GW8510 increases insulin expression in pancreatic alpha cells through activation of p53 transcriptional activity. Treatment of mouse alpha cells and human islet cells with GW8510 upregulates insulin expression and secretion. Gene expression profiling reveals upregulation of p53 target genes in alpha cells treated with GW8510. Further experiments show that GW8510 enhances p53 transcriptional activity and increases p53 phosphorylation and acetylation associated with increased activity. GW8510 treatment arrests alpha cell cycle progression, consistent with activation of p53. These results suggest that GW8510 induces insulin expression by activating the p53 signaling pathway in alpha cells.
1) Hepatitis C virus (HCV) infection inhibits the expression of insulin receptor substrate 1 (IRS-1) in hepatocytes. HCV activates the mTOR/S6K1 signaling pathway, which negatively regulates IRS-1.
2) HCV infection suppresses the tumor suppressor proteins TSC-1 and TSC-2 in hepatocytes. This leads to increased expression of the protein Rheb.
3) Increased Rheb then activates mTOR and its downstream target S6K1, which inhibits insulin signaling by degrading IRS-1. This perturbation of the insulin signaling pathway by HCV contributes to insulin resistance.
The document discusses a study investigating the effects of food restriction on gene expression. It was previously found that 15 genes were upregulated in the brain during food restriction, suggesting they are part of an ancient stress response pathway. The current study aims to test if these genes are also induced in other tissue types under food restriction. Mice were either food restricted or not for 5 days, then gene expression was analyzed using qPCR in various tissues including the kidney. It was found that Angptl4, Mertk, Arrdc2 and Cdkn1a were significantly upregulated in the kidney of food restricted mice compared to controls, providing further evidence they are part of a general stress response pathway activated by food restriction across multiple
Evaluation of the changes in the gene CYP3A4 expression in HepG2 cells under ...Angela Farngren
This study evaluated the effects of rifampicin and alpha-ketoglutarate on expression of the CYP3A4 gene in HepG2 liver cells. HepG2 cells were treated with either 10mM rifampicin or 4mM alpha-ketoglutarate for 7 days. RNA was extracted on days 0, 3, and 7 and analyzed via qPCR. Rifampicin treatment significantly increased CYP3A4 expression, peaking on day 3 and decreasing slightly by day 7. Alpha-ketoglutarate initially decreased CYP3A4 expression for the first 3 days but then increased expression levels. The study demonstrates that rifampicin and alpha-ket
- The study investigated the roles of phosphate and FGF23 in left ventricular hypertrophy (LVH) using mouse models of chronic kidney disease (CKD). Mice with CKD (Jck mutants) and low FGF23 (GALNT3 mutants) were given normal or low phosphate diets.
- While the low phosphate diet successfully reduced serum phosphate levels, mice did not develop severe CKD or high FGF23 levels. Jck and double mutants still exhibited signs of mild CKD and increased LVH gene expression despite normal heart size.
- The results suggest that factors other than FGF23 and hyperphosphatemia contribute to LVH in CKD, since LVH markers remained elevated even when serum phosphate and FGF
This study investigated how insulin signaling through Akt regulates mitochondrial oxidative phosphorylation complex V activity in cardiac muscle. The key findings were:
1) Insulin stimulation causes Akt to translocate sequentially to the mitochondrial intermembrane space, inner membrane, and matrix.
2) Only the Akt1 isoform translocates to mitochondria in response to insulin, and this translocation is blunted in models of diabetes.
3) Activating mitochondrial Akt1 increases complex V activity in normal cardiac muscle and restores its lower activity in diabetic models. Basal complex V activity is also lower in Akt1 knockout mice.
4) Insulin-stimulated complex V activity is maintained in Akt1 knockout mice due to compens
International Journal of Pharmaceutical Science Invention (IJPSI)inventionjournals
International Journal of Pharmaceutical Science Invention (IJPSI) is an international journal intended for professionals and researchers in all fields of Pahrmaceutical Science. IJPSI publishes research articles and reviews within the whole field Pharmacy and Pharmaceutical Science, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online
Antiretroviral therapy Reloaded in view of updated PPTCT guidelinesDr Kamal Sundar
Updated PPTCT guidelines of NACO are new for OBG practitioners. Pharmacotherapy can be explained in a less complex manner to meet a busy practitioner's need.
