Insulin Response Element Research Articles

Isotopic Depletion Increases the Spatial Resolution of FPOP Top-Down Mass Spectrometry Analysis.

Protein radical labeling, like fast photochemical oxidation of proteins (FPOP), coupled to a top-down mass spectrometry (MS) analysis offers an alternative analytical method for probing protein structure or protein interaction with other biomolecules, for instance, proteins and DNA. However, with the increasing mass of studied analytes, the MS/MS spectra become complex and exhibit a low signal-to-noise ratio. Nevertheless, these difficulties may be overcome by protein isotope depletion. Thus, we aimed to use protein isotope depletion to analyze FPOP-oxidized samples by top-down MS analysis. For this purpose, we prepared isotopically natural (IN) and depleted (ID) forms of the FOXO4 DNA binding domain (FOXO4-DBD) and studied the protein-DNA interaction interface with double-stranded DNA, the insulin response element (IRE), after exposing the complex to hydroxyl radicals. As shown by comparing tandem mass spectra of natural and depleted proteins, the ID form increased the signal-to-noise ratio of useful fragment ions, thereby enhancing the sequence coverage by more than 19%. This improvement in the detection of fragment ions enabled us to detect 22 more oxidized residues in the ID samples than in the IN sample. Moreover, less common modifications were detected in the ID sample, including the formation of ketones and lysine carbonylation. Given the higher quality of ID top-down MSMS data set, these results provide more detailed information on the complex formation between transcription factors and DNA-response elements. Therefore, our study highlights the benefits of isotopic depletion for quantitative top-down proteomics. Data are available via ProteomeXchange with the identifier PXD044447.

Insulin Inhibits Autophagy by Inhibiting the Binding of FoXO1 to the Promoter Region of GABARAPL1.

Hyperinsulinemia and insulin resistance in T2D have a potent suppressive effect on hepatic autophagy, however, the underlying mechanisms remain unclear. To explore the effect of insulin on hepatic autophagy and its possible signaling pathways, HL-7702 cells were treated with insulin with or without insulin signaling inhibitors. The interaction between insulin and the promoter region of GABARAPL1 was assessed through luciferase assay and EMSA. There were significant dose-dependent decreases in the number of intracellular autophagosomes and the protein levels of GABARAPL1 and beclin1 in insulin-treated HL-7702 cells. Insulin signaling inhibitors reversed the inhibitory effect of insulin on rapamycin-induced autophagy and autophagy-related gene upregulation. Insulin blocks the binding of FoxO1 to putative insulin response elements in GABARAPL1 gene promoter, leading to the repressed transcription of GABARAPL1 gene and the suppression of hepatic autophagy. Our study identified GABARAPL1 as a novel target of insulin in suppressing hepatic autophagy.

Insulin can up-regulate LC-PUFA biosynthesis with the involvement of Srebp-1c and stimulatory protein 1 (Sp1) in marine teleost Siganus canaliculatus

The rabbitfish Siganus canaliculatus is the first marine teleost found to have the biosynthetic ability of long-chain polyunsaturated fatty acids (LC-PUFA) from C18 precursors catalyzed by fatty acyl desaturases (Δ6/Δ5 Fads, Δ4 Fads) and elongases of very long chain fatty acids (Elovls). Previously, we predicted the existence of insulin (INS) response elements (IREs) including nuclear factor Y (NF-Y) and sterol regulatory element (SRE) in the core promoter region of rabbitfish Δ6/Δ5 fads and Δ4 fads. To clarify the potential regulatory effect and mechanism of INS in LC-PUFA biosynthesis, INS responding region was identified at −456 bp to + 51 bp of Δ6/Δ5 fads core promoter, but not in Δ4 fads promoter. Moreover, a unique stimulatory protein 1 (Sp1) element was predicted in the INS responding region of Δ6/Δ5 fads. Subsequently, SRE, NF-Y and Sp1 elements were proved as IREs in Δ6/Δ5 fads promoter. The up-regulation of INS on gene expression of Srebp-1c, Sp1, Δ6/Δ5 fads and elovl5 as well as the LC-PUFA biosynthesis was further demonstrated in S. canaliculatus hepatocyte line (SCHL) cells, but no influence was detected on Δ4 fads. Besides, inhibitors of transcription factors Srebp-1c (Fatostatin, PF-429242) and Sp1 (Mithramycin) could inhibit the gene expression of Srebp-1c, Δ6/Δ5 fads and elovl5, and abolish the up-regulation of INS on these genes’ expression and LC-PUFA biosynthesis. These results indicated that INS could up-regulate LC-PUFA biosynthesis with the involvement of Srebp-1c and Sp1 in rabbitfish S. canaliculatus, which is the first report in teleost.

