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Insulin regulation of glucokinase gene expression: Evidence against a role for sterol regulatory element binding protein 1 in primary hepatocytes
FEBS Letters 580 (2006) 410–414
Insulin regulation of glucokinase gene expression: Evidence against a role for sterol regulatory element binding protein 1 in primary hepatocytes Claudine Gregori, Isabelle Guillet-Deniau, Jean Girard, Jean-Franc¸ois Decaux, Anne-Lise Pichard* De´partement d’Endocrinologie, Institut Cochin, Institut National de la sante´ et de la Recherche Me´dicale (INSERM) U567, Centre National de la Recherche Scientifique (CNRS), Unite´ Mixte de Recherche 8104, Universite´ Rene´ Descartes, 24 rue du Faubourg Saint-Jacques, 75014 Paris, France Received 17 October 2005; revised 9 December 2005; accepted 9 December 2005 Available online 20 December 2005 Edited by Robert Barouki
Abstract Liver key genes for carbohydrate and lipid homeostasis are regulated by insulin and glucose. The sterol regulatoryelement binding protein-1c (SREBP-1c) has emerged as a mediator of insulin effects on gene transcription, particularly on glucokinase (GK). In this paper, we show that despite stimulation of GK promoter transcription by overexpression of mature SREBP-1c, insulin induced GK transcription at least 4 h ahead of accumulation of mature SREBP-1c in the nucleus. Importantly, the knockdown of SREBP-1 mRNA using a RNA-interference technique reduced the level of nuclear SREBP-1 protein, diminished fatty acid synthase mRNA level, but did not affect GK and L-pyruvate kinase mRNA levels. We concluded that SREBP-1 is unlikely to be the mediator of the early insulin effect on GK gene transcription. 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Glucokinase; Small interfering RNA; Gene expression; Sterol regulatory element binding protein; Insulin
1. Introduction Transcriptional activation of liver glycolytic and lipogenic genes, a relatively delayed phenomenon, requires the presence of both glucose and insulin, neither of which being active alone [1,2]. In contrast, stimulation of glucokinase (GK) gene, coding for the first enzyme involved in glucose metabolism, is a rapid strictly insulin-dependent and glucose-independent process [3,4]. When GK is overexpressed either in hepatoma cells [5] or in liver of diabetic mice [6], the transcriptional activation of glycolytic and lipogenic genes becomes insulin-independent and glucose-dependent. Similarly, in absence of GK in hepatic GK-KO mice, glycolytic and lipogenic genes are not induced after refeeding a high carbohydrate diet [7]. Therefore, insulin induction of GK gene expression is the key step to subsequent activation of glycolytic and lipogenic gene expression by glu-
* Corresponding author. Fax: +33 1 53 73 27 03. E-mail address: [email protected] (A.-L. Pichard).
Abbreviations: GK, glucokinase; L-PK, L-pyruvate kinase; FAS, fatty acid synthase; PEPCK, phosphoenolpyruvate kinase; SREBP, sterol regulatory element binding protein; CAT, chloramphenicol acetyltransferase; siRNA, small interfering RNA; SRE, sterol regulatory element; ChoRE, carbohydrate responsive element; ChREBP, carbohydrate responsive element binding protein
cose in liver. However, the molecular mechanisms by which insulin regulates GK gene expression are still controversial and the cis-acting elements mediating insulin effects remain unknown. The transcription factor sterol regulatory-element binding protein-1c (SREBP-1c), whose transcription is also insulin-dependent, was pointed out as the main mediator of insulin effect on GK gene expression [8,9], but other studies failed to confirm this finding [10,11]. Therefore, whether or not SREBP1-c mediates the action of insulin on GK gene expression is still a controversial issue. Owing to the central role of insulin in the control of liver metabolism, and of GK in glucose metabolism, the goal of this study was to reinvestigate the role of SREBP-1c in mediating insulin effect upon GK gene expression. For this purpose, we studied in primary cultured hepatocytes the kinetics of insulin upregulation of GK gene transcription as well as the level of transcriptionally active SREBP-1 protein in the nucleus. Further, SREBP-1 knockdown by small interfering RNAs (siRNAs) resulted in an impaired induction of fatty acid synthase (FAS) gene in response to glucose and insulin, but did not prevent the induction of GK and L-pyruvate kinase (L-PK) genes. We clearly demonstrated that early insulin stimulation of GK gene was SREBP-1-independent.