1) Hepatitis C virus (HCV) infection inhibits the expression of insulin receptor substrate 1 (IRS-1) in hepatocytes. HCV activates the mTOR/S6K1 signaling pathway, which negatively regulates IRS-1.
2) HCV infection suppresses the tumor suppressor proteins TSC-1 and TSC-2 in hepatocytes. This leads to increased expression of the protein Rheb.
3) Increased Rheb then activates mTOR and its downstream target S6K1, which inhibits insulin signaling by degrading IRS-1. This perturbation of the insulin signaling pathway by HCV contributes to insulin resistance.
The document discusses a study investigating the effects of food restriction on gene expression. It was previously found that 15 genes were upregulated in the brain during food restriction, suggesting they are part of an ancient stress response pathway. The current study aims to test if these genes are also induced in other tissue types under food restriction. Mice were either food restricted or not for 5 days, then gene expression was analyzed using qPCR in various tissues including the kidney. It was found that Angptl4, Mertk, Arrdc2 and Cdkn1a were significantly upregulated in the kidney of food restricted mice compared to controls, providing further evidence they are part of a general stress response pathway activated by food restriction across multiple
Evaluation of the changes in the gene CYP3A4 expression in HepG2 cells under ...Angela Farngren
This study evaluated the effects of rifampicin and alpha-ketoglutarate on expression of the CYP3A4 gene in HepG2 liver cells. HepG2 cells were treated with either 10mM rifampicin or 4mM alpha-ketoglutarate for 7 days. RNA was extracted on days 0, 3, and 7 and analyzed via qPCR. Rifampicin treatment significantly increased CYP3A4 expression, peaking on day 3 and decreasing slightly by day 7. Alpha-ketoglutarate initially decreased CYP3A4 expression for the first 3 days but then increased expression levels. The study demonstrates that rifampicin and alpha-ket
- The study investigated the roles of phosphate and FGF23 in left ventricular hypertrophy (LVH) using mouse models of chronic kidney disease (CKD). Mice with CKD (Jck mutants) and low FGF23 (GALNT3 mutants) were given normal or low phosphate diets.
- While the low phosphate diet successfully reduced serum phosphate levels, mice did not develop severe CKD or high FGF23 levels. Jck and double mutants still exhibited signs of mild CKD and increased LVH gene expression despite normal heart size.
- The results suggest that factors other than FGF23 and hyperphosphatemia contribute to LVH in CKD, since LVH markers remained elevated even when serum phosphate and FGF
This study investigated how insulin signaling through Akt regulates mitochondrial oxidative phosphorylation complex V activity in cardiac muscle. The key findings were:
1) Insulin stimulation causes Akt to translocate sequentially to the mitochondrial intermembrane space, inner membrane, and matrix.
2) Only the Akt1 isoform translocates to mitochondria in response to insulin, and this translocation is blunted in models of diabetes.
3) Activating mitochondrial Akt1 increases complex V activity in normal cardiac muscle and restores its lower activity in diabetic models. Basal complex V activity is also lower in Akt1 knockout mice.
4) Insulin-stimulated complex V activity is maintained in Akt1 knockout mice due to compens
International Journal of Pharmaceutical Science Invention (IJPSI)inventionjournals
International Journal of Pharmaceutical Science Invention (IJPSI) is an international journal intended for professionals and researchers in all fields of Pahrmaceutical Science. IJPSI publishes research articles and reviews within the whole field Pharmacy and Pharmaceutical Science, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online
Antiretroviral therapy Reloaded in view of updated PPTCT guidelinesDr Kamal Sundar
Updated PPTCT guidelines of NACO are new for OBG practitioners. Pharmacotherapy can be explained in a less complex manner to meet a busy practitioner's need.
This document discusses drug transporter polymorphism and its effects. It begins by introducing drug transporter proteins and how genetic variations can affect drug uptake, distribution, and toxicity. It then examines these effects for the cancer drug irinotecan in particular. The rest of the document details the two main classes of drug transporter proteins - ATP-binding cassette (ABC) transporters and solute carrier (SLC) proteins. It provides examples of specific transporters like P-glycoprotein, MRP1, BCRP, and discusses their tissue expression and drug substrates. In closing, it lists several important transporter genes and the substrates they transport.
This study investigated how insulin deficiency affects mitochondrial oxidative phosphorylation in the hearts of diabetic mice. The key findings were:
1) Activity of oxidative phosphorylation complex V (ATP synthase) was significantly reduced in the hearts of streptozotocin-induced diabetic mice.
