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Fraunhofer Institute of Toxicology and Experimental Medicine, Center for Drug Research and Medical Biotechnology, Hannover, Germany
Abstract
The orphan hepatic nuclear factor (HNF) HNF4 is of pivotal importance for liver development and hepatocellular differentiation and plays an essential role in a regulatory circuitry to control a wide range of metabolic processes. It also targets genes in other organs, including pancreas, kidney, intestine, and colon; promotes expression of an epithelial phenotype; triggers de novo formation of functional tight junctions; and contributes to epithelial cell polarity. In particular, HNF4 dysfunction leads to metabolic disorders, including diabetes. We used the chromatin immunoprecipitation (ChIP) cloning procedure and a bioinformatic approach to search for candidate genes associated with impaired liver, pancreas, and kidney function. We identified two novel targets regulated by HNF4, which participate in the control, at least in part, in cell-cycle regulation and are members of the mitogen-activated kinase pathway. In multiple ChIP assays, ribosomal S6 kinase 4 (RSK4) and p21-activated kinase 5 (PAK5) were confirmed, and in vitro binding of HNF4 was evidenced by electrophoretic mobility shift assays (EMSA) using oligonucleotides, which harbor novel binding sites. We also used EMSA to probe for binding sites in promoters of HNF1, apolipoprotein B, 1-antitrypsin, and angiotensinogen. We further studied RSK4 and PAK5 kinase expression in streptozotocin-induced diabetic rat kidney and brain and observed significant repression of HNF4, RSK4, and PAK5 as determined by quantitative real-time reverse transcriptase-polymerase chain reaction. RSK4 and PAK5 may provide a molecular rationale for late-stage complications in disease, and further studies are warranted to explore these targets for the treatment of diabetic nephro- and neuropathy, frequently seen in patients with HNF4 dysfunction.
HNF4 is a zinc-finger transcription factor and regulates a large number of genes involved in lipid, steroid, xenobiotic, and amino acid metabolism (Sladek and Seidel, 2001; Schrem et al., 2002). This factor is of paramount importance for hepatocyte differentiation and organ development, and HNF4 knockout mice die at approximately embryonic day 9.5 because of impaired liver organogenesis (Chen et al., 1994; Li et al., 2000; Hayhurst et al., 2001; Parviz et al., 2003). The role of HNF4 in the glucose-dependent insulin secretory pathways is well recognized. Indeed, one form of a rare monogenetic disorder, termed maturity-onset diabetes of the young (MODY), was mapped to mutations within the HNF4 gene (MODY-1), thus confirming its role in pancreatic -cell function (Sladek and Seidel, 2001; Schrem et al., 2002). Moreover, HNF4 dysfunction leads to multifactorial type 2 diabetes (Love-Gregory et al., 2004). Besides its pivotal functions in liver metabolism, HNF4 also targets genes in other tissues and organs including kidney, intestine, and colon (Sladek and Seidel, 2001). In general, HNF4 is a dominant regulator of an epithelial phenotype, triggers de novo formation of functional tight junctions, and contributes to epithelial cell polarity (Chiba et al., 2003). Because of its role in epithelial differentiation, it is probable that HNF4 functions in the control of cell proliferation as well. Little is known about disease-associated or disease-causing genes targeted by HNF4. In this study, we used the chromatin immunoprecipitation assay to search for novel HNF4 target genes in cultures of Caco-2 cells, and this colon carcinoma-derived cell line is well accepted for its usefulness in investigating HNF4 function and genes targeted by this factor (Soutoglou and Talianidis, 2002). In particular, we identified two disease-associated kinases (i.e., RSK4 and PAK5) that are regulated by HNF4. This points to novel functions of this factor, which are beyond the regulation of genes involved in liver metabolism.
Materials and Methods
Caco-2 Cell Culture. Caco-2 cells were obtained from and cultivated as recommended by Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany), seeded with a density of 4 x 106 cells/75 cm2 flask, and harvested after 11 days.
