e
t.2Previous work from our laboratory has provided support, for the concept that malonyl-CoA acts as a metabolic corrpiirrg factor in nutrient-induced insulin release (19-15). This-conl cept is teleologically attractive because
it
implies that the same metabolite, j.e. malonyl-CoA, which rises in the target tissues of insulin to decrease the oxidation of fatty acids a-ndfavor nutrient storage (16), also rises in the pancreatic B-cell to cause the release of the fuel storage hormone insulin. Since
th9-flux through ACC controls malonyl-CoA levels (16, 1Z), ACC might be like glucokinase for glucose (2), a key nut.ieni
sensor in the B-cell. Therefore, studying both the short and long term regulation of ACC in the B-cell should contribute to our understanding of the true function of this key regula_
tory enzyme in p-cell physiology.
In_the present reporL we have shown that glucose is a major regulator of ACC gene expression in clonal pancreatic p-cjtts (INS-l). The effect is relatively rapid since the lag time of ACC mRNA induction by glucose lies between B to 4 h. The action of the sugar is quantitatively important as it caused a 15-20-fold induction in both ACC prorein and nRNA. It is noteworthy that glucose caused the accumulation of ACC protein and mRNA only above b mu. This demonstrates that the action of glucose occurs only at physiological concentra_
tions of the sugar and that it does not refleôt an unspecific fuel repletion induction. Indeed at 11 mM glucose, th; ACC protein content of INS-I cells was approximately b-fold of that observed at 5 mM (Fig. 10). In addition, our unpublished work2 indicates that rat pancreatic islets obtained irom 4g-h fasted animals refed a high carbohydrate diet for 72 h show marked induction of ACC nRNA. The action of glucose on ACC protein and mRNA showed identical dose dependencies and were quantitatively similar. Moreover, glucose did not
*gdify the stability of the ACC transcript. Thus, glucose induction of ACC most likely occurs at the transcriptional level. This remains to be directly demonstrated
By simple analogy with nutrient regulation of insulin secre_
tion, we initially thought that post glycolytic metabolites might mediate the action of glucose on ACC induction and
tha! th9 Ca2*, cAMP, and diacylglycerol/C-kinase pathways might be implicated
in
the induction process (fà). Using similar thinking Collins et al. (LL, 12) have proposed ..rr.ru'i criteria to be met for a protein to qualify as a pancreatic p-cell glucose-response protein, including one staling that the effect of glucose should be mimicked by the secretagogue 2-ketoisocaproate. Surprisingly, we found a remarkable ipeci-hcity of the action of glucose on ACC mRNA/protein induc-tion. Among the many tested carbohydrates, keto acids, amino acids, and fatty acids, only mannose mimicked the action ofglucose. In addition, activation of the Ca2*, cAMp, and C_
kinase pathways with high K*, forskolin, and pMA, respec-tively, did not induce ACC mRNA. These observations imply three important conclusions:
l)
glucose does not need to be metabolized beyond glucose 6-phosphatein
the glycolytic pathway to induce ACC mRNA; 2) the metabolic-coupiing factors and signal transduction systems that mediate thé action ofglucose to cause insulin release are entirely different from those implicated in the induction of the ACC gene; and3) different nutrient secretagogues should not nôcessarily induce the same proteins.
FI-c. 8.- Effect of glucose, mannoheptulose, and glucosamine on the.glucose G-phosphate content ôf fNS_f cells. Cells were preincubated for 24 h in the presence ofpyruvate (1 mrrr) and lactate (10 mu) without glucose. Cells were thàn incubatnd for 2 h in the presence of basal medium (pyruvate
*
lactate) with the indicated concentrations of test substances. The glucose 6-phosphate content in the absence of glucose (basal medium) was 0.0?0 + 0.01b pmol/gcubated at various glucose concentrations. Cells were pieincu_
!_at9d f91 24 h at 5, 11, or 20 mu glucose. Following two washes wirh Krebs-Ringer bicarbonate mediuÀ, cells were inc,ibated for B0 min as described under "Experimental procedures." The results are ex-pressed as the percentage of immunoreactive (1Æ1) insulin released
from the total cellular content of insulin. The total celrurar IRI
mv) as indicated under "Experimental procedures." Insul'in release meâsurem^entÆ at basal glucose are similar to the values shown in Fig.
9. The ACC protein cont€nt of INS-1 cells was determined by
im:-munoblotting densitometric scanning of the autoradiogr"*r; .uun.