2) Normalizing blood glucose with phlorizin treatment did not improve complex V activity, but insulin treatment did normalize it, indicating the reduction was caused by insulin deficiency rather than hyperglycemia.
3) Acute insulin stimulation induced phosphorylation and translocation of Akt to mitochondria in heart muscle. This translocation was enhanced in diabetic mice and blocked inhibition of Akt, blunting the activation of complex V by insulin.
PPARs are transcriptional factors that regulate gene expression and belong to the nuclear receptor superfamily. The three main PPAR isoforms are α, β, and γ, which vary in ligand specificity, tissue distribution, and biological functions. PPARs control processes like cellular differentiation, development and metabolism. They are activated by ligands such as fatty acids and eicosanoids and regulate energy homeostasis by initiating transcription of genes involved. Each PPAR isoform has distinct roles such as PPAR-γ promoting adipogenesis and insulin sensitization while PPAR-α stimulates fatty acid oxidation.
The document discusses various methods for directly assessing insulin resistance, including the hyperinsulinemic euglycemic clamp, which is considered the gold standard. It involves infusing high levels of insulin while frequently measuring blood glucose to maintain normal levels, and the glucose infusion rate required is a measure of insulin sensitivity. Other direct methods discussed are the hyperglycemic clamp to measure insulin secretion, insulin tolerance test, and minimal model analysis of intravenous glucose tolerance tests.
Indole-3-carbinol (I3C) is a compound found in cruciferous vegetables that may have anticancer properties. This document summarizes I3C's sources, chemical properties, metabolism, mechanisms of action, and clinical trial data. Key points:
1) I3C is produced from glucosinolates in cruciferous vegetables and may be converted to dimers and trimers in the stomach and intestines.
2) Preclinical studies suggest I3C has anti-inflammatory, antioxidant, and anticancer effects through multiple pathways like NF-kB and Akt.
3) Phase I/II clinical trials show I3C is well tolerated at doses of 200-
A mild reduction of food intake slows disease progression in an orthologous m...Mina Rezaei
A mild 23% reduction in food intake (RFI) significantly slowed disease progression in a mouse model of polycystic kidney disease (PKD). RFI suppressed renal cyst growth, with kidney weight increasing 41% compared to 151% in controls. Proliferation of cyst-lining cells was also reduced from 15.9% in controls to 7.7% with RFI. RFI maintained kidney function and prevented progression to end-stage renal disease. RFI activated AMPK and inhibited both branches of mTOR signaling in cyst cells, more broadly than rapamycin which primarily inhibits one branch. These results suggest a mild decrease in caloric intake may slow disease progression in human ADPKD patients.
The document discusses using the Burmese python as a model to study lipid homeostasis. After eating, pythons show a doubling in size of major organs and a 52-fold increase in triglycerides without signs of lipotoxicity. This suggests a more efficient mechanism of lipid homeostasis than humans. The nuclear receptor PPARα is identified as a master regulator of lipid homeostasis and is the gene of interest. Methods are described to isolate RNA from python liver, synthesize cDNA, validate and test primers for PPARα and other genes, and perform quantitative real-time PCR to analyze gene expression levels at different time points after feeding.
1. Certain single nucleotide polymorphisms (SNPs) are associated with increased risk of type 2 diabetes mellitus by affecting glucose handling and insulin secretion or action.
2. Some SNPs have been shown to alter patient response to common antidiabetic drugs like sulfonylureas through effects on glucose handling pathways.
3. Understanding the pharmacogenomic implications of diabetes risk SNPs may help optimize treatment and achieve better glycemic control for individual patients.
This study investigated how endoplasmic reticulum (ER) stress induced by glucosamine (GlcN) is involved in pancreatic beta-cell dysfunction. The researchers found that GlcN treatment increased levels of the ER stress markers BiP/GRP78 and CHOP/GADD153 in mouse pancreatic islets and INS-1E beta cells. GlcN also decreased mRNA levels of genes important for beta-cell function like Glut2, glucokinase, and Insulin1. Pretreating cells with a chemical chaperone partially prevented GlcN-induced ER stress and gene expression changes. However, an antioxidant did not prevent ER stress, indicating oxidative stress is not involved. The results suggest
CLASS 12 BIOLOGY INVESTIGATORY PROJECT29NIRAJKUMAR
The document summarizes information about human insulin, including its structure, production in the pancreas, and production through recombinant DNA technology. It describes insulin as a peptide hormone composed of two polypeptide chains that is produced in pancreatic β-cells. Insulin regulates carbohydrate and fat metabolism, and maintains blood glucose levels. The document also outlines the process of using recombinant DNA technology to synthesize and produce insulin in E. coli cells for treatment of diabetes.
Murphy et al, AJP Cell Physiol 296_ C746–C756, 2009.Beth Murphy
Fasting enhances the response of hypothalamic arcuate nucleus neuropeptide Y (NPY) neurons that are inhibited by glucose (NPY-GI neurons) to decreases in extracellular glucose. Using hypothalamic explants from fed and fasted rats, the authors showed that fasting enhanced NPY release in response to lowered glucose concentrations. They also demonstrated that fasting caused NPY-GI neurons to depolarize, or become more electrically active, in response to smaller decreases in glucose levels. This increased responsiveness was mediated by the cellular energy sensor AMPK. During fasting, decreased leptin and glucose activate AMPK in NPY-GI neurons, making them more sensitive to fluctuations in glucose.
Tyrosine kinases are enzymes that transfer phosphate groups from ATP to tyrosine residues on proteins. There are two main families of tyrosine kinases: receptor tyrosine kinases and non-receptor tyrosine kinases. Receptor tyrosine kinases are transmembrane receptors that transmit extracellular signals to intracellular pathways. Examples include receptors for epidermal growth factor, insulin, fibroblast growth factors, platelet-derived growth factor, nerve growth factor, ephrins, and vascular endothelial growth factor.
This document summarizes a study examining the role of platelet-derived growth factor (PDGF)-evoked calcium signaling in human pulmonary fibroblasts. The study found that both acute and overnight PDGF treatment evoked calcium waves in these cells. Blocking external calcium, internal calcium stores, or phospholipase C inhibited the PDGF-evoked calcium waves and PDGF-induced expression of fibronectin and collagen genes. The results indicate that in human pulmonary fibroblasts, PDGF acts through IP3-induced calcium release from internal stores to trigger calcium waves, which then modulate expression of extracellular matrix genes.
Different Therapeutic Aspects of Peroxisomes Proliferator-Activated ReceptorsAI Publications
Peroxisome proliferator-activated receptors (PPARs) was discovered in 1990 belong to the super family of steroid hormone receptors. Three subtypes of PPAR which have been identified so far- PPARα, PPARβ/δ, and PPARγ. Human peroxisome proliferator-activated receptors (hPPARs) were initially recognized as therapeutic targets for the development of drugs to treat metabolic disorders, such as diabetes and dyslipidemia but now they have been used in energy burning, dyslipidemia, diabetes, inflammation, Hepatic steatosis, liver cancer, diabetic neuropathy, atherosclerosis also. These are included in management of NIDDM, macrophage differentiation, adipose differentiation , anti-cancer, inhibition of TH2 cytokine production and rheumatoid arthritis. PPARβ/δ can use to treat Huntington’s disease, fertility, dyslipidemia. The functions of a third PPAR isoform and its potential as a therapeutic target are currently under investigation.
1. The document describes using transgenic livestock animals as bioreactors for producing pharmaceutical proteins in their milk.
2. Specifically, it details an experiment where researchers created transgenic sheep that produced the human protein alpha-1-antitrypsin (AAT) in their milk by fusing the AAT gene to the regulatory sequences of the ovine beta-lactoglobulin gene.
3. The transgenic sheep were able to stably pass the transgene to offspring and produced over 1g of fully active, glycosylated human AAT per liter of milk.
The document discusses cellular signaling pathways and their role in diabetes. It explains that signaling pathways involve an initial signal, receptor, signaling molecules that transmit the message, and effectors that cause a cellular response. It then describes two common signaling mechanisms: (1) protein kinase cascades where one activated kinase activates another in a chain, amplifying the signal and speeding the response; and (2) second messenger pathways where a small molecule messenger amplifies and spreads the signal throughout the cell. Understanding these signaling mechanisms is important for understanding how diabetes occurs at the cellular level and for developing new diabetes treatments.
1. The study investigates the role of the regulatory subunit SUR-6 in the protein phosphatase 2A (PP2A) complex and its effect on muscle protein degradation in the nematode C. elegans.
2. Experiments show that RNAi knockdown of SUR-6 prevents severe muscle degradation in a daf-2 mutant strain at 25°C, suggesting SUR-6 is required for PP2A to activate the MAPK pathway and promote degradation.
3. The findings indicate SUR-6 allows PP2A to target and activate RAF kinase by removing inhibitory phosphates placed by AKT, thus positively regulating muscle protein degradation.
Huntington's disease is a genetic disorder caused by a CAG repeat expansion in the HTT gene on chromosome 4. It is characterized by motor dysfunction and cognitive decline that worsen over time. Symptoms appear when around 30-40 years old but can occur earlier. The mutation results in abnormal huntingtin protein that causes neuronal dysfunction and death through disruption of transcriptional pathways and induction of apoptosis. No cure exists but some treatments can help manage symptoms.
This document discusses orphan G protein-coupled receptors (GPCRs) and efforts to identify endogenous ligands for these receptors. It provides background on the IUPHAR database and criteria for designating pairings between orphan receptors and endogenous ligands. 57 class A, 28 class B, and 6 class C orphan GPCRs currently have no reported pairings. The document calls for further research to validate reported pairings and identify ligands for the remaining orphan GPCRs to advance our understanding of their functions and therapeutic potential.
The document describes research identifying a small molecule called BRD7389 that induces insulin expression in pancreatic α-cells. Key findings include:
1) BRD7389 was identified in a high-throughput screen as inducing insulin expression in mouse α-cells. It also increased expression of the β-cell marker genes Pdx1, Pax4, Iapp, and Npy.
2) Treatment with BRD7389 caused α-cells to take on a clustered morphology resembling β-cells and to express low levels of insulin protein.
3) Profiling and biochemical assays identified BRD7389 as an inhibitor of RSK kinases, and knockdown experiments supported a role for RSK kin
Set7/9 is a lysine methyltransferase that interacts with the transcription factor Pdx1. This study found that:
1) Set7/9 methylates two lysine residues (Lys-123 and Lys-131) on Pdx1. Methylation only occurred with full-length Pdx1, indicating structural requirements.
2) Lys-131 methylation by Set7/9 augments Pdx1's transcriptional activity in luciferase reporter assays.
3) Mice with beta-cell specific deletion of Set7/9 (SetΔβ mice) showed glucose intolerance and impaired insulin secretion from islets, similar to Pdx1-deficient mice. Target genes
This document discusses drug transporter polymorphism and its effects. It begins by introducing drug transporter proteins and how genetic variations can affect drug uptake, distribution, and toxicity. It then examines these effects for the cancer drug irinotecan in particular. The rest of the document details the two main classes of drug transporter proteins - ATP-binding cassette (ABC) transporters and solute carrier (SLC) proteins. It provides examples of specific transporters like P-glycoprotein, MRP1, BCRP, and discusses their tissue expression and drug substrates. In closing, it lists several important transporter genes and the substrates they transport.
This study investigated how insulin deficiency affects mitochondrial oxidative phosphorylation in the hearts of diabetic mice. The key findings were:
1) Activity of oxidative phosphorylation complex V (ATP synthase) was significantly reduced in the hearts of streptozotocin-induced diabetic mice.
2) Normalizing blood glucose with phlorizin treatment did not improve complex V activity, but insulin treatment did normalize it, indicating the reduction was caused by insulin deficiency rather than hyperglycemia.
3) Acute insulin stimulation induced phosphorylation and translocation of Akt to mitochondria in heart muscle. This translocation was enhanced in diabetic mice and blocked inhibition of Akt, blunting the activation of complex V by insulin.
PPARs are transcriptional factors that regulate gene expression and belong to the nuclear receptor superfamily. The three main PPAR isoforms are α, β, and γ, which vary in ligand specificity, tissue distribution, and biological functions. PPARs control processes like cellular differentiation, development and metabolism. They are activated by ligands such as fatty acids and eicosanoids and regulate energy homeostasis by initiating transcription of genes involved. Each PPAR isoform has distinct roles such as PPAR-γ promoting adipogenesis and insulin sensitization while PPAR-α stimulates fatty acid oxidation.
The document discusses various methods for directly assessing insulin resistance, including the hyperinsulinemic euglycemic clamp, which is considered the gold standard. It involves infusing high levels of insulin while frequently measuring blood glucose to maintain normal levels, and the glucose infusion rate required is a measure of insulin sensitivity. Other direct methods discussed are the hyperglycemic clamp to measure insulin secretion, insulin tolerance test, and minimal model analysis of intravenous glucose tolerance tests.
Indole-3-carbinol (I3C) is a compound found in cruciferous vegetables that may have anticancer properties. This document summarizes I3C's sources, chemical properties, metabolism, mechanisms of action, and clinical trial data. Key points:
1) I3C is produced from glucosinolates in cruciferous vegetables and may be converted to dimers and trimers in the stomach and intestines.
2) Preclinical studies suggest I3C has anti-inflammatory, antioxidant, and anticancer effects through multiple pathways like NF-kB and Akt.
3) Phase I/II clinical trials show I3C is well tolerated at doses of 200-
A mild reduction of food intake slows disease progression in an orthologous m...Mina Rezaei
A mild 23% reduction in food intake (RFI) significantly slowed disease progression in a mouse model of polycystic kidney disease (PKD). RFI suppressed renal cyst growth, with kidney weight increasing 41% compared to 151% in controls. Proliferation of cyst-lining cells was also reduced from 15.9% in controls to 7.7% with RFI. RFI maintained kidney function and prevented progression to end-stage renal disease. RFI activated AMPK and inhibited both branches of mTOR signaling in cyst cells, more broadly than rapamycin which primarily inhibits one branch. These results suggest a mild decrease in caloric intake may slow disease progression in human ADPKD patients.
The document discusses using the Burmese python as a model to study lipid homeostasis. After eating, pythons show a doubling in size of major organs and a 52-fold increase in triglycerides without signs of lipotoxicity. This suggests a more efficient mechanism of lipid homeostasis than humans. The nuclear receptor PPARα is identified as a master regulator of lipid homeostasis and is the gene of interest. Methods are described to isolate RNA from python liver, synthesize cDNA, validate and test primers for PPARα and other genes, and perform quantitative real-time PCR to analyze gene expression levels at different time points after feeding.
1. Certain single nucleotide polymorphisms (SNPs) are associated with increased risk of type 2 diabetes mellitus by affecting glucose handling and insulin secretion or action.
2. Some SNPs have been shown to alter patient response to common antidiabetic drugs like sulfonylureas through effects on glucose handling pathways.
3. Understanding the pharmacogenomic implications of diabetes risk SNPs may help optimize treatment and achieve better glycemic control for individual patients.
This study investigated how endoplasmic reticulum (ER) stress induced by glucosamine (GlcN) is involved in pancreatic beta-cell dysfunction. The researchers found that GlcN treatment increased levels of the ER stress markers BiP/GRP78 and CHOP/GADD153 in mouse pancreatic islets and INS-1E beta cells. GlcN also decreased mRNA levels of genes important for beta-cell function like Glut2, glucokinase, and Insulin1. Pretreating cells with a chemical chaperone partially prevented GlcN-induced ER stress and gene expression changes. However, an antioxidant did not prevent ER stress, indicating oxidative stress is not involved. The results suggest
CLASS 12 BIOLOGY INVESTIGATORY PROJECT29NIRAJKUMAR
The document summarizes information about human insulin, including its structure, production in the pancreas, and production through recombinant DNA technology. It describes insulin as a peptide hormone composed of two polypeptide chains that is produced in pancreatic β-cells. Insulin regulates carbohydrate and fat metabolism, and maintains blood glucose levels. The document also outlines the process of using recombinant DNA technology to synthesize and produce insulin in E. coli cells for treatment of diabetes.
Murphy et al, AJP Cell Physiol 296_ C746–C756, 2009.Beth Murphy
Fasting enhances the response of hypothalamic arcuate nucleus neuropeptide Y (NPY) neurons that are inhibited by glucose (NPY-GI neurons) to decreases in extracellular glucose. Using hypothalamic explants from fed and fasted rats, the authors showed that fasting enhanced NPY release in response to lowered glucose concentrations. They also demonstrated that fasting caused NPY-GI neurons to depolarize, or become more electrically active, in response to smaller decreases in glucose levels. This increased responsiveness was mediated by the cellular energy sensor AMPK. During fasting, decreased leptin and glucose activate AMPK in NPY-GI neurons, making them more sensitive to fluctuations in glucose.
Tyrosine kinases are enzymes that transfer phosphate groups from ATP to tyrosine residues on proteins. There are two main families of tyrosine kinases: receptor tyrosine kinases and non-receptor tyrosine kinases. Receptor tyrosine kinases are transmembrane receptors that transmit extracellular signals to intracellular pathways. Examples include receptors for epidermal growth factor, insulin, fibroblast growth factors, platelet-derived growth factor, nerve growth factor, ephrins, and vascular endothelial growth factor.
This document summarizes a study examining the role of platelet-derived growth factor (PDGF)-evoked calcium signaling in human pulmonary fibroblasts. The study found that both acute and overnight PDGF treatment evoked calcium waves in these cells. Blocking external calcium, internal calcium stores, or phospholipase C inhibited the PDGF-evoked calcium waves and PDGF-induced expression of fibronectin and collagen genes. The results indicate that in human pulmonary fibroblasts, PDGF acts through IP3-induced calcium release from internal stores to trigger calcium waves, which then modulate expression of extracellular matrix genes.
Different Therapeutic Aspects of Peroxisomes Proliferator-Activated ReceptorsAI Publications
Peroxisome proliferator-activated receptors (PPARs) was discovered in 1990 belong to the super family of steroid hormone receptors. Three subtypes of PPAR which have been identified so far- PPARα, PPARβ/δ, and PPARγ. Human peroxisome proliferator-activated receptors (hPPARs) were initially recognized as therapeutic targets for the development of drugs to treat metabolic disorders, such as diabetes and dyslipidemia but now they have been used in energy burning, dyslipidemia, diabetes, inflammation, Hepatic steatosis, liver cancer, diabetic neuropathy, atherosclerosis also. These are included in management of NIDDM, macrophage differentiation, adipose differentiation , anti-cancer, inhibition of TH2 cytokine production and rheumatoid arthritis. PPARβ/δ can use to treat Huntington’s disease, fertility, dyslipidemia. The functions of a third PPAR isoform and its potential as a therapeutic target are currently under investigation.
1. The document describes using transgenic livestock animals as bioreactors for producing pharmaceutical proteins in their milk.
2. Specifically, it details an experiment where researchers created transgenic sheep that produced the human protein alpha-1-antitrypsin (AAT) in their milk by fusing the AAT gene to the regulatory sequences of the ovine beta-lactoglobulin gene.
3. The transgenic sheep were able to stably pass the transgene to offspring and produced over 1g of fully active, glycosylated human AAT per liter of milk.
The document discusses cellular signaling pathways and their role in diabetes. It explains that signaling pathways involve an initial signal, receptor, signaling molecules that transmit the message, and effectors that cause a cellular response. It then describes two common signaling mechanisms: (1) protein kinase cascades where one activated kinase activates another in a chain, amplifying the signal and speeding the response; and (2) second messenger pathways where a small molecule messenger amplifies and spreads the signal throughout the cell. Understanding these signaling mechanisms is important for understanding how diabetes occurs at the cellular level and for developing new diabetes treatments.
1. The study investigates the role of the regulatory subunit SUR-6 in the protein phosphatase 2A (PP2A) complex and its effect on muscle protein degradation in the nematode C. elegans.
2. Experiments show that RNAi knockdown of SUR-6 prevents severe muscle degradation in a daf-2 mutant strain at 25°C, suggesting SUR-6 is required for PP2A to activate the MAPK pathway and promote degradation.
3. The findings indicate SUR-6 allows PP2A to target and activate RAF kinase by removing inhibitory phosphates placed by AKT, thus positively regulating muscle protein degradation.
Huntington's disease is a genetic disorder caused by a CAG repeat expansion in the HTT gene on chromosome 4. It is characterized by motor dysfunction and cognitive decline that worsen over time. Symptoms appear when around 30-40 years old but can occur earlier. The mutation results in abnormal huntingtin protein that causes neuronal dysfunction and death through disruption of transcriptional pathways and induction of apoptosis. No cure exists but some treatments can help manage symptoms.
This document discusses orphan G protein-coupled receptors (GPCRs) and efforts to identify endogenous ligands for these receptors. It provides background on the IUPHAR database and criteria for designating pairings between orphan receptors and endogenous ligands. 57 class A, 28 class B, and 6 class C orphan GPCRs currently have no reported pairings. The document calls for further research to validate reported pairings and identify ligands for the remaining orphan GPCRs to advance our understanding of their functions and therapeutic potential.
The document describes research identifying a small molecule called BRD7389 that induces insulin expression in pancreatic α-cells. Key findings include:
1) BRD7389 was identified in a high-throughput screen as inducing insulin expression in mouse α-cells. It also increased expression of the β-cell marker genes Pdx1, Pax4, