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From the Nutritional Genetics Unit (G.C.V., C.F.M.) Biomedical Research Institute, National University of Mexico, Mexico City, Mexico 04530; Hormone Research Institute (M.S.G., J.W.) University of California San Francisco, San Francisco, California 94143-0534; and Diabetes Research Center (F.M.M.), University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104-6015
Address all correspondence and requests for reprints to: Cristina Fernandez-Mejia, Unidad de Genetica de la Nutricion, Instituto de Investigaciones Biomedicas Universidad Nacional Autónoma de México/Instituto Nacional de Pediatría, Avenida del Iman 1, 4th Floor, Mexico City, C.P. 04530, Mexico. E-mail: crisfern{at}servidor.unam.mx
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Comparison of the pancreatic and hepatic glucokinase gene transcripts reveals tissue specific control of expression and the existence of two distinct promoters in a single glucokinase gene (10, 11). The existence of alternate promoters suggests that separate factors regulate glucokinase transcription in the two tissues. In the liver, glucokinase gene transcription is under multihormonal control (12, 13, 14). Less is known about the transcriptional regulation of pancreatic glucokinase by hormones. Posttranscriptional regulation have been invoked to explain glucose induction of glucokinase (15). In a previous report, we demonstrated that pancreatic glucokinase transcription and activity is also regulated by different lipophilic hormone ligands of the nuclear hormone receptor superfamily, and that this hormonal regulation differs between the hepatic and the pancreatic genes (16).
One member of this family, all-trans-retinoic acid, a derivative of vitamin A and a ligand for a member of the nuclear hormone receptor superfamily, plays an important role in cellular development, cellular growth, and differentiation (17, 18). In cultured cells, the nature of the growth and differentiation response elicited by retinoic acid depends upon the cell line. Thus, retinoic acid induces terminal differentiation of many cell types, including mouse teratocarcinoma stem cells (19), neuroblastoma cells (20), and the promyelocytic cell line, HL-60 (21). In contrast, retinoic acid inhibits the differentiation of chondrocytes (22) and adipocytes (23). The effect of retinoic acid on gene expression is also related to the differentiation state of the cell: retinoic acid induces S14 gene transcription in cultured adipocytes but not preadipocytes (24, 25).
During development, the natural source of retinoids for embryonic tissues is maternal retinol. In view of its highly pleiotropic effects, it is likely that retinoic acid is synthesized from retinol in discrete areas of the embryo, close to its sites of action. It has been suggested (26) that the function of cytosolic retinol binding protein (CRBP) is to concentrate and store retinol in sites where retinoic acid is required in relatively high concentrations, so that retinol can be converted to retinoic acid in relation to specific morphogenetic processes. Conversely, cytosolic retinoic acid binding protein (CRABP) may be expressed by cells whose normal developmental function requires low levels of retinoic acid. In fetal pancreas, homogenous expression of CRBP but not CRABP can be detected (26), suggesting that high concentrations of retinoic acid are important during pancreatic development.
Retinoic acid also affects the function of pancreatic ß-cells: 1) It restores insulin secretion in vitamin A deficient rats (27). 2) It induces both first and second phase insulin secretory responses to glucose in explants of human fetal pancreas, which are normally poorly responsive to glucose (28). 3) It increases insulin production in RINm5F cells (16) and in human islets (29). Finally, retinoic acid increases pancreatic glucokinase activity and messenger RNA (mRNA) levels in the insulinoma cell line RIN-m5F (16); but this effect could be related to the differentiating capacity of retinoic acid because this cell line is relatively undifferentiated in comparison to rat adult islets cells (30, 31) and several manipulation can cause further maturation of these cells (32, 33, 34, 35). Therefore, in this work we analyzed the effect of retinoic acid on glucokinase activity and expression in primary cultures of rat islets in two different stages of maturity to determine if the effect of the retinoid is related to its differentiating actions.