* S.E. of four separate experiments. 2 T. Brun, E. Roche, K.-H. Kim, and M. prentki, unpublished data.
of action of glucose on ACC gene induction, Namely, does glucose itself or one of its metabolites cause ACC mRNA accumulation, and what are the ACC gene regulatory se-quences and associated proteins implicated in the process?
Since 2-deoxyglucose is effective and the actions of glucose on both ACC mRNA and the glucose G-phosphate content of INS-1 ceils are suppressed by the glucokinase inhibitors man-noheptulose and glucosamine, it is likely as suggested before
in the adipocytes (37) that glucose 6-phosphate mediates the effect. Consistent with the view that glucose 6-phosphate mediates ACC gene induction by glucose, glucose caused a dose-dependent rise in the glucose 6-phosphate content of INS-I cells. Carbohydrate response elements have recently been identified in the L-type pyruvate kinase and S14 gene promoters (38-42). Comparison of the sequences conferring carbohydrate regulation revealed a segment with 9 out of l0 nucleotides identity (42). The conserved motif contained the sequence 5'-CACGTG-3' which has similarity to the core recognition site for transcription factors having a basic helix-Ioop-helix domain such as c-nLyc and MLTF (41-49). A similar motif exists in the promoter 2 regulatory region of the ACC gene (44);
it
is the sequence 5'-CACGTC-3'. In addition, a cloeer examination of sequence homologies between thereg-ulatory regions of the pyruvate kinase (41), S14 (42), and
ACC (44) gene reveals a stretch of 7 bp with 6 our of ?
nucleotides identity. Promoter 2 region of the ACC gene contains two of such sequences (Fig. 11). Whether the se-quence CTC(A/C)CGT confers glucose induction to the three genes is a possibility that needs to be examined.
Our flrndings are potentially related to several aspects of pancreatic islet physiopathology. First, increased ACC expres-sion promoted by glucose may be one of the trophic adaptive responses which are associated to the induction of B-cell proliferation by the carbohydrate. Since proteins and lipids are synthetized at
I
high rate in proliferating cells, it will be of interest to determine whether high glucose also induces thefatty acid synthase gene in f-cells. Second,
it
may be that glucose causes a coordinated expression of key regulatory enzymes in the p-cell such as glucokinase (2), pyruvate de-hydrogenase (9), and acetyl-CoA carboxylase (14, 15, 4b)which are thought to control the production of metabolic coupling factors. Increased metabolic enzyme expression may be a mechanism which contributes to insulin hypersecrelion in chronically elevated glucose conditions (7, 46).
It
is note-worthy that cultured rat pancreatic islets preincubated for along period of time at high glucose (30 mrvr) exhibited a
markedly elevated insulin release at basal glucose (5 mr"r) correlating with the islet glucokinase content (47), with little further stimulation at high glucose (47, 48). We have con-firmed this observation
in
INS-I cells and have shown in addition that this phenomenon correlates with ACC induc-tion. Thus, high ACC enzyme activity and malonyl-CoA pro-duction possibly explain, like the high glucokinase activity (4?), the high basal rate of insulin release and the relative glucose insensitivity of B-cells which have been exposed toconcept that ACC and malonyl-CoA which conlrois fatty acid oxidation (16) play pivotal role in insulin release, we previ-ously showed that inhibiting fatty acid oxidation with 2-bromopalmitate potentiates glucose-induced insulin release in HIT cells (15). Third, the ACC gene which encodes an enzyme which is rate-limiting in a metabolic pathway (lipid biogene-sis) may be, like the glucokinase gene for B-cell glycolysis (49), a candidate gene implicated in type 2 diabetes mellitus.
It can be hypothesized that either mutations or altered expres-sion of the ACC gene by environmental factors may contribute to the development of some forms of this genetically elevations in glucose and other nutrients.
Achnowbdgments-We thank Drs. Mariam Asfari, Françoise As-simacopoulos-Jeannet, Dominique Belin, Barbara Corkey, Jacques Deshusses, and Claes Wollheim for helpful discussions. We are in-debted to Dominique Duhamel for expert technical assistance.
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III. IONG CIIAIN FATTY ACIDS II{HIBIT ACETYLCOA CARBOXYLASE GEI{E
EXPRESSIONIN THE INSI]LIN-SECRETING
T}-CELLLINE INS-1.
(In preparation)
INTRODUCTION
Non-insulin-dependent