Insulin mimetic effect of D-allulose on apolipoprotein A-I gene.

Several nutrients modulate the transcriptional activity of the apolipoprotein A-I (apo A-I) gene. To determine the influence of rare sugars on apo A-I expression in hepatic (HepG2) and intestinal derived (Caco-2) cell lines, apo A-I, albumin, and SP1 were quantified with enzyme immunoassay and Western blots while mRNA levels were quantified with real-time polymerase chain reaction. The promoter activity was measured using transient transfection assays with plasmids containing various segments and mutations in the promoter. D-allulose and D-tagatose, increased apo A-I concentration in culture media while D-sorbose and D-allose did not have any measurable effects. D-allulose did not increase apo A-I levels in Caco-2 cells. These changes paralleled the increased mRNA levels and promoter activity. D-allulose-response was mapped at the insulin response core element (IRCE). Mutation of the IRCE decreased the ability of D-allulose and insulin to activate the promoter. Treatment of HepG2 cells, but not Caco-2 cells, with D-alluose and insulin increased SP1 expression relative to control cells. D-allulose augmented the expression and IRCE binding of SP1, an essential transcription factor for the insulin on apo A-I promoter activity. D-allulose can modulate some insulin-responsive genes and may have anti-atherogenic properties, in part due to increasing apo A-I production. PRACTICAL APPLICATIONS: Coronary artery disease (CAD) is the number one cause of mortality in industrialized countries. A risk factor associated with CAD is low high-density lipoprotein (HDL) cholesterol and apolipoprotein A-I (apo A-I) concentrations in plasma. Thus, novel therapeutic agents or nutrients that upregulate apo A-I production should be identified. D-allulose and D-tagatose are used as sweeteners and may have favorable effects on insulin resistance and diabetes. This study shows that D-allulose and D-tagatose increases apo A-I production through increased transcription factor SP1-binding to insulin response element of the promoter. These sweeteners modulate some insulin responsive genes, increase the production of apo-A-I, and therefore may have anti-atherogenic properties.

Ring finger protein 152-dependent degradation of TSPAN12 suppresses hepatocellular carcinoma progression

BackgroundHepatocellular carcinoma (HCC) is the third cause of cancer death in the world, and few molecularly targeted anticancer therapies have been developed to treat it. The E3 ubiquitin ligase RNF152 has been reported to regulate the activity of the mechanistic target of rapamycin complex 1 (mTORC1), induce autophagy and apoptosis. However, the relationship between RNF152 and HCC is unclear.MethodsTranscriptome RNA-sequencing data of HCC samples and normal tissues were used to detect the mRNA expression of RNF152. Luciferase reporter and chromatin immunoprecipitation (ChIP) assays were used to determine the transcriptional regulation of RNF152 in HCC by FoxO1. RNAi, cell proliferation, colony formation and transwell assays were used to determine the in vitro functions of RNF152. Mouse xenograft models were used to study the in vivo effects of RNF152. The immunoprecipitation assay was used to determine the interaction between RNF152 and TSPAN12. The in vivo ubiquitination assay was performed to determine the regulation of TSPAN12 by RNF152.ResultsWe found that RNF152 is significantly down-regulated in clinic HCC samples, and its down-regulation is associated with pool overall survival (OS), progression-free survival (PFS) and disease-specific survival (DSS) in HCC patients. The transcription factor FoxO1 was significantly positively correlated RNF152 expression in HCC tissues. FoxO1 recognizes a classic insulin response element (IRE) on the RNF152 promoter to regulate its expression in HCC. RNF152 suppressed HCC cell proliferation, clonogenic survival, invasion in vitro, and tumorigenesis in vivo. Mechanistically, RNF152 interacted with TSPAN12 and targeted it for ubiquitination and proteasomal degradation, thereby inhibiting TSPAN12-dependent CXCL6 expression and HCC progression.ConclusionCollectively, our data revealed a tumor suppressor role of RNF152 and a connection between RNF152 and FoxO1 in HCC. Our results support an important role of the FoxO1-RNF152-TSPAN12 axis in the development of HCC. Therapeutic targeting this axis may be an effective means of treating HCC.