2. Materials and methods 2.1. Primary hepatocyte culture Hepatocytes were isolated from liver of 8-week-old male Wistar rats after perfusion with collagenase. Hepatocytes were seeded at a density of 3.5 · 106 cells per dish (20 cm2) in M199 medium with Earl’s salts (Invitrogen) supplemented with 100 U/ml penicillin, 100 lg/ml streptomycin, 0.1% (w/v) BSA and 2% (v/v) Ultroser G (BioSepra). After a 4 h-incubation at 37 C under 5% (v/v) CO2, the cells were washed with PBS and switched for 16 h to a BSA–Ultroser-free medium. The medium was then supplemented with 1 lM dexamethasone, in the absence or presence of insulin and/or glucose as indicated in the figure legends. 2.2. Plasmid construction and transient transfection GK promoter construct: The mouse GK–chloramphenicol acetyltransferase (CAT) plasmid is the PeCAT vector [12] in which the fragment (960 to +19 bp) of the mouse liver GK promoter was subcloned, along with two fragments (+145 to +272 and +4035 to +4120) corresponding to the splice sequence in 5 0 and 3 0 of the first intron, respectively. These splice fragments were obtained by PCR using added restriction sites (underlined): For the 3 0 splice: 5 0 -TATGGTACCCTTGGTGTGTGGTGGCTT-3 0 and 5 0 -TTAGAGCTCTGCCAGGATCTGCTCTACC-3 0 . For the 5 0 splice: 5 0 -TTACCTTGGAG CCCAGTCGTTGACTCT-3 0 and 5 0 -TTCGGATCCTACAGGATCGCACTCA-3 0 . The PCR product for the 3 0 splice fragment was
0014-5793/$32.00 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2005.12.032
C. Gregori et al. / FEBS Letters 580 (2006) 410–414 digested by Kpn1 and Sst1, and subcloned into PeCAT digested with the same enzymes. The PCR product for the 5 0 splice and the promoter fragments were first digested by Sty1 and then ligated together. The resulting fragment was digested by HindIII and BamH1 and subcloned in the above-described plasmid digested with the same enzymes. All fragments generated by PCR were sequenced. SREBP-1c expression plasmid: SREBP-1c plasmid was the pSVSport1-ADD1 (403) coding for the nuclear transcription factor [13] (gift of Dr. B.M. Spiegelman, Harvard Medical School, Boston, MA). Transient transfections: Transfections were carried out as previously described [14]. In each experiment, 7.5 lg of the CAT plasmid and 2.5 lg of the luciferase plasmid were cotransfected. The luciferase standardization pRSV plasmid was used to monitor variations in transfection efficiency. The CAT assay and luciferase assay were performed as previously described [12]. 2.3. RNA isolation and Northern-blot analysis Total RNA extraction from cultured hepatocytes was performed using the RNAzol B reagent (Q Biogene) according to the manufacturer’s instructions. 15 lg aliquots of total RNA were electrophoretically separated in denaturating formaldehyde agarose gels and transferred to Hybond N+ membranes (Amersham). The cDNA probes were labeled with (a-32P) dATP using random prime labeling (Amersham-Pharmacia Biotech). The membranes were hybridized with the indicated 32P-labeled probe (106 cpm/ml) at 42 C overnight. The GK, L-PK, phosphoenolpyruvate kinase (PEPCK), FAS and SREBP-1 probes were obtained as previously described [15]. Northern blots were hybridized with a 32P-labeled ribosomal 18S probe to verify that equivalent amounts of total RNA were loaded in each lane.
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gene expression was induced in a concentration-dependent manner (Fig. 1). At the maximum amount tested (1.25 lg), the control expression vector had no effect (data not shown) whereas the constitutively active SREBP1-c stimulated 2.5-fold the GK promoter activity. Strikingly, insulin treatment failed to induce basal GK–CAT fusion gene expression (Fig. 1A point 0 lg). These data indicated that the SREBP binding sites may well be functional but requestioned the role of SREBP-1c as a major mediator of insulin action on GK gene expression. To ascertain these results, the well-established effects of insulin on endogenous GK mRNA level [3,18] were measured, in the same pool of cultured hepatocytes but untransfected. We observed the expected rapid (2 h) time-dependent accumulation of GK mRNA in the presence of insulin and low glucose concentration (5 mM) (Fig. 1B), indicating that insulin signaling
2.4. Immunoblot analysis Nuclear extracts and membrane fractions were prepared from cultured hepatocytes using the NE-PER Nuclear and Cytoplasmic Extraction Reagents Kit (Pierce) according to the instructions provided by the manufacturer. Protein concentration was determined using the Bradford method (Bio-Rad). Proteins (35 lg) were separated by SDS–PAGE (7% polyacrylamide gel) and transferred to a nitrocellulose membrane (Bio-Rad). Immunoblot analysis was performed using a monoclonal anti-SREBP-1 antibody (clone 2A4 from NeoMarkers), a Lamin A/C antibody from Cell Signalling and the Enhanced Chemiluminescence System (Supersignal, Pierce). 2.5. Design, synthesis and transfection of siRNA A target sequence in the rat SREBP-1 mRNA (GenBank Accession No. L16995) [13] was identified following the principles described by Elbashir et al. [16] as previously described [17]. Primary rat hepatocytes were cultured in antibiotic-free medium the day before transfection. Cells transfection was conducted using Lipofectamine 2000 according to the supplier’s instruction (Invitrogen). On day 1, hepatocytes were transfected with 200 pmol siRNA per 20-cm2 dish. On day 2, the medium was supplemented with 100 nM insulin and 25 mM glucose. Twenty-four hours later, cells were harvested and either RNA or proteins were prepared.