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Materials and Methods |
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Fetal islet culture
Twenty-one-day gestation fetal Wistar rat islets were isolated
as described previously (37). Briefly, pancreatic glands obtained from
four to six rats were finely minced and digested with collagenase Type
D and 2 µl DNase I (Boehringer Mannheim,
Mannheim, Germany). The washed digest was suspended in
supplemented RPMI 1640 medium and distributed equally into two 60-mm
tissue culture dishes (Costar). Cultures were incubated at
37 C in a humidified atmosphere of 5% CO2, after 5 h
of plating to deplete fibroblasts, islets were seeded into 60-mm tissue
culture dishes (Costar, Cambridge, MA) and treated either
with retinoic acid or vehicle (DMSO; 0.01%) for the times indicated in
the text.
Glucokinase assay
Seven hundred to 800 islets were harvested and centrifuged at
1,200 rpm. Tissue pellets were lysed in 500 µl reporter lysis buffer
(Promega Corp., Madison, WI), vortexed, and cell
membranes disrupted by three freeze-thaw cycles. Five hundred
microliters of GK buffer consisting of 50 mM Tris (pH 7.6),
4 mM EDTA, 150 mM KCl, 4 mM
Mg2SO4, and 2.5 mM dithiothreitol
were added. The lysates were then centrifuged at 4 C for 1 h at
35,000 x g, in a Beckman Coulter, Inc.
ultracentrifuge model Optima. Supernatants were recovered, and
enzymatic activity was assayed as described previously by Walker and
Parry (38), using NAD (Sigma Chemical Co., St. Louis, MO)
as coenzyme. Glucose-6-phosphate dehydrogenase from Leuconostoc
mesenteroides (Sigma Chemical Co.), was used as
coupling enzyme. Correction for low hexokinase activity was applied by
subtracting the activity measured at 0.5 mM glucose from
the activity measured at 100 mM glucose. Protein
concentrations were determined by Bradford assay (39).
mRNA analysis
Glucokinase, insulin, and actin mRNA were quantified using bDNA
technology in a 96-microwell format as previously described for
quantification of insulin preRNA (40). All components, including
buffers and DNA reagents were obtained from Chiron Corp.
(Emeryville, CA). RNA was extracted by either Trizol (Life Technologies, Inc.) or by cell lysis with 400 µl of extraction
buffer (78 mM HEPES. pH 8.0/12.5 mM EDTA, pH
8.0/6.27 mM LiCl/1.6 lithium lauryl sulfate/proteinase K (1
mg/ml)/single-stranded DNA (19 µg/ml)/7.8% formamide/0.05% sodium
azide/0.05% Proclin 300. RNA samples were mixed with 200 µl
extraction buffer, along with proteinase K and glucokinase capture and
label probes, loaded in the microwell plate, sealed with an
adhesive-backed mylar plate sealer (Microtiter Plate Sealer, Flow
Laboratory), and incubated overnight at 63 C in a plate heater to
capture the targeted nucleic acids to the oligonucleotide-modified
microwell surface. After cooling at room temperature for 10 min, cells
were washed twice with wash A (Chiron Corp.). Fifty
microliters of bDNA amplifier solution containing the bDNA amplifier
probe at 1 pmol/ml in amplifier diluent (Chiron Corp.)
were added and hybridized at 53 C for 30 min. After cooling and washing
as described above, 50 µl of a mixture containing alkaline
phosphatase-conjugated label probes (2 pmol/ml) in label diluent
(Chiron Corp.) were added and hybridized at 53 C, for 15
min. The plate was cooled and washed twice in buffer A as above and
then washed three times with wash solution B (Chiron Corp.). Finally, 50 µl of chemiluminescent substrate (Lumiphos
530), an enzyme-triggerable dioxetane substrate for alkaline
phosphatase, was added, and the plate was incubated at 37 C for 25 min.
Light emission was measured in a luminometer at 37 C. Each sample was
assayed in triplicate. Each sample was standardized to actin mRNA.
Plasmid constructs
The construction of pFOXCAT1 has been described previously (41).
The rat ß cell glucokinase -1000 promoter extends from -1000 to +14
bp and was a gift from M. Magnuson (Vanderbilt University, Nashville,
TN) (42).
