ILPR alleles fall into one of two size classes: class 1, short ILPR with an average of 40 repeats (560 bp); or class 3, long ILPR with an average of 150 repeats (2100 bp)(42) . Class 1 alleles have been associated with increased risk of type 1 (autoimmune) diabetes mellitus(43) .

The allele used for the deletion experiments was the original human insulin genomic clone HI1 isolated by Bell and co-workers(32) , which contains 34 repeats totaling 486 bp(41) , making it a class 1 allele. Because the ILPR from the HI1 allele appears to have some modest transcriptional activity (Fig. 2a), we tested the class 3 ILPR from HI3, isolated by Owerbach and Aagaard(44) . Activity of the two alleles was compared to the -1373 construct with the entire ILPR replaced with a single XbaI site (the 1373D promoter, Fig. 2c). Fig. 2b demonstrates that the class 3 allele HI13 is a more potent activator than the class 1 allele HI1. Other studies on the ILPR support this view(45) .

Substitution of the longer ILPR may increase overall activity of the promoter, but it does not increase the response to glucose (Table 1). In fact, the response to glucose appears to decrease (p value as calculated by paired Student's t test <0.1 for -2850 versus -1373 promoter and <0.05 for -2850 versus -1373D promoter), suggesting that the ILPR may not respond to glucose.

The sequence upstream of the ILPR, -1373 to -882, also appears to contribute to the overall activity of the promoter (Fig. 2a). To test whether these sequences could function independently as an enhancer, we linked the 491-bp fragment to the beta cell-specific promoter from the human islet amyloid polypeptide (hIAPP) gene(35) . The distal sequences can activate in this context (Fig. 3a), but the effect is small.


Figure 3: Function of chimeric and mutated insulin promoters. A, cultured fetal rat islets were transfected with the chimeric insulin-hIAPP promoter-CAT plasmids shown and grown in 2 mM (solid bars) or 16 mM (hatched bars) glucose. CAT enzyme activity was assayed with equal amounts of protein 36 h after transfection. Each data point represents the mean of four independent transfections. CAT activity of cultures transfected with the promoterless pFOXCAT1 plasmid and grown in 2 mM glucose was arbitrarily set at 1.0. B, cultured fetal rat islets were transfected with the wild-type and G1-mutant human insulin promoter-CAT plasmids shown and grown in 2 mM (solid bars) or 16 mM (hatched bars) glucose. CAT enzyme activity was assayed with equal amounts of protein 36 h after transfection. Each data point represents the mean of five independent transfections. CAT activity of cultures transfected with the promoterless pFOXCAT1 plasmid and grown in 2 mM glucose was arbitrarily set at 1.0. C, construction of the hIns-hIAPP promoters is demonstrated.



G1 Element

Mutation of the G1 element in the rInsI promoter causes a dramatic decrease in promoter activity in cultured islet cells (28) . To test if the human G1 element is similarly critical, we used site-directed mutagenesis to create a 10-bp replacement mutation of the G1 element. In the context of the -362 promoter, the G1 mutation (-362 mG1) does not decrease promoter activity (Fig. 3b). Interestingly, the hIns G1 element also lacks the ability to bind the transcription factor Pur1, (^2)which binds with high affinity to the rInsI and rInsII G1 elements(46) .

There is an identical copy of the G1 element upstream of the ILPR(41) . We also mutated this sequence. The mutation did not significantly reduce activity of the -1373 and -1373D promoters, either by itself or in conjunction with the proximal G1 mutation, nor did it reduce activity of the distal promoter sequences linked to the hIAPP promoter (data not shown).

Individual Sequence Elements

Using a method described before (28) , we tested individual sequence elements (Table 2) from the human insulin promoter by linking five copies of each element to a minimal promoter, the rInsI promoter from -85 to +1 bp. The -85 rInsI promoter was chosen because it retains cell specificity but cannot respond to glucose on its own(28) . These mini-enhancer constructs were made in the pFOXCAT2 plasmid, which includes the human beta-globin gene intron, resulting in approximately 5-fold higher specific CAT activities than the pFOXCAT1 plasmid used in Fig. 2, 3, and 5.