3. Results 3.1. Regulation of GK gene transcription by SREBP-1c and insulin in cultured rat hepatocytes Two potential binding sites for the transcription factor SREBP were identified, by computer analysis, in the mouse GK promoter (113 to 106 and 65 to 55). To test SREBP-1c effect upon GK fusion gene expression we used primary cultured rat hepatocytes. We transiently transfected cells with a GK–CAT fusion gene, containing the promoter sequence located between 960 and +19, and either a control expression vector or the same vector encoding nSREBP-1c [13]. The cells were cultured in the absence or presence of insulin. When SREBP-1c was overexpressed, GK–CAT fusion
Fig. 1. Regulation of GK gene transcription by SREBP-1c and insulin in cultured rat hepatocytes. (A) Primary hepatocytes cultured for 16 h in the presence of 5 mM glucose without insulin were then transfected with 7.5 lg of CAT–GK fusion gene, 2.5 lg of the reference plasmid RSV luciferase and the indicated amount of expression vector for mature SREBP-1c. Cells were cultured in medium containing 5 mM glucose, in the absence (h) or presence (d) of 100 nM insulin. CAT activity was normalized according to transfection efficiency by measuring the luciferase activity. Values are means of four separate experiments performed in duplicate. (B) Time course of GK and SREBP-1 mRNA accumulation in hepatocytes cultured in the presence of 5 mM glucose and 100 nM insulin. Total mRNA (15 lg) were subjected to electrophoresis and blot hybridization with the indicated 32 P-labeled cDNA probe. (C) Time-course appearance of SREBP-1 precursor (P) and mature (M) proteins. Membrane pellet and nuclear extract (35 lg protein) were subjected to SDS/PAGE and analyzed by immunoblotting using an antibody against the precursor and mature forms of SREBP-1. Lamin A/C was used as control for the nuclear extract. The blots are representative of three independent experiments.
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is functional in these cultured cells. In the same experiments, SREBP-1 mRNA accumulated in parallel with GK mRNA (Fig. 1B). We next measured endogenous SREBP-1 protein levels both in membrane and nuclear extracts. SREBP-1 precursor amount reflected the insulin-induced upregulation of SREBP-1 mRNA (Fig. 1C). In marked contrast, mature form of SREBP-1 protein, which serves as an active transcription factor, was undetectable during the first 6-h incubation with insulin and was highest after 24 h (Fig. 1C). If SREBP-1 was the insulin-dependent transcription factor controlling GK gene transcription, we would expect to detect SREBP-1 protein in nuclear extracts ahead to accumulation of GK mRNA. Altogether, these observations argued against SREBP-1 being the prime mediator of insulin effect upon GK gene transcription. 3.2. Glycolytic and lipogenic gene expression in hepatocytes In order to check the functionality of metabolic responses in cultured hepatocytes, we measured mRNA levels of the key glycolytic (L-PK), lipogenic (FAS) genes known to be induced by insulin and/or glucose [19] and gluconeogenic (PEPCK) gene known to be repressed by insulin. Hepatocytes were incubated for 16 h in the presence of low glucose (5 mM) and no insulin. Thereafter, the cells were incubated for 24 h with high glucose (25 mM) and insulin (100 nM). As expected, Fig. 2 shows an increase in GK and SREBP-1 mRNAs within 2 h followed by L-PK and FAS mRNAs at 24 h as well as the marked suppression of PEPCK mRNA after 2 h. These results demonstrated that insulin and glucose signalings were maintained in cultured hepatocytes. 3.3. Silencing of SREBP-1 in hepatocytes To test the direct control of GK gene transcription by SREBP-1 in another way, hepatocytes were transfected with a SREBP-1 siRNA duplex and then maintained for 24 h in the presence of insulin and high glucose concentration to induce transcription of control genes such as FAS. Both SREBP-1 mRNA and nuclear active SREBP-1 protein were severely reduced in transfected cells (Fig. 3A and B), indicating
Fig. 2. Glycolytic and lipogenic genes induction by glucose and insulin. Hepatocytes cultured 16 h in the presence of 5 mM glucose without insulin (time 0) were maintained for a further 24 h in the presence of 25 mM glucose and 100 nM insulin. Total RNA (15 lg) were subjected to blot hybridization with the indicated 32P-labeled cDNA probe. The blots are representative of three independent experiments.
