Effect of retinoic acid on glucokinase activity and gene expression in neonatal and adult cultured hepatocytes

Effect of retinoic acid on glucokinase activity and gene expression in neonatal and adult cultured hepatocytes

Life Sciences 68 (2001) 2813–2824 Effect of retinoic acid on glucokinase activity and gene expression in neonatal and adult cultured hepatocytes Gabr...

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Life Sciences 68 (2001) 2813–2824

Effect of retinoic acid on glucokinase activity and gene expression in neonatal and adult cultured hepatocytes Gabriela Cabrera-Valladaresa, Franz M. Matschinskyb, Juehu Wangc, Cristina Fernandez-Mejiaa,* a

Unidad de Genetica de la Nutricion, Instituto de Investigaciones Biomedicas, Universidad Nacional Autonoma de Mexico/Instituto Nacional de Pediatria, Mexico City, C.P. 04530, Mexico b Diabetes Research Center, University of Pennsylvania, Medical Center, 501 Stemmler Hall, 36th and Hamilton Walk, Philadelphia, PA 19104-6015, USA c Hormone Research Institute, University of California San Francisco, 505 Parnassus Av., West Tower, HSW1090, San Francisco, CA 94143-0534, USA Received 12 July 2000; accepted 17 November 2000

Abstract It has been shown that all-trans retinoic acid induces prematurely hepatic glucokinase mRNA in ten days-old neonatal rat hepatocytes, however, this effect could be related to the capacity of the retinoid to promote a more differentiated state of the hepatocyte. In this report we demonstrate that physiological concentrations of all-trans retinoic acid stimulate glucokinase activity in both mature fully differentiated hepatocytes and at the onset of the induction of the enzyme in 15 to 17 days-old neonatal hepatocytes. The effects produced by the retinoid were similar both in magnitude and in time, to those elicited by insulin, a well-known stimulator of hepatic glucokinase expression. No additive effect was observed when insulin and retinoic acid were tested together. Using the branched DNA assay, a sensitive signal amplification technique, we detected relative increases in glucokinase mRNA levels of about 70% at 3 and 24 h after the treatment with 1026 M all-trans retinoic acid, in both neonatal and adult hepatocytes. These data show that retinoic acid exerts a stimulatory effect on hepatic glucokinase independent of the hepatocyte stage of maturity and suggest a physiological role of retinoic acid on glucose metabolism. The action of retinoic acid on hepatic glucokinase might explain previous observations on the relationship between vitamin A status and liver glycogen synthesis. These findings may serve as basis for further investigations on the biological functions of retinoic acid derivatives on hepatic glucose metabolism. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Glucokinase; Hepatocytes; Retinoic acid

* Corresponding author. Unidad de Genetica de la Nutricion, Instituto de Investigaciones Biomedicas, Universidad Nacional Autonoma de Mexico/ Instituto Nacional de Pediatria, Avenida del Iman 1, 4th floor, Mexico City, C.P. 04530, Mexico. Tel.: (525) 606 35 58; fax: (525) 606 34 89. E-mail address: [email protected] (C. Fernandez-Mejia) 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 0 6 5 -7