The individual E and A elements function as weak enhancers of transcription on their own; the E1 and A1 elements have the highest activities (Fig. 4a). A striking activation results from the combination of either E1 with A1 or E1 with A2/C1. Similar synergy has been noted previously between the A2/C1 and E1 elements in the rInsII promoter(27) , as well as between the A3 or A4 elements and the E2 element in the rInsI promoter(25, 26, 28) . The E2A3 combination in the human gene, however, does not result in significant activation (Fig. 4a).


Figure 4: Function of the human insulin promoter minienhancers. A, cultured fetal rat islets were transfected with plasmids containing five tandem copies of the mini-enhancers shown or a single copy of the Rous sarcoma virus enhancer, linked to the minimal -85-bp rat insulin I promoter (RIP1) driving the CAT gene in pFOXCAT2 and grown in 2 mM (solid bars) or 16 mM (hatched bars) glucose. See Table 2for the sequences of the mini-enhancers. CAT enzyme activity was assayed with equal amounts of protein 36 h after transfection. Each data point represents the mean of at least three independent transfections. CAT activity of cultures transfected with the promoterless pFOXCAT2 plasmid and grown at 2 mM glucose was arbitrarily set at 1.0. B, cultured fetal rat islets were transfected with plasmids containing 5 tandem copies of the wild-type or mutated E1A1 mini-enhancers linked to the minimal RIP1 promoter driving the CAT gene and grown in 2 mM (solid bars) or 16 mM (hatched bars) glucose. CAT enzyme activity was assayed with equal amounts of protein 36 h after transfection. Each data point represents the mean of at least three independent transfections. CAT activity of cultures transfected with the promoterless pFOXCAT2 plasmid and grown at 2 mM glucose was arbitrarily set at 1.0. C, construction of the mini-enhancer multimers is demonstrated.



Both the active mini-enhancers, A2E1 and E1A1, are stimulated by glucose (Table 3), although the A2E1 construct appears to respond more strongly (p < 0.1 by paired Student's t test for comparison of A2E1 and E1A1 -fold stimulation by glucose). Both minienhancers contain the E1 element, which can respond to glucose on its own (Table 3). The A2/C1 element (which includes the two overlapping elements A2 and C1) also has a small response to glucose that the A1 element lacks (p < 0.05 by paired Student's t test for comparison of A2 and A1 -fold stimulation by glucose). The results from mutation of the E1 or A1 elements in the E1A1 minienhancer (Table 3) also support the conclusion that the E1 element responds to glucose.



Other Promoters

In previous studies using the transfected islet cells(1, 28) , our only nonglucose-responsive control was the RSV long terminal repeat, which could be functioning in non-beta cells in our cultured islet cell system. Because all of our insulin promoter constructs responded to glucose(28) , the possibility remained that the effect was nonspecific; glucose would stimulate any promoter limited to the beta cell.

Therefore, we obtained and tested several additional beta cell promoters, the rat beta cell glucokinase promoter (rBGK), the human islet amyloid polypeptide promoter (hIAPP), the rat liver pyruvate kinase (rLPK) promoter, and the rat branched chain ketoacid dehydrogenase E1alpha (rBCKDHA) promoter, comparing them to the rInsI and hIns promoters (Fig. 5). The two rLPK promoter constructs give the strongest response to glucose, but the hIAPP promoter constructs also respond modestly to glucose (Table 4). The rBGK promoter is not stimulated by glucose (confirming results in other systems(47, 48, 49) ), although overall activity of this promoter is quite low, only 2.2-fold higher than the promoterless pFOXCAT1 plasmid at 2 mM glucose (Fig. 5). Activity of both rBCKDHA promoter constructs are suppressed by glucose.


Figure 5: Function of other beta cell promoters in primary cultured islet cells. Cultured fetal rat islets were transfected with the promoter-CAT plasmids shown and grown in 2 mM (solid bars) or 16 mM (hatched bars) glucose. CAT enzyme activity was assayed with equal amounts of protein 36 h after transfection. Each data point represents the mean of at least four independent transfections. CAT activity of cultures transfected with the promoterless pFOXCAT1 plasmid and grown in 2 mM glucose was arbitrarily set at 1.0. Abbreviations, hIAPP, human islet amyloid polypeptide promoter; rLPK, rat L-pyruvate kinase promoter; rBGK, rat beta cell glucokinase promoter; rBCKDHA, rat branched chain ketoacid dehydrogenase E1a promoter.