Fig. 3. Effect of SREBP-1 knockdown by RNA interference (siRNA) on glycolytic and lipogenic gene expression in rat hepatocytes. Twelve hours after transfection with a SREBP-1 siRNA duplex as described in Section 2, hepatocytes were cultured for 24 h in the presence of 100 nM insulin and 25 mM glucose. Total mRNA, membrane and nuclear extracts were then prepared. (A) Western blotting was performed to detect SREBP-1 precursor (P) and mature (M) proteins. Lamin A/C was used as control for the nuclear extract. (B) Total RNA (15 lg) were subjected to blot hybridization with indicated 32P-labeled cDNA probes Northern blots were scanned and quantified. Each value represents the amount of mRNA relative to that in the untransfected cells which is arbitrarily set to 100. Values represent means ± S.E. of data from three independent experiments.
an efficient knockdown of SREBP-1 expression. Under these conditions, we observed a marked impairment of FAS mRNA accumulation, but no change in L-PK and GK mRNAs (Fig. 3B). These results confirmed SREBP-1 as being a mediator of insulin effects upon FAS gene transcription, but definitively ruled out its role in controlling GK and L-PK gene expression.
4. Discussion The SREBPs are a family of three nuclear transcription factors encoded by two genes. SREBP-1a and SREBP-1c derive from the same gene, and both appear to regulate lipogenic gene transcription [20]. Despite being a weak activator [21], SREBP-1c is the dominant isoform in liver where its regulation is insulin-dependent [22]. The purpose of the present work was to clarify the involvement of SREBP-1 in insulin-mediated induction of GK. In agreement with studies in this field [8], we have demonstrated that, when ectopically overexpressed in isolated hepatocytes, SREBP-1c enhanced the transcription of GK promoter in a dose-dependent manner. Although a role for SREBP in controlling GK transcription cannot be ruled out, the striking observation that the basal activity of GK–CAT fusion gene was insensitive to insulin put into question the role of SREBP in mediating insulin effects. Indeed, its involvement in insulin early effect seems rather unlikely since, in the same culture of hepatocytes, the accumulation of mature SREBP-1 protein was not seen in the nucleus 2 h after insulin addition, whereas GK gene was already actively transcribed. These data indicated that factor(s) other than SREBP-1 could be essential for induction of GK gene transcription following insulin chal-
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lenge. However, Kim et al. [9] demonstrated by CHIP assay the binding of SREBP-1 to GK promoter in liver of rats refed a high carbohydrate/fat-free diet for 24 h. Therefore, SREBP could play a critical role in sustaining long term GK gene transcription in the presence of insulin. Finally, SREBP-1 knockdown, using the RNA interference technique, severely reduced the effects of insulin and 25 mM glucose on FAS gene expression, but not on GK and L-PK gene expression. These results correlated with the observed response of GK to carbohydrate refeeding in SREBP-1c knockout mice [23]. Considered altogether, these data definitely established that SREBP-1c is not the early mediator of insulin effect upon GK gene transcription. The decrease, rather than the extinction of FAS gene transcription upon SREBP-1 knockdown, is in line with previous studies suggesting that SREBP-1 works in concert with other factor(s) for full induction of FAS gene transcription [24–26]. By contrast, L-PK gene transcription has been demonstrated to be SREBP-1independent [27,28]. A glucose responsive element (ChoRE) was defined within the L-PK and FAS promoters [29–31] and a transcription factor, ChRE-binding protein (ChREBP), recognizing this sequence was identified [32,33]. Its functionality was confirmed by transfection experiments [34], RNA interference [7] and finally by gene disruption [35]. In conclusion, by performing time-course studies and knockdown of SREBP-1 in cultured rat hepatocytes, we demonstrated that the induction of GK gene expression by insulin could occur in the absence of mature SREBP-1 in the nucleus. Acknowledgements: We thank Dr. Carina Prip-Buus for helpful comments on the manuscript and Aminata Kone´ for invaluable help with Western-blotting.