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Introduction Glucokinase (EC 2.7.1.1), one of the isoenzymes of the hexokinase group, also known as hexokinase type IV, is a tissue-specific enzyme expressed in hepatocytes, in pancreatic beta cells and in certain neuroendocrine cells of the brain and gut [1–3]. Glucokinase plays a key role in glucose homeostasis, regulating insulin secretion in response to glucose in the beta cells [4,5] and glucose uptake in the liver [6,7]. In humans, mutations in the glucokinase gene cause maturity-onset-diabetes of the youth (MODY 2) [8], or hyperinsulinemia [9]. The comparison of the pancreatic and hepatic glucokinase gene transcripts reveals a tissue-specific control of their expression as well as the existence of two distinct promoters in a single glucokinase gene [10,11]. The existence of alternate promoters suggests that separate factors should regulate glucokinase transcription in the two tissues. In the liver, glucokinase is regulated in response to fasting and refeeding [12], with insulin and cAMP serving as the mediators of this response [13–15], whereas in the pancreatic beta cells glucose levels modulate glucokinase activity [2,16,17]. Posttranscriptional regulations have been invoked to explain glucose induction of pancreatic glucokinase [17]. Lipophilic hormone ligands of the nuclear hormone receptor superfamily, such as glucocorticoids and thyroid hormones, have also been demonstrated to affect the expression of glucokinase both in the liver [11, 18–21] and in the pancreatic beta cells [22], however, some differences exist. The development of the enzyme in the islet and the liver also differs. In the pancreatic beta cell glucokinase is already present in fetal life [23], whereas in the liver, glucokinase appears two weeks after birth [24] and then rises rapidly reaching the adult level 10–12 days later [24,25]. Hepatic glucokinase expression depends upon the hormonal and nutritional status [24,25], and its premature induction can be promoted in vivo and in vitro by hormonal treatment [25–28]. Precocious induction of pancreatic glucokinase has not been documented. All-trans retinoic acid, a derivative of vitamin A and a ligand for a certain members of the nuclear hormone receptor superfamily, plays an important role in cellular development, cellular growth and differentiation [29,30]. 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 [31], neuroblastoma cells [32], and the promyelocytic cell line, HL-60 [33]. In contrast, retinoic acid inhibits the differentiation of chondrocytes [34] and adipocytes [35]. The effect of retinoic acid on gene expression is also related to the differentiation state of the cell: it induces S14 gene transcription in cultured adipocytes, but not preadipocytes [36,37]. It has been shown that retinoic acid induces hepatic glucokinase mRNA levels in neonatal rat hepatocytes at ten days of age, when rats normally do not express the enzyme [38], however this effect could be related to the differentiating capacity of retinoic acid. In this work we investigate whether retinoic acid is capable to modulate glucokinase in neonatal hepatocytes already expressing the enzyme and wheter this response is maintained in the fully developed hepatic cell.

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Methods Neonate hepatocyte culture Wistar rats pups from 15 to 17 days of age were separated from their mother just before the isolation of hepatocytes. Four to six animals were anesthetized by ether inhalation, liver was dissected, finely minced and incubated 15 minutes in EGTA (0.5 mM)/Saline TD (NaCl 8 g/l, KCl 3.75 g/l, Na2PO4 0.1 g/l, Trizma base 3g/l, phenol red 0.1%). The tissue was washed in Hank’s solution and digested with 0.1% collagenase type H (Boheringer Mannheim, Mannheim, Germany). Cellular suspensions were filtered through a nylon mesh and washed/ centrifuged (500 rpm) three times with Hank’s solution. The cells were resuspended in RPMI 1640 medium suplemented with 10% fetal bovine serum and 400 U/ml penicillin, and 200 mg streptomycin (Life Technologies Inc, Gaithersburg, MD) (suplemmented RPMI) and seeded at a density of one million cells in Primaria 100 mm tissue culture dishes (Falcon, Lincoln Park, NJ) in supplemented RPMI 1640 containing 1026 M dexamethasone (RPMI/ Dex) to facilitate the cell adhesion to the plates [39]. After one day of culture, the medium was replaced with fresh supplemented RPMI/Dex and the designated agents were added (i.e. insulin, all-trans retinoic acid). After different periods of incubation, as indicated in the text, the cells were washed with 5 ml PBS, detached by scrapping, counted and centrifuged at 2,000 rpm at 48 C. Adult hepatocyte culture Rat liver parenchymal cells were isolated by collagenase perfusion [40] from 24 h fed ad libitum Wistar male rats (200–250 g) injected intraperitonaly with 0.25 ml of a 6.3% solution of pentobarbital (Pfiezer, Inc. Mexico City, Mexico). The cellular suspension was filtered through a nylon mesh and washed/centrifuged (500 rpm) three times with Hank’s solution. The cells were then resuspended in supplemented RPMI 1640 and seeded at a density of one million cells in Primaria 100 mm tissue culture dishes (Falcon, NJ) in supplemented RPMI/ Dex. After one day of culture, the medium was replaced with fresh supplemented RPMI/ Dex and the designated agents were added (i.e. insulin, all-trans retinoic acid). After different periods of treatment, as indicated in the text, the cells were recovered as indicated for neonatal hepatocytes. Glucokinase assay Hepatocytes were harvested and centrifuged, as described above in culture procedures. Tissue pellets were lysed in 500 ml reporter lysis buffer (Promega, WI), vortexed and cell membranes disrupted by three freeze-thaw cycles. Five hundred microliters of a 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 48 for one hour at 35,000 3 g, in a Beckman ultracentrifuge model Optima. Supernatants were recovered, and enzymatic activity was assayed as previously described [41], using NAD (Sigma, MO) as coenzyme. Glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides (Sigma, MO), was used as coupling enzyme. Correction for low hexokinase activity was applied by subtracting the activity measured at 0.5 mM glucose from that measured at 100 mM glucose. Protein concentrations were determined by Bradford assay [42].