Forkhead transcription factor O1 (FoxO1) in torafugu pufferfish Takifugu rubripes: Molecular cloning, in vitro DNA binding, and target gene screening in fish metagenome.

The fish insulin/insulin-like growth factor (IGF) pathway has weak control over carbohydrate metabolism. To understand the molecular basis for the metabolic diversity, we characterized the forkhead box transcription factor O1A (FoxO1A), a downstream target of the insulin/IGF pathway, in torafugu Takifugu rubripes. The cloned torafugu FoxO1A cDNA contained all conserved features critical for its transcriptional activity and a unique unspliced intron encoding a poly-glutamine stretch. Torafugu FoxO1A showed the IGF-dependent nuclear exclusion and in vitro binding to the well-conserved FoxO1 binding site, DAF-16 binding element (DBE), but failed to bind to the insulin-responsive element by which mammalian FoxO1 mediates insulin effects. The subsequent in silico genomic screening provided a list of 587 potential torafugu FoxO1A target genes containing the DBE. Some carbohydrate metabolic genes regulated by FoxO1 in mammals were not included in the list. We further identified about 250 potential fish FoxO1 target genes by integrating results of the DBE screening against fish metagenome that contained 262 species. Neuronal processes appeared to be the common major function of fish FoxO1, although further annotation of the potential target genes is required. These results provide a part of the molecular basis underlying the weak association between the insulin/IGF pathway and carbohydrate metabolism in fish.

Hormonal Regulation of Glycine Decarboxylase and its Impact on Cellular Physiology

The amino acid glycine is involved in generation of multiple critical metabolites including glutathione, heme, and creatinine. Interestingly, in both humans and rodents, circulating glycine levels are significantly reduced in obesity, glucose intolerance, type II diabetes and non‐alcoholic fatty liver disease. The glycine cleavage system (GCS) is the predominant glycine degradation pathway in humans. The rate‐limiting enzyme of GCS is glycine decarboxylase (GLDC), and loss‐of‐function mutations of GLDC cause hyperglycinemia. Here, we show that GLDC gene expression is upregulated in livers of mouse models of diabetes and diet‐induced obesity as well as in the fasted state in normal animals. In exploring the hormonal signals that mediate these regulatory events we found that both glucagon and insulin stimulated GLDC gene expression. In primary rat hepatocytes, GLDC expression was strongly stimulated by glucagon and cAMP, and mildly with insulin while in a rat hepatoma cell line, insulin strongly stimulated GLDC expression as compared to cAMP. We identified the cAMP‐response element binding protein 1 (Creb1) and insulin responsive transcription factor Sterol regulatory element binding protein 1c (Srebp‐1c) as mediators of the glucagon and insulin stimulatory effect, respectively. By knocking down or overexpressing GLDC in cell lines we observed GLDC expression levels are strongly linked to intracellular glutathione and reactive oxygen species (ROS) levels. Our findings suggest that the hormonal regulation of GLDC may contribute to compensatory changes in glutathione production as a defense against metabolic disease‐associated oxidative stress.Support or Funding InformationThis work is supported by NIH R21AG050741, Grants Boost OVPR Wayne State University and the Michigan Diabetes Research and Training Center.

Zbtb7c is a critical gluconeogenic transcription factor that induces glucose-6-phosphatase and phosphoenylpyruvate carboxykinase 1 genes expression during mice fasting