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Insulin regulation of glucokinase gene expression: Evidence against a role for sterol regulatory element binding protein 1 in primary hepatocytes Claudine Gregori, Isabelle Guillet-Deniau, Jean Girard, Jean-Franc¸ois Decaux, Anne-Lise Pichard* De´partement d’Endocrinologie, Institut Cochin, Institut National de la sante´ et de la Recherche Me´dicale (INSERM) U567, Centre National de la Recherche Scientifique (CNRS), Unite´ Mixte de Recherche 8104, Universite´ Rene´ Descartes, 24 rue du Faubourg Saint-Jacques, 75014 Paris, France Received 17 October 2005; revised 9 December 2005; accepted 9 December 2005 Available online 20 December 2005 Edited by Robert Barouki
Abstract Liver key genes for carbohydrate and lipid homeostasis are regulated by insulin and glucose. The sterol regulatoryelement binding protein-1c (SREBP-1c) has emerged as a mediator of insulin effects on gene transcription, particularly on glucokinase (GK). In this paper, we show that despite stimulation of GK promoter transcription by overexpression of mature SREBP-1c, insulin induced GK transcription at least 4 h ahead of accumulation of mature SREBP-1c in the nucleus. Importantly, the knockdown of SREBP-1 mRNA using a RNA-interference technique reduced the level of nuclear SREBP-1 protein, diminished fatty acid synthase mRNA level, but did not affect GK and L-pyruvate kinase mRNA levels. We concluded that SREBP-1 is unlikely to be the mediator of the early insulin effect on GK gene transcription. 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Glucokinase; Small interfering RNA; Gene expression; Sterol regulatory element binding protein; Insulin
1. Introduction Transcriptional activation of liver glycolytic and lipogenic genes, a relatively delayed phenomenon, requires the presence of both glucose and insulin, neither of which being active alone [1,2]. In contrast, stimulation of glucokinase (GK) gene, coding for the first enzyme involved in glucose metabolism, is a rapid strictly insulin-dependent and glucose-independent process [3,4]. When GK is overexpressed either in hepatoma cells [5] or in liver of diabetic mice [6], the transcriptional activation of glycolytic and lipogenic genes becomes insulin-independent and glucose-dependent. Similarly, in absence of GK in hepatic GK-KO mice, glycolytic and lipogenic genes are not induced after refeeding a high carbohydrate diet [7]. Therefore, insulin induction of GK gene expression is the key step to subsequent activation of glycolytic and lipogenic gene expression by glu-
* Corresponding author. Fax: +33 1 53 73 27 03. E-mail address: [email protected] (A.-L. Pichard).
Abbreviations: GK, glucokinase; L-PK, L-pyruvate kinase; FAS, fatty acid synthase; PEPCK, phosphoenolpyruvate kinase; SREBP, sterol regulatory element binding protein; CAT, chloramphenicol acetyltransferase; siRNA, small interfering RNA; SRE, sterol regulatory element; ChoRE, carbohydrate responsive element; ChREBP, carbohydrate responsive element binding protein
cose in liver. However, the molecular mechanisms by which insulin regulates GK gene expression are still controversial and the cis-acting elements mediating insulin effects remain unknown. The transcription factor sterol regulatory-element binding protein-1c (SREBP-1c), whose transcription is also insulin-dependent, was pointed out as the main mediator of insulin effect on GK gene expression [8,9], but other studies failed to confirm this finding [10,11]. Therefore, whether or not SREBP1-c mediates the action of insulin on GK gene expression is still a controversial issue. Owing to the central role of insulin in the control of liver metabolism, and of GK in glucose metabolism, the goal of this study was to reinvestigate the role of SREBP-1c in mediating insulin effect upon GK gene expression. For this purpose, we studied in primary cultured hepatocytes the kinetics of insulin upregulation of GK gene transcription as well as the level of transcriptionally active SREBP-1 protein in the nucleus. Further, SREBP-1 knockdown by small interfering RNAs (siRNAs) resulted in an impaired induction of fatty acid synthase (FAS) gene in response to glucose and insulin, but did not prevent the induction of GK and L-pyruvate kinase (L-PK) genes. We clearly demonstrated that early insulin stimulation of GK gene was SREBP-1-independent.
2. Materials and methods 2.1. Primary hepatocyte culture Hepatocytes were isolated from liver of 8-week-old male Wistar rats after perfusion with collagenase. Hepatocytes were seeded at a density of 3.5 · 106 cells per dish (20 cm2) in M199 medium with Earl’s salts (Invitrogen) supplemented with 100 U/ml penicillin, 100 lg/ml streptomycin, 0.1% (w/v) BSA and 2% (v/v) Ultroser G (BioSepra). After a 4 h-incubation at 37 C under 5% (v/v) CO2, the cells were washed with PBS and switched for 16 h to a BSA–Ultroser-free medium. The medium was then supplemented with 1 lM dexamethasone, in the absence or presence of insulin and/or glucose as indicated in the figure legends. 2.2. Plasmid construction and transient transfection GK promoter construct: The mouse GK–chloramphenicol acetyltransferase (CAT) plasmid is the PeCAT vector [12] in which the fragment (960 to +19 bp) of the mouse liver GK promoter was subcloned, along with two fragments (+145 to +272 and +4035 to +4120) corresponding to the splice sequence in 5 0 and 3 0 of the first intron, respectively. These splice fragments were obtained by PCR using added restriction sites (underlined): For the 3 0 splice: 5 0 -TATGGTACCCTTGGTGTGTGGTGGCTT-3 0 and 5 0 -TTAGAGCTCTGCCAGGATCTGCTCTACC-3 0 . For the 5 0 splice: 5 0 -TTACCTTGGAG CCCAGTCGTTGACTCT-3 0 and 5 0 -TTCGGATCCTACAGGATCGCACTCA-3 0 . The PCR product for the 3 0 splice fragment was
0014-5793/$32.00 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2005.12.032
C. Gregori et al. / FEBS Letters 580 (2006) 410–414 digested by Kpn1 and Sst1, and subcloned into PeCAT digested with the same enzymes. The PCR product for the 5 0 splice and the promoter fragments were first digested by Sty1 and then ligated together. The resulting fragment was digested by HindIII and BamH1 and subcloned in the above-described plasmid digested with the same enzymes. All fragments generated by PCR were sequenced. SREBP-1c expression plasmid: SREBP-1c plasmid was the pSVSport1-ADD1 (403) coding for the nuclear transcription factor [13] (gift of Dr. B.M. Spiegelman, Harvard Medical School, Boston, MA). Transient transfections: Transfections were carried out as previously described [14]. In each experiment, 7.5 lg of the CAT plasmid and 2.5 lg of the luciferase plasmid were cotransfected. The luciferase standardization pRSV plasmid was used to monitor variations in transfection efficiency. The CAT assay and luciferase assay were performed as previously described [12]. 2.3. RNA isolation and Northern-blot analysis Total RNA extraction from cultured hepatocytes was performed using the RNAzol B reagent (Q Biogene) according to the manufacturer’s instructions. 15 lg aliquots of total RNA were electrophoretically separated in denaturating formaldehyde agarose gels and transferred to Hybond N+ membranes (Amersham). The cDNA probes were labeled with (a-32P) dATP using random prime labeling (Amersham-Pharmacia Biotech). The membranes were hybridized with the indicated 32P-labeled probe (106 cpm/ml) at 42 C overnight. The GK, L-PK, phosphoenolpyruvate kinase (PEPCK), FAS and SREBP-1 probes were obtained as previously described [15]. Northern blots were hybridized with a 32P-labeled ribosomal 18S probe to verify that equivalent amounts of total RNA were loaded in each lane.
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gene expression was induced in a concentration-dependent manner (Fig. 1). At the maximum amount tested (1.25 lg), the control expression vector had no effect (data not shown) whereas the constitutively active SREBP1-c stimulated 2.5-fold the GK promoter activity. Strikingly, insulin treatment failed to induce basal GK–CAT fusion gene expression (Fig. 1A point 0 lg). These data indicated that the SREBP binding sites may well be functional but requestioned the role of SREBP-1c as a major mediator of insulin action on GK gene expression. To ascertain these results, the well-established effects of insulin on endogenous GK mRNA level [3,18] were measured, in the same pool of cultured hepatocytes but untransfected. We observed the expected rapid (2 h) time-dependent accumulation of GK mRNA in the presence of insulin and low glucose concentration (5 mM) (Fig. 1B), indicating that insulin signaling
2.4. Immunoblot analysis Nuclear extracts and membrane fractions were prepared from cultured hepatocytes using the NE-PER Nuclear and Cytoplasmic Extraction Reagents Kit (Pierce) according to the instructions provided by the manufacturer. Protein concentration was determined using the Bradford method (Bio-Rad). Proteins (35 lg) were separated by SDS–PAGE (7% polyacrylamide gel) and transferred to a nitrocellulose membrane (Bio-Rad). Immunoblot analysis was performed using a monoclonal anti-SREBP-1 antibody (clone 2A4 from NeoMarkers), a Lamin A/C antibody from Cell Signalling and the Enhanced Chemiluminescence System (Supersignal, Pierce). 2.5. Design, synthesis and transfection of siRNA A target sequence in the rat SREBP-1 mRNA (GenBank Accession No. L16995) [13] was identified following the principles described by Elbashir et al. [16] as previously described [17]. Primary rat hepatocytes were cultured in antibiotic-free medium the day before transfection. Cells transfection was conducted using Lipofectamine 2000 according to the supplier’s instruction (Invitrogen). On day 1, hepatocytes were transfected with 200 pmol siRNA per 20-cm2 dish. On day 2, the medium was supplemented with 100 nM insulin and 25 mM glucose. Twenty-four hours later, cells were harvested and either RNA or proteins were prepared.
3. Results 3.1. Regulation of GK gene transcription by SREBP-1c and insulin in cultured rat hepatocytes Two potential binding sites for the transcription factor SREBP were identified, by computer analysis, in the mouse GK promoter (113 to 106 and 65 to 55). To test SREBP-1c effect upon GK fusion gene expression we used primary cultured rat hepatocytes. We transiently transfected cells with a GK–CAT fusion gene, containing the promoter sequence located between 960 and +19, and either a control expression vector or the same vector encoding nSREBP-1c [13]. The cells were cultured in the absence or presence of insulin. When SREBP-1c was overexpressed, GK–CAT fusion
Fig. 1. Regulation of GK gene transcription by SREBP-1c and insulin in cultured rat hepatocytes. (A) Primary hepatocytes cultured for 16 h in the presence of 5 mM glucose without insulin were then transfected with 7.5 lg of CAT–GK fusion gene, 2.5 lg of the reference plasmid RSV luciferase and the indicated amount of expression vector for mature SREBP-1c. Cells were cultured in medium containing 5 mM glucose, in the absence (h) or presence (d) of 100 nM insulin. CAT activity was normalized according to transfection efficiency by measuring the luciferase activity. Values are means of four separate experiments performed in duplicate. (B) Time course of GK and SREBP-1 mRNA accumulation in hepatocytes cultured in the presence of 5 mM glucose and 100 nM insulin. Total mRNA (15 lg) were subjected to electrophoresis and blot hybridization with the indicated 32 P-labeled cDNA probe. (C) Time-course appearance of SREBP-1 precursor (P) and mature (M) proteins. Membrane pellet and nuclear extract (35 lg protein) were subjected to SDS/PAGE and analyzed by immunoblotting using an antibody against the precursor and mature forms of SREBP-1. Lamin A/C was used as control for the nuclear extract. The blots are representative of three independent experiments.
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is functional in these cultured cells. In the same experiments, SREBP-1 mRNA accumulated in parallel with GK mRNA (Fig. 1B). We next measured endogenous SREBP-1 protein levels both in membrane and nuclear extracts. SREBP-1 precursor amount reflected the insulin-induced upregulation of SREBP-1 mRNA (Fig. 1C). In marked contrast, mature form of SREBP-1 protein, which serves as an active transcription factor, was undetectable during the first 6-h incubation with insulin and was highest after 24 h (Fig. 1C). If SREBP-1 was the insulin-dependent transcription factor controlling GK gene transcription, we would expect to detect SREBP-1 protein in nuclear extracts ahead to accumulation of GK mRNA. Altogether, these observations argued against SREBP-1 being the prime mediator of insulin effect upon GK gene transcription. 3.2. Glycolytic and lipogenic gene expression in hepatocytes In order to check the functionality of metabolic responses in cultured hepatocytes, we measured mRNA levels of the key glycolytic (L-PK), lipogenic (FAS) genes known to be induced by insulin and/or glucose [19] and gluconeogenic (PEPCK) gene known to be repressed by insulin. Hepatocytes were incubated for 16 h in the presence of low glucose (5 mM) and no insulin. Thereafter, the cells were incubated for 24 h with high glucose (25 mM) and insulin (100 nM). As expected, Fig. 2 shows an increase in GK and SREBP-1 mRNAs within 2 h followed by L-PK and FAS mRNAs at 24 h as well as the marked suppression of PEPCK mRNA after 2 h. These results demonstrated that insulin and glucose signalings were maintained in cultured hepatocytes. 3.3. Silencing of SREBP-1 in hepatocytes To test the direct control of GK gene transcription by SREBP-1 in another way, hepatocytes were transfected with a SREBP-1 siRNA duplex and then maintained for 24 h in the presence of insulin and high glucose concentration to induce transcription of control genes such as FAS. Both SREBP-1 mRNA and nuclear active SREBP-1 protein were severely reduced in transfected cells (Fig. 3A and B), indicating
Fig. 2. Glycolytic and lipogenic genes induction by glucose and insulin. Hepatocytes cultured 16 h in the presence of 5 mM glucose without insulin (time 0) were maintained for a further 24 h in the presence of 25 mM glucose and 100 nM insulin. Total RNA (15 lg) were subjected to blot hybridization with the indicated 32P-labeled cDNA probe. The blots are representative of three independent experiments.
