Role of Peroxisome Proliferator-Activated Receptor- Coactivator-1 in the Transcriptional Regulation of the Human Uncoupling Protei
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《内分泌学杂志》
Department of Laboratory Medicine (H.O., K.K., T.K.F., W.P.), Landeskliniken and Paracelsus Private Medical University Salzburg, A-5020 Salzburg, Austria
the Department of Internal Medicine (F.K.), Krankenhaus Hallein, A-5400 Hallein, Austria
Abstract
A role of uncoupling protein 2 (UCP2) as negative modulator of insulin secretion has been suggested, but the transcriptional pathways regulating -cell UCP2 gene expression have been established in rodents only. We show here that the underlying sequence motifs are not conserved in the human gene and provide evidence for regulatory mechanisms involving the transcriptional cofactor peroxisome proliferator-activated receptor- coactivator-1 (PGC-1). PGC-1 potentiates thyroid hormone (T3)-mediated transcriptional activation of the human UCP2 gene in INS-1E cells. Two thyroid hormone response elements (TREs) located at –322/–317 (TRE1) and –170/–165 (TRE2) were identified, and mutation of either TRE1 or TRE2 abrogated the stimulatory effect of T3 treatment. Furthermore, two E-box motifs at –911/–906 (E1) and –743/–738 (E2) are involved in the regulation of UCP2 gene expression by sterol regulatory element binding protein isoforms (SREBP)-1a, -1c, and -2. Mutational analysis revealed that the presence of either E1 or E2 is sufficient to mediate activation of UCP2 gene transcription by nuclear active SREBPs. PGC-1 coactivates liver X receptor-mediated expression of SREBP-1c as well as dexamethasone-stimulated SREBP-2 expression in INS-1E cells. These transcriptional responses are antagonized by orphan nuclear receptor short heterodimer partner overexpression, which might explain its positive effects on glucose-stimulated insulin secretion in -cells overexpressing UCP2. We also provide evidence that despite a lack of sequence homology within the regulatory region, the principal mechanisms regulating UCP2 gene expression are similar in rats and humans, being consistent with a role for UCP2 as a modulator of insulin secretion in humans.
Introduction
TYPE 2 DIABETES MELLITUS (T2DM) is a heterogeneous metabolic disorder characterized by marked disturbances in glucose homeostasis. -Cell dysfunction represents a central abnormality in the pathophysiology of T2DM (1). Glucose-stimulated insulin secretion (GSIS) is tightly linked to glucose metabolism through oxidative phosphorylation as an increase in the intracellular ATP to ADP ratio triggers exocytosis of insulin containing secretory vesicles (2). Mitochondrial uncoupling proteins (UCPs) have been implicated in the regulation of GSIS by facilitating reentry of protons into the mitochondrial matrix thereby decreasing ATP synthesis (3, 4).
UCP2, a member of the UCP family, is expressed in several tissues including muscle, liver, white fat, and pancreas (5). A role of UCP2 as negative modulator of insulin secretion has been demonstrated by studies in isolated pancreatic islets derived from UCP2–/– mice showing elevated ATP levels and increased insulin release (6). UCP2-deficient animals were characterized by an increased acute insulin secretion rate in response to glucose, compared with controls (7). In addition, adenoviral overexpression of UCP2 in pancreatic -cells decreased their ATP content and blunted GSIS (8). In type 2 diabetic subjects with impaired GSIS, isolated pancreatic islets showed increased UCP2 gene expression and alterations in mitochondrial morphology (9). Furthermore, a quantitative trait locus for glucose homeostasis phenotypes reflecting -cell function has been mapped to a genomic region encompassing the human UCP2 gene locus (10) and associations of a common –866G/A UCP2 promoter polymorphism with indices of -cell function and T2DM risk have been observed in different populations (11, 12, 13, 14, 15). The pancreatic transcription factor paired box domain transcription factor-6 was shown to preferentially bind to and more effectively trans activate the –866A than the –866G allele (11). In vitro studies revealed lower insulin secretion rates in response to glucose stimulation in isolated pancreatic islet cells from –866A/A nondiabetic donors (14). In addition, the –866G/A polymorphism affected -cell UCP2 gene expression and was a determinant of insulin secretion in Japanese subjects (13). Taken together these data strongly suggested a role of UCP2 as negative regulator of insulin secretion (4).
The transcriptional coactivator peroxisome proliferator-activated receptor- coactivator (PGC)-1 has been implicated in the transcriptional control of distinct metabolic pathways involved in the pathogenesis of T2DM such as hepatic gluconeogenesis, muscle glucose uptake, mitochondrial biogenesis, and respiration as well as GSIS (16). Ectopic overexpression of PGC-1 negatively regulates insulin secretion in isolated rat islets and a -cell line and suppresses membrane depolarization without affecting the basal secretory apparatus (17). It was suggested that inhibition of insulin release by PGC-1 might be mediated by up-regulation of glucose-6-phosphatase and down-regulation of GLUT2, glucokinase, and glycerol-3-phosphatase gene expression, blunting the glucose-dependent rise in cellular ATP (17). However, a coordinate up-regulation of pancreatic UCP2 and PGC-1 gene expression has been observed in several animal models of T2DM including Zucker fatty rats and ob/ob mice that are characterized by marked defects in insulin secretion (6, 17, 18). Furthermore, a role of PGC-1 in the cold-induced up-regulation of UCP2 has been suggested from studies in rat pancreatic islets (19). Our present knowledge about the transcriptional control of -cell UCP2 gene expression is solely based on studies of the rodent gene. A multipartite enhancer in the proximal mouse UCP2 promoter (–86/–44) comprising a specificity protein-1 binding site, a sterol regulatory response element, and a double E-Box motif mediates stimulatory effects of oleic acid on UCP2 gene expression (20, 21). We therefore asked whether PGC-1 might contribute to the transcriptional regulation of the human UCP2 gene. In the present study, we characterized regulatory sequences within the human UCP2 promoter region that mediate effects of PGC-1 on -cell UCP2 gene expression.
Materials and Methods
Materials
DNA restriction and modification enzymes were obtained from New England Biolabs (Beverly, MA) and Promega (Madison, WI). Cell culture media, fetal calf serum, and LipofectAMINE 2000 were obtained from Life Technologies (Groningen, Netherlands). T3, dexamethasone, 22(R)-hydroxycholesterol [22(R)HC], and 9-cis retinoic acid (9cRA) were purchased from Sigma (St. Louis, MO). The dual-luciferase assay system was purchased from Promega.
Plasmid constructs
The UCP2-Prom-Luc plasmid in which the region from –1381 to +123 of the human UCP2 gene drives the promoterless firefly luciferase gene was generated by subcloning of a SacI/XhoI fragment from a 3241-bp UCP2 promoter construct described previously (22) into the pGL3-Basic vector (Promega). Several reporter constructs originating from the original UCP2-Prom-Luc plasmid and harboring nested unidirectional 5' deletions of the UCP2 promoter region were generated using the exonuclease (Exo)III/S1 deletion kit (Fermentas, Vilnius, Lithuania). Linearized plasmid DNA was digested with ExoIII followed by treatment of timed aliquots of the ExoIII reaction with S1 nuclease to remove the 5' single-stranded overhangs. Double-stranded linear fragments were separated by gel electrophoresis and appropriately sized plasmids were religated to generate the UCP2-1 (–983/+123), UCP2-2 (–879/+123), and UCP2-3 (–351/+123) constructs, respectively.
The Mut-TRE1 construct was generated by site-directed mutagenesis of the UCP2-3 plasmid using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) and 5'-CAAGAGGTGTGTGATTTTACAAGACTTGTTTCTG-3' and 5'-CAGAAACAAGTCTTGTAAAATCACACACCTCTTG-3' as forward and reverse mutagenic primers, respectively. For the construction of the Mut-TRE2 plasmid 5'-CGCACGGAAACGGGATTTTAGAAAGTCATGGAG-3' and 5'-CTCCATGACTTTCTAAAATCCCGTTTCCGTGCG-3' were used as forward and reverse mutagenic primers, respectively. Mutated nucleotide positions are in bold. Plasmid Mut-TRE1/2 was obtained by mutagenic PCR starting from Mut-TRE1 using primers for Mut-TRE2 described above. For generation of a human thyroid hormone receptor- expression construct 5'-CAAGGCTAGCAACCTATGACTCCCAACAGTATG-3' (–5/+18) and 5'-GAAGCTCGAGGTCTAATCCTCGAACACTTCCA3–3' (+1366/+1387) were used as forward and reverse primers, respectively, to amplify a 1392-bp cDNA fragment, which was cloned into pcDNA6C-V5/His (Invitrogen, Carlsbad, CA). Numbers in parentheses refer to primer positions relative to the translational start site (GenBank accession no. NM_000461). NheI and XhoI sites introduced into the primer sequences are in bold and the ATG start codon is underlined.
The Mut-E1 construct was derived from the UCP2-1 construct by QuickChange PCR-mediated site-directed mutagenesis using 5'-TACTCCCCAAACGCTTTTGTTTGTCCCGGCCAG-3' and 5'-CTGGCCGGGACAAACAAAAGCGTTTGGGGAGTA-3' as forward and reverse primers, respectively. For the generation of the Mut-E2 plasmid 5'-CCCGGCTCTGCCTTGTCTTTTGCGGGGGCCGGC-3' and 5'-GCCGGCCCCCGCAAAAGACAAGGCAGAGCCGGG-3' were used as forward and reverse primers, respectively. Plasmid Mut-E1/2 was obtained from the Mut-E1 construct using mutagenic primers for Mut-E2 described above. Mutated nucleotides are in bold. All constructs described were verified by dye terminator cycle sequencing using the ABI Prism 310 genetic analyzer (Applied Biosystems, Foster City, CA). The hPGC-1 and LXR expression plasmids have been described previously (23, 24).
For generation of a nuclear active sterol regulatory element-binding protein (SREBP)-1a expression plasmid (naSREBP-1a) harboring amino acids 1–487 (NM_004176), a 1487-bp cDNA fragment, was amplified starting from human liver total RNA using 5'-GAGAGGATCCAAAATGGACGAGCCACCCTTCAG-3' and 5'-GAGACTCGAGCTAGCGGGAGCGGTCCAGCATGC-3' as forward and reverse primers, respectively, and cloned into pcDNA6C-V5/His (Invitrogen). For construction of naSREBP-1c containing amino acids 1–463 (NM_004176), 5'-GAGAGGATCCAAAATGGACGAGCCACCCTTCAG-3' and 5'-GAGACTCGAGCTAGCGGGAGCGGTCCAGCATGC-3' were used as forward and reverse primers, respectively. BamHI and XhoI sites introduced into the primer sequences are underlined and the ATG start codon is in bold. Plasmid naSREBP-2 harboring amino acids 1–435 (NM_004599) was cloned using NheI and XhoI sites introduced into 5'-GAGAGCTAGCATGGACGACAGCGGCGAGCT-3' and 5'-GAGACTCGAGTTAGGCTGGGGGGGACATCAGAA-3' as forward and reverse primers, respectively.
The SREBP-2-Prom-Luc construct containing a 4182-bp fragment of the human SREBP-2 promoter was generated using 5'-CAACGCGTCCAGTCCCACATCTTCCTCC-3' (+59 to +39) and 5'-ACTAGATCTGCACTCCCACCCACCCAC-3' (–4123 to –4113) as forward and reverse primers, respectively. The amplified DNA fragment was cloned into the pGL3-Basic vector (Promega). Numbers in parentheses refer to primer positions relative to the transcriptional start site (GenBank accession no. NM_004599). MluI and BlgII restriction enzymes sites introduced into the primer sequences are in bold. The SREBP-1c promoter reporter construct SREBP-1c-Prom-Luc has been described previously (25). An expression plasmid containing the coding region of the human short heterodimer partner (SHP) gene was obtained from the American Type Culture Collection (Manassas, VA; clone MGC-34176).
Promoter in silico analysis
The Basic Local Alignment Search Tool (BLAST, www.ncbi.nlm.nih.gov/BLAST) was used to identify regions of local similarity between the mouse and human UCP2 promoter sequence. For the prediction of putative transcription factor binding sites, the public domain MatInspector professional software (www.genomatix.gsf.de/cgi-bin/matinspector) version 2.2 was used together with the TRANSFAC 4.0 database containing a large library of predefined matrix descriptions for known transcription factor recognition sequences. A core similarity of 0.85 or greater and a matrix similarity of 0.90 or greater was used as a cut-off for consideration of potential query sequence matches.
Cell culture, transfections, and reporter-gene assays
INS-1E cells were cultured in 24-well dishes and transfected using LipofectAMINE 2000 reagent as described (11). Before transfection, cells were washed twice with Hanks’ balanced salt solution (Sigma) and drugs at concentrations of 10 μM for T3, dexamethasone, 22(R)HC and 9cRA were added. Unless otherwise indicated, we used 1 μg of reporter plasmids, 0.5 μg of expression plasmids, and 20 ng of pRL-TK plasmid (Promega) as transfection control. Cells were collected 24 h after transfection and firefly and renilla luciferase activities were measured in a luminometer (Anthos Labtec Instruments, Salzburg, Austria) using the dual-luciferase reporter assay system (Promega). Graphs show means of three experiments, each performed in quadruplicates. Results are given as means ± SD. For post hoc comparisons of means, Tukey’s honest significant difference test was used. Two-way ANOVA was used to compare transactivation of reporter gene constructs by trans factors.
EMSAs
Nuclear extracts from INS-1E cells were prepared, and EMSAs were performed as described previously (23, 24). INS-1E cells were either incubated without serum for cholesterol depletion or stimulated with T3 at a concentration of 10 μM for binding studies with E-Box1 or -2 and TRE1 or -2, respectively. Sense strand sequences of oligonucleotides E-Box1 and E-Box2 were 5'-aattTTCTACTCCCCAAACGACACGTGTTTGTCCCGGCCAGAGG-3' and 5'-aattGCCCGGCTCTGCCTTGTCACGTGCGGGGGCCGGCCCGTTT-3', respectively. Sense strand sequences of oligonucleotides TRE1 and TRE2 were 5'-aattAAGGCAAGAGGTGTGTGACTGGACAAGACTTGTTTCTGGC-3' and 5'-aattGGACGCACGGAAACGGGAGTGGAGAAAGTCATGGAGAGAA-3', respectively. Unlabeled competitor oligonucleotides were added at 20- and 100-fold molar excess. Overhangs are represented by lowercase letters.
Quantification of UCP2 gene expression and insulin secretion levels in INS-1E cells
Total RNA was isolated from INS-1E cells incubated for 48 h with T3, a combination of 22(R)HC and 9cRA or dexamethasone at a concentration of 10 μM for each drug as indicated. Equal amounts of RNA were reverse transcribed and cDNAs were quantified by real-time PCR using the SYBR Green PCR master mix (Applied Biosystems) as described (25). For quantification of UCP2 mRNA expression 5'-GACAGATGACCTCCCTTGCCAC-3' (+630 to +651) and 5'-CAGGAACCCAAGCGGAGAAAG-3' (+828 to +848) were used as forward and reverse primers, respectively. Numbers in parentheses refer to primer positions relative to the translational start site (GenBank accession no. NM_003355). cycle threshold values for UCP2 gene expression were normalized for the expression of acidic ribosomal protein p 0 as described (25).
INS-1E cells grown in 6-well plates were incubated for 48 h with T3, a combination of 22(R)HC and 9cRA or dexamethasone at a concentration of 10 μM for each drug as indicated. Cells were then washed with Krebs-Ringer bicarbonate buffer containing 129 mM NaCl, 4.8 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 2.5 mM CaCl2, 5 mM NaHCO3, 0.2% BSA, and 10 mM HEPES (pH 7.4). After a preincubation period of 30 min in Krebs-Ringer bicarbonate buffer containing 1 mM glucose, INS-1E cells were incubated for 60 min with the concentrations of glucose as indicated. Insulin secreted into the supernatant was measured using a rat insulin ELISA kit (Linco Research Inc., St. Charles, MO). Graphs show means of three experiments each performed in triplicate. Results are given as means ± SD.
Results
In silico analysis of the human UCP2 gene promoter was conducted in an attempt to identify potential regulatory regions. A homology search between the proximal human and mouse UCP2 promoters as well as sequence comparisons with an isolated mouse enhancer region (–86/–44) failed to provide significant alignment scores with higher-than-average sequence homology. Overall sequence identity along 1000 nucleotides of the mouse and human 5' regulatory region upstream of exon 1 was less than 55% with more than 22% gaps introduced to achieve optimal alignment results. Best hits obtained with the mouse enhancer region revealed two or more mismatches within the core region of the respective transcription factor binding sites. Analysis of the proximal UCP2 promoter region for potential cis regulatory binding sites using the MatInspector software revealed a high probability score for the presence of two potential thyroid hormone response elements (TREs) at –322/–317 (TRE1) and –170/–165 (TRE2) as well as two E-Box consensus binding sites at –911/–906 (E1) and –743/–738 (E2), respectively (Fig. 1).
The thyroid hormone receptor (TR) is a well-established target of PGC-1 action (26) and thyroid hormone is known to inhibit insulin secretion (27, 28). To determine direct effects of PGC-1 on -cell UCP2 gene expression via coactivation of TR, we performed cotransfection studies in INS-1E cells. An UCP2 promoter reporter construct (UCP2-Prom-Luc) was transiently transfected into INS-1E cells together with a PGC-1 expression plasmid (hPGC-1) in both the absence and presence of T3 (Fig. 2A). hPGC-1 as well as T3 alone stimulated basal transcriptional activity of the UCP2-Prom-Luc construct, and cotransfection with hPGC-1 further augmented T3-mediated reporter gene expression. Ectopic overexpression of the TR had only minor effects on basal UCP2 reporter gene expression but significantly increased UCP2-Prom-Luc transcriptional activity in T3-treated cells in both the absence and presence of hPGC-1 (Fig. 2A). A similar response was observed with the UCP2-3 reporter construct that was generated by ExoIII deletion and encompassed a 351-bp UCP2 promoter fragment retaining both TREs (data not shown). Possible effects of TRE1 and TRE2 were determined using site directed mutagenesis. Mutation of TRE1 or TRE2 (Mut-TRE1 or Mut-TRE2) as well as disruption of both response elements (Mut-TRE1/2) completely abolished the stimulatory effect of T3 and PGC-1 on UCP2 gene expression (Fig. 2B).
Studies in mice suggested a pivotal role of SREBPs in the transcriptional regulation of the UCP2 gene via a promoter element not conserved in humans (20, 21). We therefore determined the effects of SREBP isoforms on human UCP2 gene expression. Transient cotransfections were performed in INS-1E cells using UCP2-Prom-Luc together with expression plasmids for nuclear active SREBP-1a, -1c, and -2 isoforms (Fig. 3). Cells were incubated with cholesterol/25(R)-hydroxycholesterol to suppress endogenous SREBP activity. Overexpression of SREBP-1a and -1c resulted in a approximately 2-fold increase in UCP2 promoter activity, whereas overexpression of SREBP-2 exhibited an even more pronounced effect (6-fold induction). ExoIII mapping was used to address the role of the two putative E-Box motifs (E1 and E2). Deletion of the genomic region upstream of E1 and E2 (UCP2-1) did not affect SREBP-2 mediated transactivation. A reporter construct devoid of E1 (UCP2-2) elicited slightly reduced luciferase activity, whereas removal of both E-Boxes (UCP2-3) resulted in complete loss of SREBP-2-dependent stimulation of UCP2 promoter activity (Fig. 4). Similar results were obtained with SREBP-1a and -1c isoforms (data not shown). Site-directed mutagenesis was used to establish the individual contributions of E1 and E2 in the regulation of the human UCP2 gene. Both E-Boxes, E1 and E2, mediated effects of SREBP-2 on UCP2 gene expression, with E1 eliciting a stronger effect. Simultaneous disruption of E1 and E2 completely abolished SREBP-2-dependent stimulation of UCP2 promoter activity (Fig. 4).
EMSAs were performed to study possible interactions of nuclear proteins with regulatory elements within the UCP2 promoter region. Specific binding of proteins derived from nuclear extracts isolated from T3-treated INS-1E cells to TRE1 and TRE2 is shown (Fig. 5). Increasing amounts of unlabeled double-stranded oligonucleotides encompassing TRE1 and TRE2 competed effectively with the labeled probe for binding. These results are consistent with our transfection studies and establish a direct role of PGC-1 in the TR-mediated regulation of -cell UCP2 gene expression involving two essential TREs. Similarly, EMSA studies suggest specific binding of nuclear proteins isolated from cholesterol starved INS-1E cells to E1 and E2 (Fig. 5), again corroborating our reporter gene studies and arguing for an important contribution of SREBPs to the control of UCP2 gene transcription.
Up-regulation of UCP2 and inhibition of GSIS has been demonstrated in -cells overexpressing SREBP-1c (29). We have previously demonstrated that PGC-1 potentiates liver X receptor (LXR)-mediated transcriptional activation of the SREBP-1c isoform in HepG2 cells (25). Indirect effects of PGC-1 on -cell UCP2 gene expression via up-regulation of SREBP-1c were therefore determined using a SREBP-1c promoter construct (SREBP-1c-Luc). Basal activity of SREBP-1c-Luc in INS-1E cells was stimulated by the LXR-specific ligand 22(R)HC and the retinoid X receptor (RXR) ligand 9cRA or a combination of both. Cotransfections with a PGC-1 expression plasmid also resulted in increased basal transcriptional activity, and additional increases in activity of SREBP-1c-Luc were observed upon stimulation of cotransfected cells with 22(R)HC or 9cRA alone or a combination of both drugs (Fig. 6). LXR/RXR overexpression stimulated basal SREBP-1c promoter-reporter gene expression and significantly augmented the effects of hPGC-1 on SREBP-1c-Luc transcriptional activity in cells stimulated with 22(R)HC/9cRA. The transcriptional corepressor SHP binds to LXR, thereby diminishing its transcriptional activity (30). Recently it has been shown that SHP overexpression rescues impaired GSIS in -cells overexpressing UCP2 (31), although the underlying mechanisms remained largely unknown. Cotransfection of a SHP expression plasmid significantly reduced SREBP-1c-Luc transcriptional activity in the presence of hPGC-1 and LXR/RXR in 22(R)HC/9cRA-treated INS-1E cells (Fig. 6). Our observations are consistent with a competition of PGC1 and SHP for binding to LXR resulting in a decrease in SREBP-1c gene expression.
We show that PGC-1 enhanced basal SREBP-2 promoter activity upon cotransfection in INS-1E cells (Fig. 7). Therefore computational analysis of the human SREBP-2 promoter was performed to identify potential binding sites for transcription factors coactivated by PGC-1. Two putative glucocorticoid hormone response elements were predicted at –3805/–3822 and –2338/–2355 using the MatInspector software. Cotransfection studies in INS-1E cells revealed that dexamethasone treatment increased the basal transcriptional activity of the human SREBP-2 promoter, which was further enhanced by the addition of the hPGC-1 expression plasmid. SHP has previously been shown to antagonize PGC-1-mediated coactivation of the glucocorticoid receptor (GR) on the phosphoenolpyruvate carboxykinase promoter (32). We observed that cotransfection of hPGC-1 together with a SHP expression plasmid abrogated dexamethasone-stimulated SREBP-2 promoter activity in INS-1E cells (Fig. 7).
Because insulin-secreting human -cell models were not available, we assessed the effects of TR, LXR/RXR, and GR ligands on UCP2 gene expression and insulin release in rat INS-1E cells. Quantitative real-time PCR analyses showed that stimulation of INS-1E cells with T3, 22(R)HC/9cRA, or dexamethasone increased endogenous UCP2 mRNA levels (Fig. 8A), suggesting similar mechanisms in the regulation of the rat and human UCP2 gene expression involving PGC-1. The sequence motifs involved in human UCP2 gene transcription are not conserved. We therefore performed in silico analyses of the rat UCP2 promoter and identified putative steroid-responsive elements at positions –89/–81, –898/–890 and –920/–914 as well as TREs at –464/–459, –1164/–1159, –1190/–1185, –2443/–2438 and –2488/–2483 but failed to identify E-Box motifs.
To determine whether the effects of drugs on UCP2 gene expression translate into changes in glucose-stimulated insulin secretion rates, insulin release from INS-1E cells in response to the drugs was measured. We observed a significantly decreased insulin release from INS-1E cells cultured in 22(R)HC/9cRA or dexamethasone-supplemented medium in the presence of both 5.5 and 25 mM glucose, whereas no effect was observed in T3-stimulated cells (Fig. 8B).
Discussion
Analyses of UCP2 knockout mice, adenoviral overexpression of UCP2 in isolated pancreatic islets and -cell lines as well as association studies and functional analysis of sequence variations in humans have established UCP2, by virtue of its proton-leak activity, as a critical determinant in the regulation of insulin secretion (6, 7, 8, 11, 12, 13, 14, 15, 33). The transcriptional control of the murine UCP2 gene has been studied in detail (20, 21), but little is known about the regulation of the human UCP2 gene in -cells. Computational analyses failed to reveal sequence homologies between a well-characterized mouse enhancer region and the human UCP2 promoter. A link between PGC-1 and UCP2 gene expression has been suggested from animal studies (6, 17, 18, 19), and a role of PGC-1 in T2DM is supported by human studies showing associations of single-nucleotide polymorphisms and haplotypes across the PGC-1 gene locus with T2DM risk, carbohydrate metabolism, insulin sensitivity, and surrogate markers of -cell function in several populations (34, 35, 36, 37, 38, 39, 40).
We show here that PGC-1 stimulates TR-mediated human UCP2 gene expression via two TREs located in the proximal UCP2 promoter region. PGC-1 has been previously shown to coactivate the TR in a ligand-dependent and -independent manner (26), and an effect of thyroid hormone on insulin secretion has been suggested from animal studies. T4 and T3 treatment of Sprague Dawley rats reduced their insulin secretory response to glucose (41). T3 decreased insulin secretion from cultured fetal pancreatic islets (42) in a rat model of hyperthyroidism. In humans, hyperthyroidism was associated with postprandial hyperglycemia and an inability to mount an adequate insulin response to glucose (43, 44). These effects of thyroid hormone might therefore be mediated, at least in part, by effects of PGC-1 on UCP2 gene expression.
Increased expression of the lipogenic transcription factor SREBP-1c has been reported in islets of animal models of T2DM such as obese (fa/fa) Zucker diabetic fatty rats (45), and several studies established SREBP-1c as a negative regulator of insulin secretion. Tetracycline-inducible expression of a nuclear active fragment of SREBP-1c in INS-1 cells impaired insulin secretion and induced excess formation of lipid droplets (46). Similar effects on GSIS and lipid metabolism were observed upon adenoviral-mediated overexpression of SREBP-1c accompanied by enhanced expression of UCP2 (29). Introduction of UCP2 small interfering RNA increased the ATP to ADP ratio and partially rescued defects in insulin secretion observed in these cells. Isolated rat islets infected with an adenovirus-encoding nuclear active SREBP-1c also showed an impaired insulin release (47). In addition, transgenic mice expressing nuclear SREBP-1c under the control of the insulin promoter were characterized by reduced GSIS associated with lipid accumulation and up-regulation of UCP2 gene expression (48). SREBP isoforms increase the activity of the mouse UCP2 promoter in -cells through binding to a steroid-responsive element in the –86/–44 enhancer region (21). An adjacent double E-Box motif has been demonstrated to play a supportive role in the response of UCP2 to free fatty acids (20). We show now that SREBP isoforms are also involved in the transcriptional control of the human UCP2 gene in INS-1E cells. Two E-Box motifs were identified that mediate the stimulatory effects of nuclear active SREBPs on UCP2 gene expression. The SREBP-2 isoform elicited the highest transactivation potency on the UCP2 promoter in INS-1E cells. Site-directed mutagenesis within the core region of the two E-Boxes abrogated transcriptional activation by SREBPs. SREBP-mediated induction of UCP2 gene expression may therefore represent a critical component of lipotoxicity in human T2DM as has been originally suggested from animal studies.
We have previously established PGC-1 as a potent coactivator of LXRs (24) and showed that PGC-1 potentiates LXR-dependent SREBP-1c gene expression in hepatocytes (25). Cotransfection studies revealed that PGC-1 also efficiently stimulates SREBP-1c promoter activity in the presence of LXR ligands in a -cell environment and might therefore indirectly activate UCP2 gene expression. The transcriptional corepressor SHP has been implicated in the regulation of nuclear hormone receptor function (49). SHP has been demonstrated to interact with LXRs and repress their transcriptional activity (30). We observed a marked decrease in LXR-mediated SREBP-1c gene expression upon ectopic overexpression of SHP. PGC-1 is also involved in the regulation of SREBP-2 gene expression in INS-1E cells and increases basal as well as glucocorticoid-mediated transcription from the SREPB-2 promoter. Computational analyses identified two putative glucocorticoid hormone response elements at –3805/–3822 and –2338/–2355 within the SREBP-2 5' regulatory region. Cotransfection of a SHP expression plasmid completely abolished dexamethasone-stimulated SREBP-2 gene expression, probably via competition with PGC-1 for binding to the GR, which is consistent with previous studies (32). Taken together our observations might help to explain the positive effects of SHP overexpression on GSIS in normal and UCP2 overexpressing islets reported recently (31) via down-regulation of SREBP-1c and/or SREBP-2 gene expression.
Glucocorticoids exert their diabetogenic effects by decreasing glucose uptake, increasing hepatic gluconeogenesis, and interfering with insulin secretion (50, 51). Long- and short-term exposure of -cells to glucocorticoids reduced GSIS in several studies (52, 53), but the underlying mechanisms are a matter of debate. Transgenic mice with increased glucocorticoid sensitivity due to overexpression of the glucocorticoid receptor under the control of the rat insulin promoter developed hyperglycemia and augmented inhibition of insulin secretion (54). It was suggested that the underlying mechanisms might involve up-regulation of 2-adrenergic receptor (2-AR) expression (55). Blockage of 2-AR signaling improved GSIS in type 2 diabetics (56), and targeted overexpression of 2-ARs in -cells of transgenic mice impaired insulin secretion and increased glucose intolerance (57). A recent study provides evidence that dexamethasone mediated up-regulation of the serum- and glucocorticoid-inducible kinase 1 increases the activity of a voltage-gated K+ channel, which in turn hyperpolarizes the -cell plasma membrane and inhibits insulin release (58). We propose an additional pathway in that PGC-1 potentiates glucocorticoid-dependent induction of SREBP-2 gene transcription in turn increasing -cell UCP2 mRNA expression.
Our studies on the role of PGC-1 in the regulation of human UCP2 gene expression do not include a physiological end point in a human -cell system. Nevertheless, we could demonstrate stimulatory effects of the nuclear hormone receptor ligands used in this study on UCP2 mRNA levels in rat INS-1E cells and showed that 22(R)HC/RA and dexamethasone treatment significantly decreased insulin release from rat pancreatic -cells. The lack of T3 treatment to alter insulin secretion rates in INS-1E cells might reflect limitations in the relative abundance of essential components involved in TR-mediated transcriptional activation and requires further studies. Taken together our results indicate that the principal mechanisms regulating UCP2 gene expression are similar in rats and humans and are conceivable with a role of UCP2 as modulator of insulin secretion in humans, although the underlying regulatory sequences are not evolutionary conserved.
In conclusion, a central role of UCP2 and PGC-1 in the pathophysiology of T2DM has been suggested from animal and cell culture models as well as gene expression and association studies in humans. We provide evidence for a possible link between these two diabetogenic factors and describe several specific mechanisms of PGC-1-mediated transcriptional control of the human UCP2 gene (Fig. 9). Thus, PGC-1 might exert its negative effects on insulin secretion, at least in part, via up-regulation of UCP2 gene expression, and modulation of PGC-1 expression and action may help to reverse certain aspects of -cell dysfunction in T2DM.
Footnotes
This work was supported by grants from the Oesterreichische Nationalbank (projects 10678 and 10932), the Medizinische Forschungsgesellschaft Salzburg, and a grant from the Land Salzburg.
First Published Online November 10, 2005
Abbreviations: 2-AR, 2-Adrenergic receptor; 9cRA, 9-cis retinoic acid; Exo, exonuclease; GR, glucocorticoid receptor; GSIS, glucose-stimulated insulin secretion; LXR, liver X receptor; PGC-1, peroxisome proliferator-activated receptor- coactivator-1; RA, retinoic acid; 22(R)HC, 22(R)-hydroxycholesterol; RXR, retinoid X receptor; SHP, short heterodimer partner; SREBP, sterol regulatory element binding protein; T2DM, type 2 diabetes mellitus; TR, thyroid hormone receptor; TRE, thyroid hormone response element; UCP2, uncoupling protein 2.
Accepted for publication November 3, 2005.
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the Department of Internal Medicine (F.K.), Krankenhaus Hallein, A-5400 Hallein, Austria
Abstract
A role of uncoupling protein 2 (UCP2) as negative modulator of insulin secretion has been suggested, but the transcriptional pathways regulating -cell UCP2 gene expression have been established in rodents only. We show here that the underlying sequence motifs are not conserved in the human gene and provide evidence for regulatory mechanisms involving the transcriptional cofactor peroxisome proliferator-activated receptor- coactivator-1 (PGC-1). PGC-1 potentiates thyroid hormone (T3)-mediated transcriptional activation of the human UCP2 gene in INS-1E cells. Two thyroid hormone response elements (TREs) located at –322/–317 (TRE1) and –170/–165 (TRE2) were identified, and mutation of either TRE1 or TRE2 abrogated the stimulatory effect of T3 treatment. Furthermore, two E-box motifs at –911/–906 (E1) and –743/–738 (E2) are involved in the regulation of UCP2 gene expression by sterol regulatory element binding protein isoforms (SREBP)-1a, -1c, and -2. Mutational analysis revealed that the presence of either E1 or E2 is sufficient to mediate activation of UCP2 gene transcription by nuclear active SREBPs. PGC-1 coactivates liver X receptor-mediated expression of SREBP-1c as well as dexamethasone-stimulated SREBP-2 expression in INS-1E cells. These transcriptional responses are antagonized by orphan nuclear receptor short heterodimer partner overexpression, which might explain its positive effects on glucose-stimulated insulin secretion in -cells overexpressing UCP2. We also provide evidence that despite a lack of sequence homology within the regulatory region, the principal mechanisms regulating UCP2 gene expression are similar in rats and humans, being consistent with a role for UCP2 as a modulator of insulin secretion in humans.
Introduction
TYPE 2 DIABETES MELLITUS (T2DM) is a heterogeneous metabolic disorder characterized by marked disturbances in glucose homeostasis. -Cell dysfunction represents a central abnormality in the pathophysiology of T2DM (1). Glucose-stimulated insulin secretion (GSIS) is tightly linked to glucose metabolism through oxidative phosphorylation as an increase in the intracellular ATP to ADP ratio triggers exocytosis of insulin containing secretory vesicles (2). Mitochondrial uncoupling proteins (UCPs) have been implicated in the regulation of GSIS by facilitating reentry of protons into the mitochondrial matrix thereby decreasing ATP synthesis (3, 4).
UCP2, a member of the UCP family, is expressed in several tissues including muscle, liver, white fat, and pancreas (5). A role of UCP2 as negative modulator of insulin secretion has been demonstrated by studies in isolated pancreatic islets derived from UCP2–/– mice showing elevated ATP levels and increased insulin release (6). UCP2-deficient animals were characterized by an increased acute insulin secretion rate in response to glucose, compared with controls (7). In addition, adenoviral overexpression of UCP2 in pancreatic -cells decreased their ATP content and blunted GSIS (8). In type 2 diabetic subjects with impaired GSIS, isolated pancreatic islets showed increased UCP2 gene expression and alterations in mitochondrial morphology (9). Furthermore, a quantitative trait locus for glucose homeostasis phenotypes reflecting -cell function has been mapped to a genomic region encompassing the human UCP2 gene locus (10) and associations of a common –866G/A UCP2 promoter polymorphism with indices of -cell function and T2DM risk have been observed in different populations (11, 12, 13, 14, 15). The pancreatic transcription factor paired box domain transcription factor-6 was shown to preferentially bind to and more effectively trans activate the –866A than the –866G allele (11). In vitro studies revealed lower insulin secretion rates in response to glucose stimulation in isolated pancreatic islet cells from –866A/A nondiabetic donors (14). In addition, the –866G/A polymorphism affected -cell UCP2 gene expression and was a determinant of insulin secretion in Japanese subjects (13). Taken together these data strongly suggested a role of UCP2 as negative regulator of insulin secretion (4).
The transcriptional coactivator peroxisome proliferator-activated receptor- coactivator (PGC)-1 has been implicated in the transcriptional control of distinct metabolic pathways involved in the pathogenesis of T2DM such as hepatic gluconeogenesis, muscle glucose uptake, mitochondrial biogenesis, and respiration as well as GSIS (16). Ectopic overexpression of PGC-1 negatively regulates insulin secretion in isolated rat islets and a -cell line and suppresses membrane depolarization without affecting the basal secretory apparatus (17). It was suggested that inhibition of insulin release by PGC-1 might be mediated by up-regulation of glucose-6-phosphatase and down-regulation of GLUT2, glucokinase, and glycerol-3-phosphatase gene expression, blunting the glucose-dependent rise in cellular ATP (17). However, a coordinate up-regulation of pancreatic UCP2 and PGC-1 gene expression has been observed in several animal models of T2DM including Zucker fatty rats and ob/ob mice that are characterized by marked defects in insulin secretion (6, 17, 18). Furthermore, a role of PGC-1 in the cold-induced up-regulation of UCP2 has been suggested from studies in rat pancreatic islets (19). Our present knowledge about the transcriptional control of -cell UCP2 gene expression is solely based on studies of the rodent gene. A multipartite enhancer in the proximal mouse UCP2 promoter (–86/–44) comprising a specificity protein-1 binding site, a sterol regulatory response element, and a double E-Box motif mediates stimulatory effects of oleic acid on UCP2 gene expression (20, 21). We therefore asked whether PGC-1 might contribute to the transcriptional regulation of the human UCP2 gene. In the present study, we characterized regulatory sequences within the human UCP2 promoter region that mediate effects of PGC-1 on -cell UCP2 gene expression.
Materials and Methods
Materials
DNA restriction and modification enzymes were obtained from New England Biolabs (Beverly, MA) and Promega (Madison, WI). Cell culture media, fetal calf serum, and LipofectAMINE 2000 were obtained from Life Technologies (Groningen, Netherlands). T3, dexamethasone, 22(R)-hydroxycholesterol [22(R)HC], and 9-cis retinoic acid (9cRA) were purchased from Sigma (St. Louis, MO). The dual-luciferase assay system was purchased from Promega.
Plasmid constructs
The UCP2-Prom-Luc plasmid in which the region from –1381 to +123 of the human UCP2 gene drives the promoterless firefly luciferase gene was generated by subcloning of a SacI/XhoI fragment from a 3241-bp UCP2 promoter construct described previously (22) into the pGL3-Basic vector (Promega). Several reporter constructs originating from the original UCP2-Prom-Luc plasmid and harboring nested unidirectional 5' deletions of the UCP2 promoter region were generated using the exonuclease (Exo)III/S1 deletion kit (Fermentas, Vilnius, Lithuania). Linearized plasmid DNA was digested with ExoIII followed by treatment of timed aliquots of the ExoIII reaction with S1 nuclease to remove the 5' single-stranded overhangs. Double-stranded linear fragments were separated by gel electrophoresis and appropriately sized plasmids were religated to generate the UCP2-1 (–983/+123), UCP2-2 (–879/+123), and UCP2-3 (–351/+123) constructs, respectively.
The Mut-TRE1 construct was generated by site-directed mutagenesis of the UCP2-3 plasmid using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) and 5'-CAAGAGGTGTGTGATTTTACAAGACTTGTTTCTG-3' and 5'-CAGAAACAAGTCTTGTAAAATCACACACCTCTTG-3' as forward and reverse mutagenic primers, respectively. For the construction of the Mut-TRE2 plasmid 5'-CGCACGGAAACGGGATTTTAGAAAGTCATGGAG-3' and 5'-CTCCATGACTTTCTAAAATCCCGTTTCCGTGCG-3' were used as forward and reverse mutagenic primers, respectively. Mutated nucleotide positions are in bold. Plasmid Mut-TRE1/2 was obtained by mutagenic PCR starting from Mut-TRE1 using primers for Mut-TRE2 described above. For generation of a human thyroid hormone receptor- expression construct 5'-CAAGGCTAGCAACCTATGACTCCCAACAGTATG-3' (–5/+18) and 5'-GAAGCTCGAGGTCTAATCCTCGAACACTTCCA3–3' (+1366/+1387) were used as forward and reverse primers, respectively, to amplify a 1392-bp cDNA fragment, which was cloned into pcDNA6C-V5/His (Invitrogen, Carlsbad, CA). Numbers in parentheses refer to primer positions relative to the translational start site (GenBank accession no. NM_000461). NheI and XhoI sites introduced into the primer sequences are in bold and the ATG start codon is underlined.
The Mut-E1 construct was derived from the UCP2-1 construct by QuickChange PCR-mediated site-directed mutagenesis using 5'-TACTCCCCAAACGCTTTTGTTTGTCCCGGCCAG-3' and 5'-CTGGCCGGGACAAACAAAAGCGTTTGGGGAGTA-3' as forward and reverse primers, respectively. For the generation of the Mut-E2 plasmid 5'-CCCGGCTCTGCCTTGTCTTTTGCGGGGGCCGGC-3' and 5'-GCCGGCCCCCGCAAAAGACAAGGCAGAGCCGGG-3' were used as forward and reverse primers, respectively. Plasmid Mut-E1/2 was obtained from the Mut-E1 construct using mutagenic primers for Mut-E2 described above. Mutated nucleotides are in bold. All constructs described were verified by dye terminator cycle sequencing using the ABI Prism 310 genetic analyzer (Applied Biosystems, Foster City, CA). The hPGC-1 and LXR expression plasmids have been described previously (23, 24).
For generation of a nuclear active sterol regulatory element-binding protein (SREBP)-1a expression plasmid (naSREBP-1a) harboring amino acids 1–487 (NM_004176), a 1487-bp cDNA fragment, was amplified starting from human liver total RNA using 5'-GAGAGGATCCAAAATGGACGAGCCACCCTTCAG-3' and 5'-GAGACTCGAGCTAGCGGGAGCGGTCCAGCATGC-3' as forward and reverse primers, respectively, and cloned into pcDNA6C-V5/His (Invitrogen). For construction of naSREBP-1c containing amino acids 1–463 (NM_004176), 5'-GAGAGGATCCAAAATGGACGAGCCACCCTTCAG-3' and 5'-GAGACTCGAGCTAGCGGGAGCGGTCCAGCATGC-3' were used as forward and reverse primers, respectively. BamHI and XhoI sites introduced into the primer sequences are underlined and the ATG start codon is in bold. Plasmid naSREBP-2 harboring amino acids 1–435 (NM_004599) was cloned using NheI and XhoI sites introduced into 5'-GAGAGCTAGCATGGACGACAGCGGCGAGCT-3' and 5'-GAGACTCGAGTTAGGCTGGGGGGGACATCAGAA-3' as forward and reverse primers, respectively.
The SREBP-2-Prom-Luc construct containing a 4182-bp fragment of the human SREBP-2 promoter was generated using 5'-CAACGCGTCCAGTCCCACATCTTCCTCC-3' (+59 to +39) and 5'-ACTAGATCTGCACTCCCACCCACCCAC-3' (–4123 to –4113) as forward and reverse primers, respectively. The amplified DNA fragment was cloned into the pGL3-Basic vector (Promega). Numbers in parentheses refer to primer positions relative to the transcriptional start site (GenBank accession no. NM_004599). MluI and BlgII restriction enzymes sites introduced into the primer sequences are in bold. The SREBP-1c promoter reporter construct SREBP-1c-Prom-Luc has been described previously (25). An expression plasmid containing the coding region of the human short heterodimer partner (SHP) gene was obtained from the American Type Culture Collection (Manassas, VA; clone MGC-34176).
Promoter in silico analysis
The Basic Local Alignment Search Tool (BLAST, www.ncbi.nlm.nih.gov/BLAST) was used to identify regions of local similarity between the mouse and human UCP2 promoter sequence. For the prediction of putative transcription factor binding sites, the public domain MatInspector professional software (www.genomatix.gsf.de/cgi-bin/matinspector) version 2.2 was used together with the TRANSFAC 4.0 database containing a large library of predefined matrix descriptions for known transcription factor recognition sequences. A core similarity of 0.85 or greater and a matrix similarity of 0.90 or greater was used as a cut-off for consideration of potential query sequence matches.
Cell culture, transfections, and reporter-gene assays
INS-1E cells were cultured in 24-well dishes and transfected using LipofectAMINE 2000 reagent as described (11). Before transfection, cells were washed twice with Hanks’ balanced salt solution (Sigma) and drugs at concentrations of 10 μM for T3, dexamethasone, 22(R)HC and 9cRA were added. Unless otherwise indicated, we used 1 μg of reporter plasmids, 0.5 μg of expression plasmids, and 20 ng of pRL-TK plasmid (Promega) as transfection control. Cells were collected 24 h after transfection and firefly and renilla luciferase activities were measured in a luminometer (Anthos Labtec Instruments, Salzburg, Austria) using the dual-luciferase reporter assay system (Promega). Graphs show means of three experiments, each performed in quadruplicates. Results are given as means ± SD. For post hoc comparisons of means, Tukey’s honest significant difference test was used. Two-way ANOVA was used to compare transactivation of reporter gene constructs by trans factors.
EMSAs
Nuclear extracts from INS-1E cells were prepared, and EMSAs were performed as described previously (23, 24). INS-1E cells were either incubated without serum for cholesterol depletion or stimulated with T3 at a concentration of 10 μM for binding studies with E-Box1 or -2 and TRE1 or -2, respectively. Sense strand sequences of oligonucleotides E-Box1 and E-Box2 were 5'-aattTTCTACTCCCCAAACGACACGTGTTTGTCCCGGCCAGAGG-3' and 5'-aattGCCCGGCTCTGCCTTGTCACGTGCGGGGGCCGGCCCGTTT-3', respectively. Sense strand sequences of oligonucleotides TRE1 and TRE2 were 5'-aattAAGGCAAGAGGTGTGTGACTGGACAAGACTTGTTTCTGGC-3' and 5'-aattGGACGCACGGAAACGGGAGTGGAGAAAGTCATGGAGAGAA-3', respectively. Unlabeled competitor oligonucleotides were added at 20- and 100-fold molar excess. Overhangs are represented by lowercase letters.
Quantification of UCP2 gene expression and insulin secretion levels in INS-1E cells
Total RNA was isolated from INS-1E cells incubated for 48 h with T3, a combination of 22(R)HC and 9cRA or dexamethasone at a concentration of 10 μM for each drug as indicated. Equal amounts of RNA were reverse transcribed and cDNAs were quantified by real-time PCR using the SYBR Green PCR master mix (Applied Biosystems) as described (25). For quantification of UCP2 mRNA expression 5'-GACAGATGACCTCCCTTGCCAC-3' (+630 to +651) and 5'-CAGGAACCCAAGCGGAGAAAG-3' (+828 to +848) were used as forward and reverse primers, respectively. Numbers in parentheses refer to primer positions relative to the translational start site (GenBank accession no. NM_003355). cycle threshold values for UCP2 gene expression were normalized for the expression of acidic ribosomal protein p 0 as described (25).
INS-1E cells grown in 6-well plates were incubated for 48 h with T3, a combination of 22(R)HC and 9cRA or dexamethasone at a concentration of 10 μM for each drug as indicated. Cells were then washed with Krebs-Ringer bicarbonate buffer containing 129 mM NaCl, 4.8 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 2.5 mM CaCl2, 5 mM NaHCO3, 0.2% BSA, and 10 mM HEPES (pH 7.4). After a preincubation period of 30 min in Krebs-Ringer bicarbonate buffer containing 1 mM glucose, INS-1E cells were incubated for 60 min with the concentrations of glucose as indicated. Insulin secreted into the supernatant was measured using a rat insulin ELISA kit (Linco Research Inc., St. Charles, MO). Graphs show means of three experiments each performed in triplicate. Results are given as means ± SD.
Results
In silico analysis of the human UCP2 gene promoter was conducted in an attempt to identify potential regulatory regions. A homology search between the proximal human and mouse UCP2 promoters as well as sequence comparisons with an isolated mouse enhancer region (–86/–44) failed to provide significant alignment scores with higher-than-average sequence homology. Overall sequence identity along 1000 nucleotides of the mouse and human 5' regulatory region upstream of exon 1 was less than 55% with more than 22% gaps introduced to achieve optimal alignment results. Best hits obtained with the mouse enhancer region revealed two or more mismatches within the core region of the respective transcription factor binding sites. Analysis of the proximal UCP2 promoter region for potential cis regulatory binding sites using the MatInspector software revealed a high probability score for the presence of two potential thyroid hormone response elements (TREs) at –322/–317 (TRE1) and –170/–165 (TRE2) as well as two E-Box consensus binding sites at –911/–906 (E1) and –743/–738 (E2), respectively (Fig. 1).
The thyroid hormone receptor (TR) is a well-established target of PGC-1 action (26) and thyroid hormone is known to inhibit insulin secretion (27, 28). To determine direct effects of PGC-1 on -cell UCP2 gene expression via coactivation of TR, we performed cotransfection studies in INS-1E cells. An UCP2 promoter reporter construct (UCP2-Prom-Luc) was transiently transfected into INS-1E cells together with a PGC-1 expression plasmid (hPGC-1) in both the absence and presence of T3 (Fig. 2A). hPGC-1 as well as T3 alone stimulated basal transcriptional activity of the UCP2-Prom-Luc construct, and cotransfection with hPGC-1 further augmented T3-mediated reporter gene expression. Ectopic overexpression of the TR had only minor effects on basal UCP2 reporter gene expression but significantly increased UCP2-Prom-Luc transcriptional activity in T3-treated cells in both the absence and presence of hPGC-1 (Fig. 2A). A similar response was observed with the UCP2-3 reporter construct that was generated by ExoIII deletion and encompassed a 351-bp UCP2 promoter fragment retaining both TREs (data not shown). Possible effects of TRE1 and TRE2 were determined using site directed mutagenesis. Mutation of TRE1 or TRE2 (Mut-TRE1 or Mut-TRE2) as well as disruption of both response elements (Mut-TRE1/2) completely abolished the stimulatory effect of T3 and PGC-1 on UCP2 gene expression (Fig. 2B).
Studies in mice suggested a pivotal role of SREBPs in the transcriptional regulation of the UCP2 gene via a promoter element not conserved in humans (20, 21). We therefore determined the effects of SREBP isoforms on human UCP2 gene expression. Transient cotransfections were performed in INS-1E cells using UCP2-Prom-Luc together with expression plasmids for nuclear active SREBP-1a, -1c, and -2 isoforms (Fig. 3). Cells were incubated with cholesterol/25(R)-hydroxycholesterol to suppress endogenous SREBP activity. Overexpression of SREBP-1a and -1c resulted in a approximately 2-fold increase in UCP2 promoter activity, whereas overexpression of SREBP-2 exhibited an even more pronounced effect (6-fold induction). ExoIII mapping was used to address the role of the two putative E-Box motifs (E1 and E2). Deletion of the genomic region upstream of E1 and E2 (UCP2-1) did not affect SREBP-2 mediated transactivation. A reporter construct devoid of E1 (UCP2-2) elicited slightly reduced luciferase activity, whereas removal of both E-Boxes (UCP2-3) resulted in complete loss of SREBP-2-dependent stimulation of UCP2 promoter activity (Fig. 4). Similar results were obtained with SREBP-1a and -1c isoforms (data not shown). Site-directed mutagenesis was used to establish the individual contributions of E1 and E2 in the regulation of the human UCP2 gene. Both E-Boxes, E1 and E2, mediated effects of SREBP-2 on UCP2 gene expression, with E1 eliciting a stronger effect. Simultaneous disruption of E1 and E2 completely abolished SREBP-2-dependent stimulation of UCP2 promoter activity (Fig. 4).
EMSAs were performed to study possible interactions of nuclear proteins with regulatory elements within the UCP2 promoter region. Specific binding of proteins derived from nuclear extracts isolated from T3-treated INS-1E cells to TRE1 and TRE2 is shown (Fig. 5). Increasing amounts of unlabeled double-stranded oligonucleotides encompassing TRE1 and TRE2 competed effectively with the labeled probe for binding. These results are consistent with our transfection studies and establish a direct role of PGC-1 in the TR-mediated regulation of -cell UCP2 gene expression involving two essential TREs. Similarly, EMSA studies suggest specific binding of nuclear proteins isolated from cholesterol starved INS-1E cells to E1 and E2 (Fig. 5), again corroborating our reporter gene studies and arguing for an important contribution of SREBPs to the control of UCP2 gene transcription.
Up-regulation of UCP2 and inhibition of GSIS has been demonstrated in -cells overexpressing SREBP-1c (29). We have previously demonstrated that PGC-1 potentiates liver X receptor (LXR)-mediated transcriptional activation of the SREBP-1c isoform in HepG2 cells (25). Indirect effects of PGC-1 on -cell UCP2 gene expression via up-regulation of SREBP-1c were therefore determined using a SREBP-1c promoter construct (SREBP-1c-Luc). Basal activity of SREBP-1c-Luc in INS-1E cells was stimulated by the LXR-specific ligand 22(R)HC and the retinoid X receptor (RXR) ligand 9cRA or a combination of both. Cotransfections with a PGC-1 expression plasmid also resulted in increased basal transcriptional activity, and additional increases in activity of SREBP-1c-Luc were observed upon stimulation of cotransfected cells with 22(R)HC or 9cRA alone or a combination of both drugs (Fig. 6). LXR/RXR overexpression stimulated basal SREBP-1c promoter-reporter gene expression and significantly augmented the effects of hPGC-1 on SREBP-1c-Luc transcriptional activity in cells stimulated with 22(R)HC/9cRA. The transcriptional corepressor SHP binds to LXR, thereby diminishing its transcriptional activity (30). Recently it has been shown that SHP overexpression rescues impaired GSIS in -cells overexpressing UCP2 (31), although the underlying mechanisms remained largely unknown. Cotransfection of a SHP expression plasmid significantly reduced SREBP-1c-Luc transcriptional activity in the presence of hPGC-1 and LXR/RXR in 22(R)HC/9cRA-treated INS-1E cells (Fig. 6). Our observations are consistent with a competition of PGC1 and SHP for binding to LXR resulting in a decrease in SREBP-1c gene expression.
We show that PGC-1 enhanced basal SREBP-2 promoter activity upon cotransfection in INS-1E cells (Fig. 7). Therefore computational analysis of the human SREBP-2 promoter was performed to identify potential binding sites for transcription factors coactivated by PGC-1. Two putative glucocorticoid hormone response elements were predicted at –3805/–3822 and –2338/–2355 using the MatInspector software. Cotransfection studies in INS-1E cells revealed that dexamethasone treatment increased the basal transcriptional activity of the human SREBP-2 promoter, which was further enhanced by the addition of the hPGC-1 expression plasmid. SHP has previously been shown to antagonize PGC-1-mediated coactivation of the glucocorticoid receptor (GR) on the phosphoenolpyruvate carboxykinase promoter (32). We observed that cotransfection of hPGC-1 together with a SHP expression plasmid abrogated dexamethasone-stimulated SREBP-2 promoter activity in INS-1E cells (Fig. 7).
Because insulin-secreting human -cell models were not available, we assessed the effects of TR, LXR/RXR, and GR ligands on UCP2 gene expression and insulin release in rat INS-1E cells. Quantitative real-time PCR analyses showed that stimulation of INS-1E cells with T3, 22(R)HC/9cRA, or dexamethasone increased endogenous UCP2 mRNA levels (Fig. 8A), suggesting similar mechanisms in the regulation of the rat and human UCP2 gene expression involving PGC-1. The sequence motifs involved in human UCP2 gene transcription are not conserved. We therefore performed in silico analyses of the rat UCP2 promoter and identified putative steroid-responsive elements at positions –89/–81, –898/–890 and –920/–914 as well as TREs at –464/–459, –1164/–1159, –1190/–1185, –2443/–2438 and –2488/–2483 but failed to identify E-Box motifs.
To determine whether the effects of drugs on UCP2 gene expression translate into changes in glucose-stimulated insulin secretion rates, insulin release from INS-1E cells in response to the drugs was measured. We observed a significantly decreased insulin release from INS-1E cells cultured in 22(R)HC/9cRA or dexamethasone-supplemented medium in the presence of both 5.5 and 25 mM glucose, whereas no effect was observed in T3-stimulated cells (Fig. 8B).
Discussion
Analyses of UCP2 knockout mice, adenoviral overexpression of UCP2 in isolated pancreatic islets and -cell lines as well as association studies and functional analysis of sequence variations in humans have established UCP2, by virtue of its proton-leak activity, as a critical determinant in the regulation of insulin secretion (6, 7, 8, 11, 12, 13, 14, 15, 33). The transcriptional control of the murine UCP2 gene has been studied in detail (20, 21), but little is known about the regulation of the human UCP2 gene in -cells. Computational analyses failed to reveal sequence homologies between a well-characterized mouse enhancer region and the human UCP2 promoter. A link between PGC-1 and UCP2 gene expression has been suggested from animal studies (6, 17, 18, 19), and a role of PGC-1 in T2DM is supported by human studies showing associations of single-nucleotide polymorphisms and haplotypes across the PGC-1 gene locus with T2DM risk, carbohydrate metabolism, insulin sensitivity, and surrogate markers of -cell function in several populations (34, 35, 36, 37, 38, 39, 40).
We show here that PGC-1 stimulates TR-mediated human UCP2 gene expression via two TREs located in the proximal UCP2 promoter region. PGC-1 has been previously shown to coactivate the TR in a ligand-dependent and -independent manner (26), and an effect of thyroid hormone on insulin secretion has been suggested from animal studies. T4 and T3 treatment of Sprague Dawley rats reduced their insulin secretory response to glucose (41). T3 decreased insulin secretion from cultured fetal pancreatic islets (42) in a rat model of hyperthyroidism. In humans, hyperthyroidism was associated with postprandial hyperglycemia and an inability to mount an adequate insulin response to glucose (43, 44). These effects of thyroid hormone might therefore be mediated, at least in part, by effects of PGC-1 on UCP2 gene expression.
Increased expression of the lipogenic transcription factor SREBP-1c has been reported in islets of animal models of T2DM such as obese (fa/fa) Zucker diabetic fatty rats (45), and several studies established SREBP-1c as a negative regulator of insulin secretion. Tetracycline-inducible expression of a nuclear active fragment of SREBP-1c in INS-1 cells impaired insulin secretion and induced excess formation of lipid droplets (46). Similar effects on GSIS and lipid metabolism were observed upon adenoviral-mediated overexpression of SREBP-1c accompanied by enhanced expression of UCP2 (29). Introduction of UCP2 small interfering RNA increased the ATP to ADP ratio and partially rescued defects in insulin secretion observed in these cells. Isolated rat islets infected with an adenovirus-encoding nuclear active SREBP-1c also showed an impaired insulin release (47). In addition, transgenic mice expressing nuclear SREBP-1c under the control of the insulin promoter were characterized by reduced GSIS associated with lipid accumulation and up-regulation of UCP2 gene expression (48). SREBP isoforms increase the activity of the mouse UCP2 promoter in -cells through binding to a steroid-responsive element in the –86/–44 enhancer region (21). An adjacent double E-Box motif has been demonstrated to play a supportive role in the response of UCP2 to free fatty acids (20). We show now that SREBP isoforms are also involved in the transcriptional control of the human UCP2 gene in INS-1E cells. Two E-Box motifs were identified that mediate the stimulatory effects of nuclear active SREBPs on UCP2 gene expression. The SREBP-2 isoform elicited the highest transactivation potency on the UCP2 promoter in INS-1E cells. Site-directed mutagenesis within the core region of the two E-Boxes abrogated transcriptional activation by SREBPs. SREBP-mediated induction of UCP2 gene expression may therefore represent a critical component of lipotoxicity in human T2DM as has been originally suggested from animal studies.
We have previously established PGC-1 as a potent coactivator of LXRs (24) and showed that PGC-1 potentiates LXR-dependent SREBP-1c gene expression in hepatocytes (25). Cotransfection studies revealed that PGC-1 also efficiently stimulates SREBP-1c promoter activity in the presence of LXR ligands in a -cell environment and might therefore indirectly activate UCP2 gene expression. The transcriptional corepressor SHP has been implicated in the regulation of nuclear hormone receptor function (49). SHP has been demonstrated to interact with LXRs and repress their transcriptional activity (30). We observed a marked decrease in LXR-mediated SREBP-1c gene expression upon ectopic overexpression of SHP. PGC-1 is also involved in the regulation of SREBP-2 gene expression in INS-1E cells and increases basal as well as glucocorticoid-mediated transcription from the SREPB-2 promoter. Computational analyses identified two putative glucocorticoid hormone response elements at –3805/–3822 and –2338/–2355 within the SREBP-2 5' regulatory region. Cotransfection of a SHP expression plasmid completely abolished dexamethasone-stimulated SREBP-2 gene expression, probably via competition with PGC-1 for binding to the GR, which is consistent with previous studies (32). Taken together our observations might help to explain the positive effects of SHP overexpression on GSIS in normal and UCP2 overexpressing islets reported recently (31) via down-regulation of SREBP-1c and/or SREBP-2 gene expression.
Glucocorticoids exert their diabetogenic effects by decreasing glucose uptake, increasing hepatic gluconeogenesis, and interfering with insulin secretion (50, 51). Long- and short-term exposure of -cells to glucocorticoids reduced GSIS in several studies (52, 53), but the underlying mechanisms are a matter of debate. Transgenic mice with increased glucocorticoid sensitivity due to overexpression of the glucocorticoid receptor under the control of the rat insulin promoter developed hyperglycemia and augmented inhibition of insulin secretion (54). It was suggested that the underlying mechanisms might involve up-regulation of 2-adrenergic receptor (2-AR) expression (55). Blockage of 2-AR signaling improved GSIS in type 2 diabetics (56), and targeted overexpression of 2-ARs in -cells of transgenic mice impaired insulin secretion and increased glucose intolerance (57). A recent study provides evidence that dexamethasone mediated up-regulation of the serum- and glucocorticoid-inducible kinase 1 increases the activity of a voltage-gated K+ channel, which in turn hyperpolarizes the -cell plasma membrane and inhibits insulin release (58). We propose an additional pathway in that PGC-1 potentiates glucocorticoid-dependent induction of SREBP-2 gene transcription in turn increasing -cell UCP2 mRNA expression.
Our studies on the role of PGC-1 in the regulation of human UCP2 gene expression do not include a physiological end point in a human -cell system. Nevertheless, we could demonstrate stimulatory effects of the nuclear hormone receptor ligands used in this study on UCP2 mRNA levels in rat INS-1E cells and showed that 22(R)HC/RA and dexamethasone treatment significantly decreased insulin release from rat pancreatic -cells. The lack of T3 treatment to alter insulin secretion rates in INS-1E cells might reflect limitations in the relative abundance of essential components involved in TR-mediated transcriptional activation and requires further studies. Taken together our results indicate that the principal mechanisms regulating UCP2 gene expression are similar in rats and humans and are conceivable with a role of UCP2 as modulator of insulin secretion in humans, although the underlying regulatory sequences are not evolutionary conserved.
In conclusion, a central role of UCP2 and PGC-1 in the pathophysiology of T2DM has been suggested from animal and cell culture models as well as gene expression and association studies in humans. We provide evidence for a possible link between these two diabetogenic factors and describe several specific mechanisms of PGC-1-mediated transcriptional control of the human UCP2 gene (Fig. 9). Thus, PGC-1 might exert its negative effects on insulin secretion, at least in part, via up-regulation of UCP2 gene expression, and modulation of PGC-1 expression and action may help to reverse certain aspects of -cell dysfunction in T2DM.
Footnotes
This work was supported by grants from the Oesterreichische Nationalbank (projects 10678 and 10932), the Medizinische Forschungsgesellschaft Salzburg, and a grant from the Land Salzburg.
First Published Online November 10, 2005
Abbreviations: 2-AR, 2-Adrenergic receptor; 9cRA, 9-cis retinoic acid; Exo, exonuclease; GR, glucocorticoid receptor; GSIS, glucose-stimulated insulin secretion; LXR, liver X receptor; PGC-1, peroxisome proliferator-activated receptor- coactivator-1; RA, retinoic acid; 22(R)HC, 22(R)-hydroxycholesterol; RXR, retinoid X receptor; SHP, short heterodimer partner; SREBP, sterol regulatory element binding protein; T2DM, type 2 diabetes mellitus; TR, thyroid hormone receptor; TRE, thyroid hormone response element; UCP2, uncoupling protein 2.
Accepted for publication November 3, 2005.
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