Gluconeogenesis is essential for blood glucose homeostasis during fasting and is regulated by various enzymes, which are encoded by gluconeogenic genes. Those genes are controlled by various transcription factors. Zinc finger and BTB domain–containing 7c (Zbtb7c, also called Kr-pok) is a BTB-POZ family transcription factor with proto-oncogenic activity. Previous findings have indicated that Zbtb7c is involved in the regulation of fatty acid biosynthesis, suggesting an involvement also in primary metabolism. We found here that fasting induced Zbtb7c expression in the mouse liver and in primary liver hepatocytes. We also observed that Zbtb7c-knockout mice have decreased blood glucose levels, so we investigated whether Zbtb7c plays a role in gluconeogenesis. Indeed, differential gene expression analysis of Zbtb7c-knockout versus wild type mouse livers showed downregulated transcription of gluconeogenic genes encoding the glucose 6-phosphatase catalytic subunit (G6pc) and phosphoenolpyruvate carboxykinase 1 (Pck1), while Zbtb7c expression upregulated these two genes, under fasting conditions. Mechanistically, we found that when complexed with histone deacetylase 3 (Hdac3), Zbtb7c binds insulin response elements (IREs) within the G6pc and Pck1 promoters. Moreover, complexed Zbtb7c deacetylated forkhead box O1 (Foxo1), thereby increasing Foxo1 binding to the G6pc and Pck1 IREs, resulting in their transcriptional activation. These results demonstrate Zbtb7c to be a crucial metabolic regulator of blood glucose homeostasis, during mammalian fasting.

A Potential Link between Glycine Metabolism, Glutathione and Diabetes

Type 2 diabetes (T2D) is accompanied by alterations in amino acid metabolism. Interestingly, in both humans and rodents, circulating glycine levels are significantly reduced in obesity, glucose intolerance and T2D identifying glycine as a biomarker of T2D and raising the possibility that altered glycine metabolism plays an active role in the etiology of the disease. The goals of our study are to determine if altered glycine metabolism plays a functional role in the development of diabetes. The glycine cleavage system (GCS) is the only biochemical route that degrades glycine in humans and loss‐of‐function mutations of GCS cause hyperglycinemia. Here, we show that the expression of the rate‐limiting enzyme of GCS, Glycine Decarboxylase (GLDC), is upregulated in livers of hyperinsulinemic mouse models of diabetes, and in hepatoma cell lines insulin stimulates transcription of the GLDC gene. We identified the insulin responsive transcription factor Sterol regulatory element binding protein 1c (SREBP‐1c) as the primary mediator of this insulin stimulatory effect. We also observed that altering GLDC expression levels strongly affected intracellular glutathione levels and levels of reactive oxygen species (ROS). Our findings suggest a working model in which Srebp1c‐mediated regulation of GLDC is responsible for the reduction of glycine levels seen in insulin resistant states and may play function as a compensatory mechanism to increase glutathione production as a defense against diabetes‐induced oxidative stress.Support or Funding InformationThis work is supported by NIH R21AG050741, and the Michigan Diabetes Research and Training Center.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Transcriptional Activation of Lipogenic Genes by Insulin/Feeding

Insulin treatment or feeding a high carbohydrate diet causes a marked increase in fatty acid and fat synthesis and enzymes involved in lipogenesis are coordinately induced. We are studying transcriptional activation of lipogenic genes, employing fatty acid synthase (FAS) as a model system. We defined insulin response element at ‐65 E‐box and USF binding at this site for transcriptional activation by insulin. USF and SREBP‐1c, by binding at adjacent sites, directly interact and function together. We identified various other components of the USF complex and their posttranslational modifications by feeding/insulin treatment. USF‐1 recruits and is phosphorylated by DNA‐PK allowing recruitment and acetylation by P/CAF, resulting in the FAS promoter activation, whereas, in fasting, USF‐1 is deacetylated by HDAC9. USF also recruits Brg1/Brm‐associated factor (BAF) 60c that functions as a specific chromatin‐remodeling component for lipogenic gene transcription. In response to insulin, BAF60c is phosphorylated at S247 by atypical PKCζ/λ, which causes translocation of BAF60c to the nucleus, allowing a direct interaction with USF‐1. Thus, BAF60c is recruited to form the lipoBAF complex to remodel chromatin structure. Recently, we also found that by direct interaction USF‐1 recruits specific histone demethylase for histone modification and transcriptional activation of various lipogenic genes.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of Beclin 1 overexpression on aggressive phenotypes of colon cancer cells.

Beclin 1 is involved in autophagy, differentiation, apoptosis and cancer progression, and functions as a haploinsufficient tumor suppressor gene. The aim of the present study was to elucidate the function of Beclin 1 in colon cancer. A Beclin 1-expressing plasmid was transfected into HCT-15 and HCT-116 cells, and the phenotypes and associated molecules were determined. Beclin 1 transfectants were subcutaneously injected into nude mice to determine tumor growth, and proliferation and apoptosis levels using Ki-67 immunostaining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), respectively. Beclin 1 overexpression inhibited viability as determined using a Cell Counting Kit-8 assay, inhibited migration and invasion as determined using a wound healing assay or Transwell assay, and lamellipodia formation by filamentous actin staining, induced autophagy as determined using electron microscopy, and light chain 3B (LC-3B) expression, and apoptosis as determined using Annexin V staining in the two cell lines (P<0.05). Beclin 1 induced G2 arrest of HCT-15 transfectants as determined using propidium iodide staining (P<0.05), whereas HCT-116 transfectants were arrested in G1 phase (P<0.05). The two transfectants exhibited increased expression of c-Myc, cyclin D1, β-catenin, insulin-response element 1 and 78 kDa glucose-regulated protein compared with the control and mock cells as determined using the reverse transcription-quantitative polymerase chain reaction (P<0.05). Beclin 1 overexpression upregulated LC-3B and cyclin-dependent kinase 4 expression, but downregulated cyclin E expression of the cancer cell lines as determined using western blot analysis (P<0.05). Beclin 1 expression in vivo significantly suppressed the proliferation of colon cancer cells in xenograft models via inducing apoptosis by TUNEL, and inhibiting proliferation by Ki-67 expression (P<0.05). Beclin 1 overexpression may reverse aggressive phenotypes and suppress colon cancer tumor growth, and be employed as a target molecule for gene therapy of patients with colon cancer.

Crystal structures reveal a new and novel FoxO1 binding site within the human glucose-6-phosphatase catalytic subunit 1 gene promoter.

Human glucose-6-phosphatase plays a vital role in blood glucose homeostasis and holds promise as a therapeutic target for diabetes. Expression of its catalytic subunit gene 1 (G6PC1) is tightly regulated by metabolic-response transcription factors such as FoxO1 and CREB. Although at least three potential FoxO1 binding sites (insulin response elements, IREs) and one CREB binding site (cAMP response element, CRE) within the proximal region of the G6PC1 promoter have been identified, the interplay between FoxO1 and CREB and between FoxO1 bound at multiple IREs has not been well characterized. Here we present the crystal structures of the FoxO1 DNA binding domain in complex with the G6PC1 promoter. These complexes reveal the presence of a new non-consensus FoxO1 binding site that overlaps the CRE, suggesting a mutual exclusion mechanism for FoxO1 and CREB binding at the G6PC1 promoter. Additional findings include (i) non-canonical FoxO1 recognition sites, (ii) incomplete FoxO1 occupancies at the available IRE sites, and (iii) FoxO1 dimeric interactions that may play a role in stabilizing DNA looping. These findings provide insight into the regulation of G6PC1 gene transcription by FoxO1, and demonstrate a high versatility of target gene recognition by FoxO1 that correlates with its diverse roles in biology.

Over-expression of insulin-response element binding protein-1 (IRE-BP1) in mouse pancreatic islets increases expression of RACK1 and TCTP: Beta cell markers of high glucose sensitivity

BackgroundA targeted analysis of the 50kDa C-terminal fragment of insulin-response element binding protein-1 (IRE-BP1) activation of target genes through the insulin receptor substrate receptor/PI-3 kinase/Akt pathway has been demonstrated for the insulin growth factor-1 receptor. The broader effects of 50kDa C-terminal IRE-BP1 fragment over-expression on protein abundance in pancreatic islet beta cells have not been determined. ResultsLiquid-chromatography coupled to tandem mass spectrometry (LC-MS/MS) analyses of replicate lysates of pancreatic islets isolated from background strain animals and transgenic animals, overexpressing IRE-BP1 in pancreatic islet beta cells, demonstrated statistically significant increases in the expression of proteins involved in protein synthesis, endoplasmic reticulum (ER) stress and scaffolding proteins important for protein kinase C signaling; some of which were confirmed by immunoblot analyses. Bioinformatic analysis of protein expression network patterns suggested IRE-BP1 over-expression leads to protein expression patterns indicative of activation of functional protein networks utilized for protein post-translational modification, protein folding, and protein synthesis. Co-immunoprecipitation experiments demonstrate a novel interaction between two differentially regulated proteins receptor for activated protein kinase C (RACK1) and translationally controlled tumor protein (TCTP). ConclusionsProteomic analysis of IRE-BP1 over-expression in pancreatic islet beta cells suggest IRE-BP1 (a) directly or indirectly through establishing hyperglycemia results in increased expression of ribosomal proteins and markers of ER stress and (b) leads to the enhanced and previously un-described interaction of RACK1 and TCTP. SignificanceThis study identified C-terminal 50kDa domain of IRE-BP1 over-expression results in increased markers of ER-stress and a novel interaction between the scaffolding proteins RACK1 and TCTP.

The hepatic FOXQ1 transcription factor regulates glucose metabolism in mice.

Hepatic forkhead box q1 (FOXQ1) expression levels are regulated by nutritional and pathophysiological status. In this study we investigated the role of FOXQ1 in the regulation of hepatic gluconeogenesis. We used multiple mouse and cell models to study the role of FOXQ1 in regulating expression of gluconeogenic genes, and cellular and hepatic glucose production. Expression of hepatic FOXQ1 was regulated by fasting in normal mice and was dysregulated in diabetic mice. Overexpression of FOXQ1 in primary hepatocytes inhibited expression of gluconeogenic genes and decreased cellular glucose output. Hepatic FOXQ1 rescue in db/db and high-fat diet-induced obese mice markedly decreased blood glucose level and improved glucose intolerance. In contrast, wild-type C57 mice with hepatic FOXQ1 deficiency displayed increased blood glucose levels and impaired glucose tolerance. Interestingly, studies into molecular mechanisms indicated that FOXQ1 interacts with FOXO1, thereby blocking FOXO1 activity on hepatic gluconeogenesis, preventing it from directly binding to insulin response elements mapped in the promoter region of gluconeogenic genes. FOXQ1 is a novel factor involved in regulating hepatic gluconeogenesis, and the decreased FOXQ1 expression in liver may contribute to the development of type 2 diabetes.

Examining FOXO1 and DNA Interactions through Computational and Direct Methods

FOXO transcription factors are a subgroup of the FOX (forkhead) family and are important for regulation of cellular processes, gene expression, apoptosis, cell‐ cycle progression, and oxidative stress resistance. Several FOXO translocations have also been implicated in cancer development, with the most common being the PAX3‐FOXO1 translocation linked to the development of Alveolar Rhabdomyosarcoma (ARMS). All FOXO proteins share sequence similarity and a highly conserved DNA‐ binding domain (DBD). This DNA‐binding domain consists of three alpha helices accompanied by two winged domains; this domain binds the Daf‐16 binding element (DBE) and the insulin response element (IRE), with the crystal structure indicating that the majority of the DNA contacts are located within Helix 3. There have been reports in the literature of sequences flanking the IRE causing an increase in binding affinity; however, no mechanism has been reported to explain this effect. Using Molecular Dynamics Simulation (MDS) we observed that the number of direct interactions between protein and DNA increases over time. Interestingly, MDS showed that wing 1 has no contact with DNA at 0 ns but eventually makes contact with a region flanking the IRE consensus sequence at 10 ns. In order to verify the new interactions observed in the 10 ns model, we mutated several bases within the IRE and observed the effect of these mutations on the interaction between protein and DNA through Electrophoretic Mobility Shift Assays (EMSA) and intrinsic fluorescence. Mutation of several bases in the IRE consensus sequence as well as in the immediate 5′ and 3′ flanking regions decreased the interaction between FOXO1 and DNA. In support of our model, mutation of the new base contact identified with Wing 1 also decreased FOXO1 DNA‐binding. The truncated FOXO1 DNA‐binding domain found in the PAX3‐FOXO1 fusion was also examined by MDS, and was predicted to retain several contacts within Helix 3 as well as the newly identified contact in Wing 1. Using this information, we can further examine the DNA‐binding of PAX3‐FOXO1 to determine if the presence of flanking sequences surrounding the IRE consensus sequence also affect the affinity of the fusion protein for DNA. Knowledge of the contribution of the FOXO1 domain to PAX3‐FOXO1 DNA targeting is important for understanding the activity of this fusion protein and its role in ARMS development.Support or Funding InformationNIH SC2GM095430RCMI‐Molecular Structure and Modeling core facility (NIH‐RCMI 2G12MD007595‐06 )NIH 5RL5GM118966‐03RISE (R25GM060926)

Loss of Interdependent Binding by the FoxO1 and FoxA1/A2 Forkhead Transcription Factors Culminates in Perturbation of Active Chromatin Marks and Binding of Transcriptional Regulators at Insulin-sensitive Genes

FoxO1 binds to insulin response elements located in the promoters of insulin-like growth factor-binding protein 1 (IGFBP1) and glucose-6-phosphatase (G6Pase), activating their expression. Insulin-mediated phosphorylation of FoxO1 promotes cytoplasmic translocation, inhibiting FoxO1-mediated transactivation. We have previously demonstrated that FoxO1 opens and remodels chromatin assembled from the IGFBP1 promoter via a highly conserved winged helix motif. This finding, which established FoxO1 as a "pioneer" factor, suggested a model whereby FoxO1 chromatin remodeling at regulatory targets facilitates binding and recruitment of additional regulatory factors. However, the impact of FoxO1 phosphorylation on its ability to bind chromatin and the effect of FoxO1 loss on recruitment of neighboring transcription factors at its regulatory targets in liver chromatin is unknown. In this study, we demonstrate that an amino acid substitution that mimics insulin-mediated phosphorylation of a serine in the winged helix DNA binding motif curtails FoxO1 nucleosome binding. We also demonstrate that shRNA-mediated loss of FoxO1 binding to the IGFBP1 and G6Pase promoters in HepG2 cells significantly reduces binding of RNA polymerase II and the pioneer factors FoxA1/A2. Knockdown of FoxA1 similarly reduced binding of RNA polymerase II and FoxO1. Reduction in acetylation of histone H3 Lys-27 accompanies loss of FoxO1 and FoxA1/A2 binding. Interdependent binding of FoxO1 and FoxA1/A2 possibly entails cooperative binding because FoxO1 and FoxA1/A2 facilitate one another's binding to IGFPB1 promoter DNA. These results illustrate how transcription factors can nucleate transcriptional events in chromatin in response to signaling events and suggest a model for regulation of hepatic glucose metabolism through interdependent FoxO/FoxA binding.

Direct regulation of IGF-binding protein 1 promoter by interleukin-1β via an insulin- and FoxO-1-independent mechanism.

The level of insulin-like growth factor-binding protein 1 (IGFBP1), a liver-produced serum protein that regulates insulin-like growth factor-I bioactivity, glucose homeostasis, and tissue regeneration, increases during inflammation. This manuscript describes a novel pathway for the regulation of hepatic IGFBP1 mRNA and protein levels by interleukin (IL)-1β. Experiments with the luciferase reporter system show that IL-1β stimulates transcriptional activity from the 1-kb promoter region of IGFBP1. Although IL-1β stimulation suppresses the insulin activation of protein kinase B, the major upstream regulator of IGFBP1 mRNA transcription, the induction of IGFBP1 by IL-1β did not require an intact insulin response element. Furthermore, neither overexpression nor silencing of FoxO-1 had any effect on the IL-1β-induced increase in IGFBP1 mRNA levels and promoter activity. However, inhibition of the ERK MAP kinases effectively prevented the IL-1β effects. Inhibition of neutral sphingomyelinase, a key player in the IL-1β signaling cascade that acts upstream of ERK, also suppressed the IL-1β effects, while increasing the ceramide, through the addition of C2-ceramide or via treatment with exogenous sphingomyelinase, was sufficient to induce IGFBP1 promoter-driven luciferase activity. Studies in primary rat hepatocytes where the levels of neutral sphingomyelinase were either elevated or suppressed using adenoviral constructs affirmed the key role of neutral sphingomyelinase and ceramide (exerted likely through ERK activation) in the IL-1β-induced IGFBP1 production. Finally, the IL-1β effects on IGFBP1 mRNA production and protein secretion could be abolished by the addition of insulin, either at very late time points or at very high doses.