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Messenger RNA analysis Glucokinase and actin mRNA levels were quantified using branched DNA technology (bDNA), a sensitive and rapid method previously described for quantification of insulin preRNA [43]. All components, including buffers and DNA reagents were obtained from Chiron, Corp. (Emeryville CA). RNA was extracted by either Trizol (Gibco BRL, Grand Island, NY) or by cell lysis with 400 ml 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 mg/ml) / 7.8% formamide / 0.05% sodium azide / 0.05% Proclin 300]. RNA samples were mixed with 200 ml extraction buffer, 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 638 C in a plate heater to capture the targeted nucleic acids to the oligonucleotide-modified microwell surface. After cooling at room temperature for 10 minutes, cells were washed twice with wash A (Chiron, CA). Fifty microliters of bDNA amplifier solution containing the bDNA amplifier probe at 1 pmol/ml in amplifier diluent (Chiron,CA) was added and hybridized at 538 C, for 30 min. After cooling and washing as described above, 50 ml of a mixture containing alkaline phosphatase-conjuged label probes (2 pmol/ml) in label diluent (Chiron, CA) was added and hybridized at 538 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). Finally, 50 ml of chemiluminescent substrate (Lumiphos 530), an enzyme-triggerable dioxetane substrate for alkaline phosphatase, was added and the plate was incubated at 378 C for 25 min. Light emission was measured in a luminometer at 378 C. Each sample was assayed in triplicate. Each sample was standardized to actin mRNA. Statistics Data are presented as mean 6 S.E. Multiple comparisons were evaluated by one-way ANOVA. The significance level chosen was P , 0.05. Results Effect of retinoic acid on hepatic neonatal glucokinase activity We analyzed the effect of all-trans retinoic acid on hepatocytes from 15 to 17 day-old pups. This is the earliest age in which glucokinase is expressed in the liver of our rats. This time period of enzyme detection is similar to that reported by other investigators [1,27]. As shown in Figure 1, incubation with retinoic acid as low as 1028 M significantly increased (P, 0.05) glucokinase activity, further increases were achieved at concentrations of 1027 and 1026 M. The microscopic analysis revealed no morphological differences between the control and the retinoic acid treated cells. Since dexamethasone was present in the hepatocyte cultures to facilitate cell adhesion to the plates [39] we investigated whether the effect of retinoic acid was due to a permissive effect of dexamethasone. Similar increases were produced by the retinoid (1026 M) both, in the presence (81.9 6 4.0%) and absence (85.8 6 21%) of dexamethasone.

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Fig. 1. Effect of different concentrations of all-trans retinoic acid on glucokinase activity in 15–17 days neonatal hepatocytes. Hepatocytes were isolated from normal rats by collagenase digestion as described in Methods. Hepatocytes were cultured for 48 h in the absence or in the presence of the designated concentrations of all-trans retinoic acid or vehicle. Each bar represents the mean percentages 6 SE of glucokinase activity of 4 independent experiments. Significance was assessed by one-way ANOVA analysis of variance (*); P # 0.05, (**) P # 0.005.

We also investigated the effect of retinoic acid after different incubation times, our results showed that retinoic acid concentrations of 1026 M significantly increased (P, 0.05) glucokinase activity by 66.1 6 17 % and 70.7 6 11 % at 3 and 24 h respectively (Figure 2A). The effect of retinoic acid was also observed after 48 h incubation (67.8 6 16%). The increases produced by the retinoid after 3 h incubation were similar to those produced by insulin 10.2 nM, a well-known stimulator of hepatic glucokinase expression. However, at 24 h insulin produced slightly higher induction than retinoic acid (P $ 0.05). We also investigated the effect of retinoic acid on insulin induced glucokinase activity. As can be seen in Figure 2A, no significant differences (P $ 0.05) were observed between the increases produced by the treatment with insulin alone and retinoic acid plus insulin. Effect of retinoic acid on adult liver glucokinase activity We also analyzed whether the stimulatory effect of retinoic acid is maintained in the fullydeveloped adult hepatocyte. Figure 2B depicts the effect of the retinoid on adult glucokinase. After 3 h of treatment retinoic acid increased glucokinase activity by 97.3 6 20%. This increase is similar to that produced by insulin. After 24 h of treatment the stimulatory effect of