The rLPK promoter also responds to glucose in hepatocytes(50) , and that response has been mapped to a small region of the promoter with a dual architecture similar to the EA minienhancers(51, 52) . We tested this rLPK minienhancer and found that it also responds to glucose in cultured beta cells (Fig. 4a and Table 3).


DISCUSSION

Functional Elements in the hIns Promoter

Most previous studies of the insulin promoter have been performed in insulin-producing tumor cell lines. Although these cells produce and secrete insulin, tumor cells differ in many important regards from normal beta cells. Glucose metabolism and sensing is altered in most beta cell tumor lines relative to normal beta cells(53, 54) , and the levels of insulin mRNA are lower(53, 55) , possibly reflecting lower rates of insulin gene transcription and altered promoter function. We have previously shown in studies of the rInsI promoter that the relative importance of promoter sequence elements differs strikingly between cultured beta cells and beta cell tumor lines(28) .

It is not surprising, therefore, that our present results with the hIns promoter do not agree completely with previous studies in beta cell tumor lines. The -260-bp truncated promoter demonstrates one of the most conspicuous differences. In beta cell tumor lines, Boam et al.(11) found that deletion of sequences between -281 and -260 bp caused a 25-fold activation of the hIns promoter relative to the -341 bp or -281 bp promoter and concluded that a negative regulatory element lies between -281 and -260 bp in the insulin promoter. We found instead that removal of this region caused a large decrease in promoter activity. The repressor effect of the negative regulatory element may be limited to tumor cells, although no significant relief of repression was noted by Walker et al.(2) with the -260 deletion in the hamster beta cell tumor line HIT-T15. Our results suggest that the region from -260 to -341 makes a significant positive contribution to the overall activity of the hIns promoter in normal beta cells.

The individual contributions of isolated sequence elements are difficult to quantify because none of the isolated elements that we tested had much activity on their own. Instead, these elements function largely by synergistic interactions. The interactions between E1 and the two elements A1 and A2/C1 may explain a large part of the activity proximal to -171 bp, but a significant portion of the activity of the promoter must be contributed by sequences that lie distal to -171.

How important are the E1-A2/C1 and E1-A1 interactions if very little of the activity of the hIns promoter resides proximal to -171 bp? This question goes to the heart of how the insulin promoter, as well as many other potent cell-specific promoters, function. The insulin promoter is not a simple summation of individual functional elements; it is the interactions among elements that give the full function of the promoter, and these interactions occur at several levels. The strongest evidence for this point is the fact that mutation of any one of several different elements in the insulin promoter can nearly completely disable the promoter (just as the -171 deletion did)(7, 9, 10, 28, 30) .

In the case of the E1 element, it has been shown that mutation of the E1 element in the insulin promoter results in near complete loss of promoter activity(7, 9, 10) . How can this be reconciled with the apparent unimportance of the -171 bp promoter? The explanation is that the importance of the E1 region (including A1, C1, and A2) is evident in the longer promoter because full expression of the inherent activity in the E1 region requires interaction with other regions of the promoter. It is this complexity, this dependence on multiple interacting elements and the nuclear protein complexes that bind them that gives the insulin promoter both its exquisite specificity and its broad potential for regulation. Because of this complexity, however, it is difficult to study these interactions in the intact promoter (as the -171 bp result shows). Therefore, what we have done in this study is to break down the promoter into the smallest elements with which we can study these interactions.

In this regard, it is interesting that A3 does not synergize with the poorly conserved E box E2. Since the A3 site is critically important to overall function of the intact hIns promoter (30) and it has little activity on its own, it must interact with other elements. It could interact with the E1 element or with other elements that are not E boxes. Furthermore, there are three potential E boxes distal to E2: at -263 (CAGCTG), at -272 (CATTTG), and at -299 (CAGGTG). Any of these elements could interact with A3, A5 or with other as yet unidentified sequence elements. Finally, it should be noted that we only studied the E and A elements and their interactions. Although these elements are critically important, other sequence elements certainly contribute to the overall function of the promoter.

Metabolic Response Elements

The hIns promoter minienhancer results corroborate our earlier evidence that the rInsI E2 element (which shares an 8-bp sequence identity with the hIns E1 element) functions as a metabolic response element(28) . From our results, we can conclude that the human E1 element is a metabolic response element. Furthermore, the rLPK glucose response maps to a mini-enhancer that also appears to contain an E box that binds basic helix-loop-helix proteins(51, 52, 56, 57) .