Forkhead Box P1 (FOXP1) Transcription Factor Regulates Hepatic Glucose Homeostasis

Dysregulation of hepatic gluconeogenesis contributes to the pathogenesis of diabetes, yet the detailed molecular mechanisms remain to be fully elucidated. Here we show that FOXP1, a transcriptional repressor, plays a key role in the regulation of systemic glucose homeostasis. Hepatic expression levels of FOXP1 are decreased in diabetic mice. Modest hepatic overexpression of FOXP1 in mice inhibited the expression of gluconeogenic genes, such as peroxisome proliferators-activated receptor γ coactivator-1α (PGC-1α), phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6-phosphatase (G6PC), leading to a decrease in hepatic glucose production and fasting blood glucose levels in normal mice and different mouse models of diabetes, including db/db diabetic and high-fat diet-induced obese mice. FOXP1 physically interacted with FOXO1 in vivo and competed with FOXO1 for binding to the insulin response element in the promoter region of gluconeogenic genes, thereby interfering expression of these genes. These results identify a previously unrecognized role for FOXP1 in the transcriptional control of hepatic glucose homeostasis.

Macrophage CGI-58 deficiency promotes IL-1β transcription by activating the SOCS3-FOXO1 pathway.

Over-nutrition induces low-grade inflammation that dampens insulin sensitivity, but the underlying molecular mediators are not fully understood. Comparative gene identification-58 (CGI-58) is an intracellular lipolytic activator. In the present study, we show that in mouse visceral fat-derived macrophages or human peripheral blood monocytes, CGI-58 negatively and interleukin (IL)-1β positively correlate with obesity. Saturated non-esterified fatty acid (NEFA) suppresses CGI-58 expression in macrophages and this suppression activates FOXO1 (forkhead box-containing protein O subfamily-1) through inhibition of FOXO1 phosphorylation. Activated FOXO1 binds to an insulin-responsive element in IL-1β promoter region to potentiate IL-1β transcription. Gain- and loss-of-function studies demonstrate that NEFA-induced CGI-58 suppression activates FOXO1 to augment IL-1β transcription by dampening insulin signalling through induction of SOCS3 (suppressor of cytokine signalling 3) expression. CGI-58 deficiency-induced SOCS3 expression is NLRP3 (nucleotide-binding oligomerization domain-like receptor family, pyrin domain containing 3) inflammasome-dependent. Our data thus identified a vicious cycle (IL-1β-SOCS3-FOXO1-IL-1β) that amplifies IL-1β secretion and is initiated by CGI-58 deficiency-induced activation of the NLRP3 inflammasome in macrophages. We further show that blocking this cycle with a FOXO1 inhibitor, an antioxidant that inhibits FOXO1 or IL-1 receptor antagonist alleviates chronic inflammation and insulin resistance in high-fat diet (HFD)-fed mice. Collectively, our data suggest that obesity-associated factors such as NEFA and lipopolysaccharide (LPS) probably adopt this vicious cycle to promote inflammation and insulin resistance.

FOXO1-mediated epigenetic modifications are involved in the insulin-mediated repression of hepatocyte aquaporin 9 expression.

Aquaporin (AQP) 9 transports glycerol and water, and belongs to the aquaglyceroporin subfamily. Insulin acts as a negative regulator of AQP9, and FOXO1 has the ability to mediate the regulatory effects of insulin on target gene expression. The aim of the present study was to determine whether insulin‑induced repression of AQP9 involved an epigenetic mechanism. HepG2 human hepatocyte cells were treated with 500 µM insulin for different durations. AQP9 mRNA expression levels were determined by quantitative polymerase chain reaction (qPCR), and histone H3 acetylation, phosphorylation and methylation at the insulin responsive element (IRE) of the AQP9 promoter was assessed using chromatin immunoprecipitation coupled with qPCR. The effects of lentiviral FOXO1 overexpression on AQP9 expression levels and H3 modifications at the AQP9 promoter were also determined. The insulin treatment resulted in a significant and time‑dependent reduction in AQP9 mRNA expression levels in HepG2 cells, as compared with untreated cells (P<0.05). In the insulin‑treated cells, the levels of H3 acetylation and phosphorylation were significantly reduced (P<0.05), but the level of H3 methylation was increased. Enforced expression of FOXO1 increased AQP9 mRNA and protein expression levels in HepG2 cells. Furthermore, FOXO1 overexpression promoted H3 acetylation and phosphorylation, and reduced H3 methylation at the IRE locus of the AQP9 promoter. These data provide, to the best of our knowledge, the first evidence that insulin‑induced transcriptional suppression of AQP9 expression in hepatocytes involves FOXO1‑mediated H3 modifications at the IRE locus in the promoter.