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Fig. 2. Effect of all-trans retinoic acid on insulin induced glucokinase in 15–17 days neonatal and adult hepatocytes. Hepatocytes were isolated by collagenase digestion as described in Methods. Hepatocytes were cultured for the indicated periods of time in the presence of vehicle (DMSO 0.01%), 1026 M all-trans retinoic acid, insulin 10.2 nM or all-trans retinoic acid 1026 M plus insulin 10.2 nM,. Panel A: neonatal hepatocytes; panel B: adult hepatocytes. Data are expressed as mean percentages 6 SE of glucokinase activity (Control; neonatal5 1.93 6 0.41; adult 5 2.9 6 0.28 mU/ 106 cells); n5 5 or 4 experiments. Multiple comparisons were evaluated by oneway ANOVA analysis of variance. (*); P # 0.05, (**) P # 0.005.

the retinoid (79.1 6 8.3%) was slightly decreased compared to that observed at 3 h, alhough this difference was not significant (p . 0.05). At 24 h insulin produced higher induction (P $ 0.05) than retinoic acid. We also tested the effect of retinoic acid in the presence of insulin. No differences were observed between the increases produced by the treatment with insulin alone and retinoic acid plus insulin (Figure 2B). Effect of retinoic acid on glucokinase mRNA We determined whether the effect of retinoic acid was related to an increase in glucokinase gene expression. We adapted the bDNA assay, a sensitive signal amplification technique, to measure glucokinase mRNA levels. Relative increases of 78.1 6 14 % and 66.6 6 15% at 3 and 24 h, respectively, were observed in neonatal hepatocytes treated with retinoic

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Fig. 3. Effect of all-trans retinoic acid on glucokinase mRNA levels. Glucokinase and actin mRNA were quantified using bDNA technology. Hepatocytes were cultured for the indicated periods of time in the presence of vehicle (DMSO; 0.01%) or 1026 M all-trans retinoic acid. Panel A: neonatal hepatocytes; panel B: adult hepatocytes. Each sample was standardized to actin. Data are expressed as relative to that measured in cells incubated with vehicle. Each value represents the mean 6 SE of the number of experiments indicated in the figure. (*); P # 0.05.

acid concentrations of 1026 M (Figure 3A). Similar responses were observed (Figure 3B) in adult hepatocytes (62.83 % and 85.7% at 3 and 24 h, n52). Discussion In the present study, we demonstrate that retinoic acid can stimulate glucokinase activity in mature fully-differentiated hepatocytes as well as in neonatal hepatocytes, and that this effect is related to an increase in glucokinase mRNA levels. In a previous study [38] it was shown that retinoic acid prematurely induces hepatic glucokinase mRNA levels in neonatal rat hepatocytes at ten-days of age, however, this effect could be related to the capacity of the retinoid to promote a more differentiated hepatocyte phenotype. Although retinoic acid may be involved in the maturation of hepatocytes, as has been previously proposed [44], our studies demonstrate that the fully-developed adult hepatocytes clearly retain the capacity to increase glucokinase activity in response to the retinoid. Furthermore, the rapid increase (3h) of glucokinase activity and gene expression in response to the retinoid and the similar or greater responses elicited in the fully developed adult hepatocyte compared to the 15–17 days-old neonatal cell still developing [24,25], indicate that the stimulatory effect of the ret-