Fetal islet transfection
Experiments were performed as previously described by German
et al. (37); briefly, 21-day gestation fetal Wistar rat
islets were isolated and digested for 5 min with collagenase Type P and
2 µl DNase I (Boehringer Mannheim). After 3 h of
plating to deplete fibroblasts, islets were recovered and dispersed
with trypsin 0.05%, 0.53 mM EDTA (Life Technologies, Inc.). Dispersed islets were incubated at 37 degree C in a
humidified atmosphere of 5% CO2, after 3 h of plating
to deplete from fibroblasts, cells were harvested by rinsing the
plates, pelleted and washed twice in room temperature PBS, resuspended
in 0.8 ml PBS with 25 µg of double cesium-purified plasmid DNA and
electroporated with a discharge of 175 V, 2,000 µF in a BTX
electroporation system model 600 (San Diego, CA). The transfected cells
were grown in supplemented RPMI with retinoic acid (10-6
M) or vehicle (DMSO; 0.01%) for approximately 48 h
before harvesting and protein extraction. CAT-TLC was performed using
25 µg of protein and to generate enough CAT product, the sample was
incubated for 3 h. Acetylated chloramphenicol signals were
analyzed using a Phosphoimager 425 (Molecular Dynamics, Inc., Costa Mesa, CA).
Insulin secretion
Adult or fetal islets were isolated and cultured as described
above. After 24 h of treatment with retinoic acid
(10-6 M or vehicle, DMSO 0.01%), cells were
washed twice with secretion buffer containing 20 mM HEPES,
115 mM NaCl, 5 mM NaHCO3, 4.7
mM KCL, 2.6 mM CaCl2, 1.2
mM KH2PO4, and 1.2 mM
MgSO4 (pH 7.4). Islets were then incubated for 1 h in
secretion buffer containing D-glucose as indicated in the
text and figure legends. Insulin concentration was analyzed by RIA
(ICN Biomedicals, Inc., CA).
Statistics
Data are presented as mean + SE. Individual
comparisons were evaluated by Student’s paired two tailed t
test. Multiple comparisons were evaluated using one-way ANOVA. The
significance level chosen was P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, retinoic acid doses of
10-6 M increased glucokinase activity by
103 ± 6.4% at 24 h. This effect is slightly decreased at
48 h when an increase of 85 ± 4.1% was observed. The
responsiveness of adult islet pancreatic glucokinase to different doses
of retinoic acid is shown in Table 1; a
stimulatory effect was observed at doses as low as 10-8
M, the minimal concentration tested.
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) that after 24 h treatment with retinoic acid
doses of 10-6 M, glucokinase activity
increased to 196.6 ± 13.7% (P < 0.005). A
weaker stimulatory effect was also observed at 48 h (44.2 ±
3.7%) (P < 0.005). As shown in Table 1, the retinoid
affects glucokinase activity in the fetal tissue at all the doses
tested.
Effect of retinoic acid on glucokinase mRNA levels
We determined if the effect of retinoic acid was related to an
increase in glucokinase gene expression. Because glucokinase mRNA
levels are low, we adapted the branched DNA (bDNA) assay, a sensitive
signal amplification technique, to measure glucokinase mRNA levels.
Relative increases of 51.8 ± 13.3 and 62.8 ± 16.1% at 12
and 24 h, respectively, were observed in adult islets treated with
retinoic acid doses of 10-6 M (Fig. 2A). The effect was detectable after
6 h incubation (18.2 ± 10.7%). In fetal islets increases of
55 ± 14.9% and 107 ± 30.5% at 12 and 24 h,
respectively, were observed (Fig. 2B).
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), was observed after 48 h of
treatment with retinoic acid doses of 10-6 M.
No effect was observed on the empty pFOX-CAT1 vector (Control =
100%, retinoic acid = 95 ± 9.3%).
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, 24 h of treatment with
(10-6 M) of the retinoid increased insulin
release in either adult islets or in fetal islets. In the fully mature
islets (panel A), retinoic acid increased insulin secretion by
139.1 ± 39 and 60.1 ± 17% at 5.5 and 16 mM
glucose, respectively (P < 0.05). In the fetal islets,
which are normally poorly responsive to glucose, retinoic acid modestly
increased insulin release by 33.1 ± 7 and 23.7 ± 7% at 5.5
and 16 mM of glucose, respectively (P <
0.05) (panel B). We also determined the effect of retinoic acid on
insulin mRNA by the bDNA method. Treatment with 10-6
M of retinoic acid for 24 h increased insulin message
by 48.2 ± 9.1% (P < 0.05) n = 5 in the
adult islets and by 79.7 ± 20% in the fetal islets
(P < 0.05) n = 5.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Retinoic acid effects on differentiation, cellular growth, and gene
expression are mediated through the activation of its nuclear receptors
(7, 18). The presence of retinoic acid nuclear receptors RXR, RAR-
,
and RAR-
as well as high levels of CRBP and CRABP has been reported
in fetal and adult islets as well as in pancreatic cell lines (26, 29, 43, 44). Doses of retinoic acid that correspond to the doses required
for activation of its nuclear receptor (10-9 to
10-8 M) (45) increased glucokinase activity in
either fetal or adult islets, suggesting a physiological effect of the
retinoid on pancreatic glucokinase.
Retinoic acid regulation of ß-cell genes is not unprecedented. Clark et al. (29) have demonstrated that the human insulin promoter activity is also activated by retinoic acid. In this work we demonstrated the ability of retinoic acid to activate the -1000 kb ß-cell glucokinase promoter, suggesting that retinoids increase glucokinase activity by activating transcription of the gene. To our knowledge, the studies presented here represent the first report on the physiological regulation of the glucokinase promoter. Current studies in our laboratory are investigating the mechanisms by which retinoic acid activate the pancreatic glucokinase promoter.
In a previous study (16), we have shown that retinoic acid increases insulin mRNA levels in the undifferentiated cell line RIN-m5F; however, we could not rule out the possibility that the effect of the retinoid was related to its differentiating capacity or a response limited to the particular cell line. In the present study, we demonstrate that, similar to its effect on glucokinase, retinoic acid can stimulate insulin gene expression in mature, fully differentiated pancreatic islets as well as in immature fetal islets. Retinoic acid has been shown to increase human insulin mRNA through a tandem repeat of three half sites of the retinoic acid/thyroid hormone regulatory elements (RARE/TRE) closely matching the RARE/TRE consensus AGGTCA at -1037 to -1006 bp in the human insulin promoter. No similar sequences are present in the rat genes. It has been reported that glucose increases insulin mRNA levels by signals coming from the glycolytic pathway (46). Because glucokinase activity governs glycolysis, it is possible that increases of metabolic signals from glycolysis due to the increased activity of glucokinase accounted for the increases observed on insulin mRNA levels. Other plausible explanations are that retinoic acid increases insulin transcriptional factor(s), which in turn increase insulin expression, or that the retinoid increases insulin mRNA stability. However, further studies will be required to determine the mechanisms by which retinoic acid regulate insulin gene expression in the rat.
The studies presented here also demonstrate that retinoic acid treatment can increase insulin secretion in either the adult and the fetal islets; however, a stronger effect was observed in the fully mature adult islets where the glycolitic flux regulated by glucokinase initiate the cascade of events in the signal transduction required for insulin secretion (4, 5). In contrast, in the fetal islets, retinoic acid produced only a modest increase on insulin secretion, in spite of the larger increases produced by the retinoid on glucokinase activity and insulin mRNA levels. Taken together, these data support previous observations suggesting that glucokinase activity and insulin secretion is uncoupled in the fetal islets (47). A functional role of retinoic acid on insulin secretion was suggested in vitamin A-deficient rats (27), the studies presented herein, demonstrate that retinoic acid can affect, as well, insulin secretion in islets from normal rats.
Defects in the normal regulation of islet function by retinoids could contribute to some forms of diabetes in humans. In malnutrition-related diabetes mellitus, vitamin A is reduced in all malnourished diabetic patients when compared with malnourished controls (48). Furthermore, it has been proposed (49) that poor nutrition in fetal and early infant life is detrimental to the development and function of the pancreatic ß cells and predisposes to the later development of type 2 diabetes. The role of vitamin A deficiency as a risk factor in diabetes underlines the significance of understanding the role of retinoids in pancreatic development and function.
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Acknowledgments |
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Footnotes |
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Received August 31, 1998.
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