Interestingly, the A2/C1 region responds to glucose as well, and the A2E1 minienhancer gives a larger response to glucose than either elements E1 or A2/C1 by themselves or than the E1A1 mini-enhancer. These experiments do not distinguish between the overlapping A2 and C1 sites, which appear to bind different nuclear proteins. The A2 site, like the other A sites, binds the homeodomain protein IPF1, (^3)while the C1 site binds an unknown nuclear complex that is selectively expressed (15) and increases in response to glucose treatment in cultured insulin-producing tumor cells(31) .

Like basal transcription of the promoter, glucose stimulation of the insulin promoter appears to require multiple interacting elements(28) . In this context, it is interesting that other investigators have mapped glucose responsiveness to other elements: the A3 element in the hIns promoter(29, 30) , the A3/A4 element in the rInsI promoter(29) , and the A2/C1 element in the rInsII promoter(31) . The A3 element is bound by IPF1 (30) (^4)and other homeodomain proteins(20) , and the major nuclear complex that binds to the A3 element may increase in response to glucose(29, 59) , although we were unable to confirm this observation(28) . Given the importance of glucose in beta cell intracellular signaling, it is not surprising that more than one transcription factor in beta cells is regulated by glucose.

Other beta Cell Promoters

The insulin promoter is not unique in its response to glucose. Among other promoters, the L-pyruvate kinase promoter has been shown previously to respond to glucose, both in hepatocytes(50, 51, 52) , in beta cell tumor lines(48) , and in transgenic animals(60) . The biological importance, however, of glucose regulation of the L-pyruvate kinase promoter in beta cells is unclear. L-Pyruvate kinase mRNA (60, 61) and protein (62) has been detected in beta cells, but pyruvate kinase activity in rat islet extract is predominantly the M2 isoenzyme, not the L isoenzyme(63) . Furthermore, since glucokinase controls the rate-limiting step for glucose catabolism in beta cells(64, 65, 66) , increases in L-pyruvate kinase activity would be expected to lower the levels of glycolytic intermediates without significantly increasing the flux through glycolysis. Because these glycolytic intermediates are important for glucose signaling in the beta cells(65, 67) , any increase in L-pyruvate kinase activity secondary to an increase in transcription rate might tend to reduce the response to glucose.

In addition to glucose, beta cells respond to other energy sources including amino acids, and leucine is one of the best non-glucose secretagogues. Leucine catabolism is regulated at the step controlled by branched chain ketoacid dehydrogenase. Leucine catabolism is reduced in beta cells exposed to glucose, due to a glucose-induced decrease in the mRNA encoding the branched chain ketoacid dehydrogenase E1alpha subunit (branched chain ketoacid dehydrogenase A)(58) . Although part of the decrease in mRNA levels may be due to the decrease in promoter activity that we have demonstrated here, the full 10-fold reduction in mRNA levels (58) induced by glucose may require other factors such as a decrease in mRNA half-life.

The ability of the beta cell to differentially regulate gene transcription in response to metabolic signals is critical for its response to both short and long term changes in nutritional status. This differential regulation depends on the interactions among sequence elements along each promoter and the beta cell nuclear proteins that bind these elements. By mapping the metabolic regulatory elements, we are starting to learn how the beta cell distinguishes and responds to the information encoded in the promoters.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant DK-21344 (to W. J. R.), a University of California at San Francisco Research, Evaluation, and Allocation Committee Springer Fund grant, and a Juvenile Diabetes Foundation Career Development Award (to M. S. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: University of California at San Francisco, Hormone Research Institute, HSW 1090, Third and Parnassus Aves., San Francisco, CA 94122-0534. Tel.: 415-476-9262; Fax: 415-731-3612; :german{at}cgl.ucsf.edu.

(^1)
The abbreviations used are: ILPR, insulin-linked polymorphic region; bp, base pair(s); CAT, chloramphenicol acetyltransferase; RSV, Rous sarcoma virus.

(^2)
G. C. Kennedy, personal communication.

(^3)
H. Odagiri and M. German, unpublished results.

(^4)
H. Odagiri and M. German, unpublished results.


ACKNOWLEDGEMENTS

We thank Leslie Spector for preparation of the manuscript and G. Kennedy, J. Johnson, and W. J. Rutter for helpful discussions.


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