Isolation of Nuclear Extracts, Western Blot Analysis, and Electrophoretic Mobility Shift Assays. The use of animals was approved by the local government of Hannover with project license 02-548. Sprague-Dawley rats (n = 3) were treated with a single intraperitoneal dose of 100 mg of Aroclor 1254 per kilogram of body weight and killed 72 h later. Nuclear extracts from rat liver were prepared as described by Gorski et al. (1986), whereas nuclear extracts from Caco-2 cells were isolated by the method used by Dignam et al. (1983) with minor modifications as detailed previously (Niehof et al., 2001). For Western blot analysis, nuclear extracts were separated on a 12% SDS-polyacrylamide gel and blotted onto a polyvinylidene difluoride membrane. Antibody directed against HNF4 was purchased from Santa Cruz Biotechnology (Heidelberg, Germany). The antigen-antibody complexes were visualized using the ECL detection system as recommended by the manufacturer (PerkinElmer Life and Analytical Sciences, Rodgau-Juegesheim, Germany), and chemiluminescence was recorded with Kodak IS 440 CF (Biostep GmbH, Jahnsdorf, Germany). Electrophoretic mobility shift assays were performed as described previously (Niehof et al., 2001). Binding buffer consisted of 25 mM HEPES, pH 7.6, 5 mM MgCl2, 34 mM KCl, 2 mM dithiothreitol, 2 mM Pefabloc, 2% aprotinin, 40 ng/e poly(dI-dC), and 100 ng/e bovine serum albumin. The oligonucleotides were purchased from MWG Biotech (Ebersberg/Munich, Germany) and used as 32P-labeled probes; for sequence information, see Table 1. Supershift experiments were done with an HNF4-specific antibody (Santa Cruz Biotechnology).
Cross-Linking and Chromatin Immunoprecipitation. All chromatin immunoprecipitation (ChIP) procedures were carried out as described by Weinmann et al. (2001) with some modifications. The samples were sonicated on ice until cross-linked chromatin was fragmented to approximately 0.2 to 1.6 kilobase pairs. Protein A-Sepharose CLB4 (Amersham Biosciences, Freiburg, Germany) was blocked with 1 mg/ml bovine serum albumin and 1 mg/ml herring sperm DNA (Promega, Mannheim, Germany) and washed extensively before use. Chromatin preparations were precleared by incubation with "blocked" Protein A-Sepharose for 1 h at 4°C. Precleared chromatin from 2.5 x 107 cells was incubated with 1 e of HNF4 antibody or no antibody and rotated at 4°C overnight. After recovering of immunocomplexes, extensive washing, and elution, two samples were pooled for a second immunoprecipitation step with the HNF4 antibody. PCR was done in a mixture containing 2 e of purified DNA or 2 e of a 1:200 dilution of the total input sample, 1 e of each primer, 0.25 mM dNTP mixture, 0.625 U Thermostart-Taq (ABgene, Hamburg, Germany), and 1x PCR buffer (ABgene; with 1.5 mM MgCl2) in a total volume of 20 e. PCRs were carried out with a T3 Thermocycler (Biometra, Gttingen, Germany) with the following conditions: initial denaturation at 95°C for 15 min (Thermostart activation), denaturation at 94°C for 30 s, annealing at different temperatures for 45 s (Table 2), extension at 72°C for 45 s, final extension at 74°C for 10 min, 45 cycles. A detailed account of PCR primers to analyze immunoprecipitated target genes is given in Table 2. We used two rounds of sequential chromatin immunoprecipitations (Weinmann et al., 2001) to increase purity and specificity of target DNA for ChIP-cloning and for validation of ChIP-derived clones. Other investigators used a single immunoprecipitation step with obvious limitations for validation of targets (Horak et al., 2002; Tomaru et al., 2003). In numerous independent ChIP experiments, immunoprecipitated DNA contained the full complement of selected HNF4 target genes (HNF1, ApoB, AAT, and ANG), which served as our positive controls. For a novel candidate gene, we demanded at least three independent confirmations from a series of independent ChIP experiments until a candidate gene was considered to be confirmed.
Cloning and Sequence Analysis. The immunoprecipitated DNA was treated with T4 DNA polymerase (New England Biolabs, Frankfurt, Germany) to create blunt ends, purified, and cloned into the zero-blunt vector (Invitrogen, Karlsruhe, Germany) using the zero-blunt PCR cloning kit (Invitrogen) according to the manufacturer's recommendations. Colonies having inserts were identified by restriction-enzyme digestion using enzymes in the polylinker. Plasmid DNA was purified with QIAquick PCR Purification Kit (QIAGEN GmbH, Hilden, Germany), subjected to cycle sequencing with vector-specific primers using BigDyeTerminator v3.1 Kit, and injected into ABI 3100 Genetic Analyzer (Applied Biosystems, Darmstadt, Germany). Sequences were identified and annotated by database searches (GenBank version build 34, maintained by NCBI). Detailed sequence information is given in Table 3.
Bioinformatic Searching for HNF4 Binding Sites. The transcription start site (+1) of the NCBI mRNA reference sequence was used for promoter annotation of the respective clones. Cloned fragments and respective proximal promoters (eC1 to eC3000 bp) were checked for putative HNF4 binding sites with two different bioinformatic weight matrix-based tools: V$HNF4_01 with cut-off core similarity 0.75 and matrix similarity 0.78, Transfac matrix (Biobase, Wolfenbetel, Germany), and V$HNF4_01 with cut-off core similarity 0.75 and matrix similarity 0.82 or V$HNF4_02 with cut-off core similarity 0.75 and matrix similarity 0.76, Genomatix matrix (Genomatix Software GmbH, Mechen, Germany).
RT-PCR and Real-Time RT-PCR. Total RNA was isolated using the nucleospin RNA Isolation Kit (Macherey-Nagel, Deen, Germany) according to the manufacturer's recommendations. Total RNA from each sample (4 e) was used for reverse transcription (Omniscript Reverse Transcriptase, QIAGEN GmbH). PCR was done in a mixture containing a cDNA equivalent to 25 ng of total RNA, 1 e concentrations of each primer, 0.25 mM dNTP mixture, 0.625 U Thermostart-Taq (ABgene), and 1x PCR buffer (ABgene; with 1.5 mM MgCl2) in a total volume of 20 e of PCR. PCRs were carried out with a thermocycler (T3; Biometra) with the following conditions: initial denaturation at 95°C for 15 min (Thermostart activation), denaturation at 94°C for 30 s, annealing at different temperatures for 45 s (Table 4), extension at 72°C for 45 s, and final extension at 74°C for 10 min. Various cycle numbers were used to demonstrate linearity, and 40 cycles were used for tissue comparison. A detailed oligonucleotide sequence information is given in Table 4.
Real-Time Semiquantitative PCR. Real-time RT-PCR measurement was done with the Lightcycler (Roche Diagnostics, Mannheim, Germany) with the following conditions (Table 5): denaturation at 94°C for 120 s, annealing at different temperatures for 8 s, extension at 72°C for different times, and fluorescence at different temperatures. The PCR reaction was stopped after a total of 30 to 50 cycles, and at the end of each extension phase, fluorescence was observed and used for quantitative measurements within the linear range of amplification. Exact quantification was achieved by serial dilution with cDNA produced from total RNA extracts using 1:5 dilution steps. A detailed oligonucleotide sequence information is given in Table 5. Gene expression levels were normalized to mitATPase6, which we found to be stably expressed.
Diabetic Disease Model. Streptozotocin (STZ) is a well-known -cell toxin that results in diabetes. Kidneys of STZ-treated Sprague-Dawley rats (6 months of treatment) were kindly provided by R. Amann (Institute of Pathology, University of Erlangen, Erlangen, Germany). Conclusive evidence for this treatment to result in diabetic nephropathy was published recently (Gross et al., 2003). Experimental diabetes was induced by single intravenous injection of 65 mg of STZ/kg body weight. All injected animals developed hyperglycemia on day 2 after STZ administration. Thereafter, diabetic animals were treated daily with 4.1 ± 1.4 IU/kg of body weight of long-acting insulin (Gross et al., 2003). Diabetic rats had stable, moderate hyperglycemia throughout the 6 months (mean blood glucose concentrations, 650 ± 104 mg/dl) (Gross et al., 2003). Furthermore, liver, kidney, and brain of STZ-treated Wistar rats (2 months of treatment) were kindly provided by P. Rsen (German Diabetes Research Institute, De箂seldorf, Germany). Experimental diabetes was induced by single intraperitoneal injection of 60 mg of STZ/kg of body weight. All animals developed hyperglycemia until 60 h after STZ injection. The animals did not receive an antidiabetic treatment. Diabetic rats had stable, moderate hyperglycemia throughout the 2 months (mean blood glucose concentrations, >400 mg/dl) (Dhein et al., 2003). After 2 months, the experiment was terminated.
Results and Discussion
We used the ChIP cloning procedure to identify novel HNF4 target genes after formaldehyde cross-linking of nucleoprotein complexes in highly differentiated Caco-2 cell cultures (Hu and Perlmutter, 1999) (Fig. 1A). At day 11 in culture, the HNF4 protein expression was abundant (Fig. 1B), as determined by Western blotting experiments. Furthermore, EMSA experiments evidenced a marked increase in HNF4 DNA binding to the A site of the HNF1 promoter (HNF1pro) (Fig. 1C). The A site is an established recognition site for HNF4 (Sladek and Seidel, 2001; Schrem et al., 2002). The ability of the HNF4 antibody to immunoprecipitate HNF4 was confirmed by Western blotting (Fig. 1D), and specificity of the ChIP assay was tested for by screening of immunoprecipitated DNA for enrichment of promoter sequences of well-known HNF4 target genes (Sladek and Seidel, 2001; Schrem et al., 2002). In particular, we confirmed by PCR amplification that immunoprecipitated DNA contains HNF1, apolipoprotein-B (ApoB), 1-antitrypsin (AAT), and angiotensinogen (ANG) (Fig. 1E). We therefore provide evidence for the enrichment of known HNF4 target genes in immunoprecipitated DNA and document specificity for the experimental procedure. We further evidence expression of HNF4 target genes by RT-PCR (Fig. 1F), and for each ChIP assay, detection of the HNF1 prosite served as a positive control. We thoroughly validated our experimental approach before the cloning of immunoprecipitated DNA and ChIP assays yielded clones with inserts of up to 1800 bp. The inserts were sequenced with vector-specific primers, and the genomic sequences were identified by database searches (GenBank, maintained by NCBI). We found novel HNF4 target genes distributed among different chromosomes. This demonstrates the usefulness of ChIP cloning procedure to identify multiple chromosomal targets for this factor. Some of the cloned fragments were within intronic regions, and this agrees well with findings by other investigators (Greenbaum and Zhuang, 2002; Martone et al., 2003; Solano et al., 2003). We also analyzed proximal promoter sequences. Cloned fragments as well as promoter sequences were checked for putative HNF4 binding sites, and primer pairs were designed to confirm experimentally predicted sites. Independent ChIP experiments followed by PCR analyses with clone-specific or promoter-specific primers enabled robust identification of HNF4 target genes.
Here, we report in detail the identification of two novel kinases targeted by HNF4. Binding in vivo of HNF4 was confirmed for two recognition sites of clone 113 (Fig. 2A). In addition, by using a bioinformatic approach, we predicted promoter binding sites within clones 113 and 23, which were specifically bound by HNF4 in vivo (Fig. 2A). We further studied the ability of HNF4 to bind to cognate recognition sites by EMSA with 32P-labeled probes encompassing the predicted HNF4 sites located in clone 113 (GS09, GS16), in the promoter of clone 113 (GS04, GS29), and in the promoter of clone 23 (GS26, GS27) (Fig. 2B). Supershift experiments with a specific HNF4 antibody resulted in strong binding of HNF4 with probes GS09, GS16, and GS26 and weaker binding with probe GS27. No binding of HNF4 was detected with probes GS04 and GS29. Competition and supershift experiments with probes specific for several HNF4 target genes were carried out to estimate binding affinity of HNF4 to known and newly identified targets (Fig. 2, B and C). Again, the HNF1 prosite was used as labeled probe, and competition was first analyzed with different known cognate recognition sites, namely the HNF1 prosite itself and HNF4 binding sites within AATpro, ApoBpro, and ANGpro. These sites were distinguishable in their binding affinity (Fig. 2C). With the AAT prosite (100x, reduction to 10%) and the ApoB site (100x, reduction to 2.5%), binding was comparable with the HNF1 prosite itself (100x, reduction to 2.2%), whereas competition with the ANG prosite (100x, reduction to 34.6%) was less efficient. In comparison, competition with the GS09 probe (100x, reduction to 13.7%) and the GS26 probe (100x, reduction to 9.5%) resulted in strong binding, whereas competition with the GS16 probe (100x, reduction to 32.5%) was less efficient, and binding affinity with the GS27 probe (100x, reduction to 85.8%) was minimal. In addition, we performed experiments with GS08 as the 32P-labeled probe, and competition was performed with probes for the known cognate recognition sites, namely HNF1pro, ApoBpro, AATpro, and ANGpro (Fig. 2C). All probes competed successfully for HNF4 binding (100x, with HNF1pro reduction to 4.7%, with ApoBpro reduction to 4.5%, with AATpro reduction to 6.6%, and with ANGpro reduction to 8.6%). Competition experiments were complemented by supershift assays. Clone 113 itself and two sites in the promoter of clone 23 were thus confirmed for in vivo and in vitro binding of HNF4.
It is noteworthy that we did not confirm HNF4 in vitro binding for certain putative sites (prosites a and c) in the promoter of clone 113. Nonetheless, the surrounding regions of 126 and 232 bp contained in vivo binding sites (Fig. 2A). The promoter thus harbors an adjacent HNF4 binding site (prosite b, GS46, localized approximately 800 bp upstream to site a and approximately 400 bp downstream to site c), exhibiting strong in vitro binding of HNF4 (Fig. 2, B and C), which would allow for immunoprecipitation of prosite a and prosite c through protein-protein cross-links. Indeed, HNF4 may contact DNA through various protein-protein interactions, particularly through cooperative binding with synergized factors, because formaldehyde cross-links leads to both protein-DNA and protein-protein complexes. Therefore in ChIP experiments, a three-dimensional, higher-order structure can be cross-linked. In addition, HNF4 may also contact another site within the in vivo confirmed fragments, which was not predicted by our computational approach.
It is noteworthy that gene regulation of a broad range of cytochrome P450 isozymes does depend on promoter activation by HNF4 (Jover et al., 2001), and treatment of hepatocytes with Aroclor 1254 resulted in the induction of HNF4 and several cytochrome P450 mono-oxgenases (Borlak and Thum, 2001). We therefore analyzed HNF4 binding in liver nuclear extracts of control and Aroclor 1254-treated rats by EMSA. Binding of HNF4 to HNF1pro as well as to the newly identified binding sites (GS09, GS16, GS46, GS26, and GS27) was significantly increased after Aroclor 1254 treatment (Fig. 2D), thus providing additional evidence for our novel targets to be strictly regulated by HNF4.
A summary of the cloned HNF4 targets is given in Table 6. Clone 23 contained two ChIP-verified HNF4 binding sites in the promoter region (around eC1430 and eC2053) and was identified as RSK4, a novel member of the ribosomal S6 kinase subfamily (Yntema et al., 1999; Kohn et al., 2003). Because RSK4 gene deletion was found in some patients with X-linked mental retardation, a role for this kinase in neuronal development was suggested (Yntema et al., 1999). RSK4 expression during mouse development, however, is ubiquitous (Kohn et al., 2003), which suggests additional roles for the gene product in development. Clone 113 contained two ChIP-verified HNF4 promoter binding sites around eC951 and eC2181 and was conclusively annotated as PAK5. This kinase was recently cloned and characterized as a novel member of mammalian p21cdc42/rac1-activated kinase subfamily (Dan et al., 2002; Pandey et al., 2002). Until now, its role was confined to the induction of neurite outgrowth (Dan et al., 2002), whereas PAK kinases, in general, play a role in neurodegenerative diseases (Kumar and Vadlamudi, 2002).
It is of considerable importance that HNF4 targets kinases important in neuronal development and function. Thus, besides its role in liver metabolism, HNF4 may also play a role in brain function. In particular, HNF4 regulates several genes involved in glucose metabolism (Sladek and Seidel, 2001; Schrem et al., 2002) and participates in the glucose-dependent insulin secretory pathways. HNF4 dysfunction, however, does lead to multifactorial type 2 diabetes (Love-Gregory et al., 2004) with patients developing diabetic neuropathies. Moreover, one form of a rare monogenetic disorder, MODY, was mapped to mutations within the HNF4 gene (MODY-1) (Sladek and Seidel, 2001; Schrem et al., 2002). Patients with diabetes harbor a high risk for progressive neuropathies for uncertain reasons (Vinik et al., 2000). Although the precise role of HNF4 in brain function is unknown, we demonstrate gene expression of this transcription factor in human and rat brain (Fig. 2E). It is interesting that expression of the splice variant HNF47 was also reported for mouse brain (Nakhei et al., 1998). Next to its expression in brain tissue, RSK4 is expressed at a similar level in kidney but lesser in pancreas and placenta (Yntema et al., 1999). We provide strong evidence for RSK4 to be expressed in Caco-2 cells, in human and rat liver, and in rat kidney (Fig. 2E). Unlike RSK4, PAK5 is abundantly expressed in pancreas, but the level of expression is minimal in liver and kidney (Dan et al., 2002). We demonstrate expression of PAK5 in RNA extracts of human and rat liver and in rat kidney (Fig. 2E) but not in cultures of Caco-2 cells. Failure to detect PAK5 mRNA transcripts in Caco-2 cells suggests lack of synergistic transcription factors acting in concert, even though in vivo binding of HNF4 to PAK5 recognition sites was confirmed (Fig. 2A).
HNF4 is a dominant regulator of the epithelial phenotype and is highly expressed in kidney (Sladek and Seidel, 2001). Furthermore, in diabetic patients, nephropathy is a frequently observed complication, and treatment of rats with STZ results in diabetic nephro- and neuropathy (Gross et al., 2003; Bianchi et al., 2004). We observed significant reduction of HNF4 transcript level in liver. Likewise, PAK5 gene expression in brain extracts of STZ-induced diabetic rats was repressed, as was HNF4 itself, RSK4, and PAK5 in total RNA extracts of rat kidney (Table 7). We therefore demonstrate diabetic neuropathy and nephropathy to be strongly associated with repressed RSK4 and PAK5 gene expression levels, as a result of HNF4 dysfunction. It is noteworthy that treatment of rats with Aroclor 1254 led to significant induction of RSK4 mRNA in rat kidneys (Table 7), thus providing further evidence for a coordinate regulation of RSK4 and HNF4 gene expression.
HNF4, RSK4, and PAK5 gene expression was measured with real-time RT-PCR. Gene expression levels were normalized to mitATPase6.
In general, members of the RSK family function as downstream mediators of mitogen-activated protein/extracellular signal-regulated kinase signal transducers of cell survival (Nebreda and Gavin, 1999) and cell-cycle regulation (Roux et al., 2003), whereas PAK kinases play key roles in the stimulation of mitogen-activated protein kinase signaling pathways (Kumar and Vadlamudi, 2002). RSKs phosphorylate an array of transcription factors (e.g., cAMP response element-binding protein, cAMP response element-binding protein/p300, estrogen receptor , IB/nuclear factor-B, c-Fos), take part in chromatin remodeling through phosphorylation of histone H3, and down-regulate p34cdc2 inhibitory kinase, which may be important for progression through G2/M phase of mitosis (Nebreda and Gavin, 1999). PAK kinases regulate cytoskeletal dynamics by disassembly of stress fibers and focal adhesions (Kumar and Vadlamudi, 2002). Its role in specific cellular cytoskeletal reorganization leads to the inhibition of cell spreading (Sanders et al., 1999). Thus, HNF4 targets two kinases that are indirect regulators of cell cycle, presumably with the aim of fostering cellular differentiation. In conclusion, we report a unique role for HNF4 in targeting RSK and PAK family members. This suggests a novel role for this liver-enriched transcription factor in repressing cell-cycle progression to enable cellular differentiation (Fig. 3). Recently, Chiba et al. (2005) reported that overexpression of HNF4 inhibited cell growth in F9 cells, and this was attributed to enhanced expression of cyclin-dependent kinase inhibitor p21CIP1/WAF1, which supports the concept that HNF4 plays fundamental roles in the control of epithelial proliferation as well. Our data point to novel functions of HNF4 that are beyond the regulation of genes involved in hepatic metabolism. Further studies are now on the way to delineate the role of HNF4 in cell-cycle regulation and neuronal function. This may provide a missing link between HNF4 dysfunction and late-stage complications in diabetes frequently seen in patients with progressive stages of disease.
Acknowledgements
We thank S. Froese and A. Pfanne for valuable technical assistance, R. Zemlin for assistance in bioinformatics and advice on design of PCR primers, and K. Amann and P. Rsen for providing tissue of STZ-treated diabetic rats.
doi:10.1124/mol.104.008672.
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