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inoid is unlikely due to its effect to promote a more differentiated cell state. Interestingly, the increase produced by the retinoid at 3 h was similar to that produced by insulin, a wellknown stimulator of hepatic glucokinase, however it appears that retinoic acid is less effective than insulin at 24 h. It has been suggested that retinoic acid may potentiate the effect of other hormones such as insulin [38]. However, our data do not support this, since we did not find significant differences between the effect of insulin alone and that of insulin in the presence of retinoic acid, suggesting that the retinoid does not potentiate insulin action. Taken together these data suggest that retinoic acid has a physiological role in glucose phosphorylation as suggested in our previous studies on pancreatic glucokinase [45]. The expression of a given gene represents the balance of its mRNA transcription, processing and stability. It has been shown that retinoic acid affects all these steps in mammalian gene expression [46–48]. Studies by Decaux et al. [38] have demonstrated that the effect of retinoic acid on hepatic glucokinase mRNA level is due to an increase of gene transcription, however these authors did not report the effect of the retinoid on glucokinase activity. Since it has been documented [22,49] that modifications of glucokinase mRNA levels are not necessarily reflected in glucokinase activity, we also analyzed the effect of the retinoid on enzyme activity. Our results demonstrate that the increases of glucokinase mRNA levels were, indeed reflected in glucokinase activity. We have recently found [45], that retinoic acid can also stimulate pancreatic glucokinase in mature, fully differentiated pancreatic islets as well as in immature fetal islets. Nevertheless, we found differences between the time requirements of retinoic acid to produce its action: In hepatocytes, glucokinase mRNA and activity were increased by the retinoid as rapidly as in 3 h. In contrast, in the pancreatic islets no effect was observed after 3 h incubation (Cabrera-Valladares, unpublished data). In the pancreatic beta cell the response to retinoic acid is slow, requiring 24 h to produce its maximal action [45]. These time differences between hepatic and pancreatic glucokinase induction are also evident for other compounds, such as biotin: this vitamin increased hepatic glucokinase as rapidly as in 2 h [50, 51], in contrast, in the pancreatic islets maximal effects required 48 h incubation [52]. The rapid effect of retinoic acid on hepatic glucokinase observed in our experiments, and the previous demonstration that retinoic acid increased glucokinase transcriptional rates [38] suggest that the retinoid exerts its effect by a direct effect on transcription. Puzzling, no classical retinoic acid element (RARE) consensus appears to be present on the hepatic glucokinase promoter. This is in contrast to the presence of a putative RARE at 2196 to 2154 on the glucokinase pancreatic promoter [53]. The regulation of the liver promoter is more complex and less understood than that of the beta cell promoter. The attempts to demonstrate the cis-regulatory elements on the glucokinase liver promoter that determine the hormone-regulated expression to a reporter gene have been unssuccesful [54, 55]. New strategies are necessary in order to gain an insight into the hormonal regulation of the liver promoter. Over 30 years ago a relationship between vitamin A and carbohydrate metabolism has been documented [56–60]. It is now well established that the pleiotropic effects of vitamin A, with exception of the vision process, are mediated by its acid derivatives. In recent years, acid derivatives of vitamin A, rexinoids have been shown to be promising candidates for the development of new therapeutics for diabetes [61–63]. Most studies have been focused on the effect of rexinoids on peripheral tissues but little is known about their effects on glucose

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metabolism in the liver [62]. Several investigations have been shown a direct relationship between vitamin A status and liver glycogen synthesis: In vitamin A deficiency, low glycogen levels exist [57, 58], on the contrary, high levels of glycogen have been reported when vitamin A is in excess [56, 59, 60]. Glucokinase (EC 2.7.1.1) is a key step for the direct pathway of glycogen synthesis in the liver: increased glucokinase activity has a potent enhancing effect on glycogen synthesis [64,65], on the contrary impaired hepatic glycogen synthesis is observed in glucokinase deficiency [66]. In the present study we demonstrated that retinoic acid can stimulate hepatic glucokinase activity and expression. These data suggest that retinoic acid could participate in the regulation of glycogen through the direct pathway of glycogen synthesis in addition to its previously suggested effect on the indirect pathway of this process [56–58, 67]: An in vivo functional role of retinoic acid on the indirect pathway of glycogen synthesis has been suggested by the observation that the decreased expression of phosphoenolpyruvate carboxykinase and 6-phosphofructo-2-kinase/ fructose-2,6-biphosphatase present in vitamin A deficiency is restored to its normal levels with the administration of retinoic acid [67]. The results in the whole animal indicating that retinoic acid is physiologically important on gluconeogenesis and the data presented here on hepatic glucokinase, suggest that retinoic acid is able to affect glucose metabolism in the liver. These findings may serve as basis for further studies investigating the biological functions of retinoic acid derivatives on hepatic glucose metabolism. The understanding of the biological functions and mechanisms of action of retinoid derivatives and discovery of novel retinoids are likely to result in improved treatments for existing responsive indications. Acknowledgments We are indebted to Dr. Michael S. German from the Hormone Research Institute, University of California San Francisco; who generously provided the reagents and laboratory facilities for the measure of bRNA. The authors are grateful to Dr. Martha Zentella de Piña for her advice on the isolation of adult hepatocytes. We are also indebted to M.S. Alberto Rojas Ochoa for teaching in the isolation of neonatal hepatocytes. We would like to thank Dr. Ignacio Camacho-Arroyo for his critical reading of this manuscript. This work was supported by DGAPA IN210894, DGAPA 212997 and UC Mexus (CF-M).

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