Fig. 3. Effect of SREBP-1 knockdown by RNA interference (siRNA) on glycolytic and lipogenic gene expression in rat hepatocytes. Twelve hours after transfection with a SREBP-1 siRNA duplex as described in Section 2, hepatocytes were cultured for 24 h in the presence of 100 nM insulin and 25 mM glucose. Total mRNA, membrane and nuclear extracts were then prepared. (A) Western blotting was performed to detect SREBP-1 precursor (P) and mature (M) proteins. Lamin A/C was used as control for the nuclear extract. (B) Total RNA (15 lg) were subjected to blot hybridization with indicated 32P-labeled cDNA probes Northern blots were scanned and quantified. Each value represents the amount of mRNA relative to that in the untransfected cells which is arbitrarily set to 100. Values represent means ± S.E. of data from three independent experiments.
an efficient knockdown of SREBP-1 expression. Under these conditions, we observed a marked impairment of FAS mRNA accumulation, but no change in L-PK and GK mRNAs (Fig. 3B). These results confirmed SREBP-1 as being a mediator of insulin effects upon FAS gene transcription, but definitively ruled out its role in controlling GK and L-PK gene expression.
4. Discussion The SREBPs are a family of three nuclear transcription factors encoded by two genes. SREBP-1a and SREBP-1c derive from the same gene, and both appear to regulate lipogenic gene transcription [20]. Despite being a weak activator [21], SREBP-1c is the dominant isoform in liver where its regulation is insulin-dependent [22]. The purpose of the present work was to clarify the involvement of SREBP-1 in insulin-mediated induction of GK. In agreement with studies in this field [8], we have demonstrated that, when ectopically overexpressed in isolated hepatocytes, SREBP-1c enhanced the transcription of GK promoter in a dose-dependent manner. Although a role for SREBP in controlling GK transcription cannot be ruled out, the striking observation that the basal activity of GK–CAT fusion gene was insensitive to insulin put into question the role of SREBP in mediating insulin effects. Indeed, its involvement in insulin early effect seems rather unlikely since, in the same culture of hepatocytes, the accumulation of mature SREBP-1 protein was not seen in the nucleus 2 h after insulin addition, whereas GK gene was already actively transcribed. These data indicated that factor(s) other than SREBP-1 could be essential for induction of GK gene transcription following insulin chal-
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lenge. However, Kim et al. [9] demonstrated by CHIP assay the binding of SREBP-1 to GK promoter in liver of rats refed a high carbohydrate/fat-free diet for 24 h. Therefore, SREBP could play a critical role in sustaining long term GK gene transcription in the presence of insulin. Finally, SREBP-1 knockdown, using the RNA interference technique, severely reduced the effects of insulin and 25 mM glucose on FAS gene expression, but not on GK and L-PK gene expression. These results correlated with the observed response of GK to carbohydrate refeeding in SREBP-1c knockout mice [23]. Considered altogether, these data definitely established that SREBP-1c is not the early mediator of insulin effect upon GK gene transcription. The decrease, rather than the extinction of FAS gene transcription upon SREBP-1 knockdown, is in line with previous studies suggesting that SREBP-1 works in concert with other factor(s) for full induction of FAS gene transcription [24–26]. By contrast, L-PK gene transcription has been demonstrated to be SREBP-1independent [27,28]. A glucose responsive element (ChoRE) was defined within the L-PK and FAS promoters [29–31] and a transcription factor, ChRE-binding protein (ChREBP), recognizing this sequence was identified [32,33]. Its functionality was confirmed by transfection experiments [34], RNA interference [7] and finally by gene disruption [35]. In conclusion, by performing time-course studies and knockdown of SREBP-1 in cultured rat hepatocytes, we demonstrated that the induction of GK gene expression by insulin could occur in the absence of mature SREBP-1 in the nucleus. Acknowledgements: We thank Dr. Carina Prip-Buus for helpful comments on the manuscript and Aminata Kone´ for invaluable help with Western-blotting.
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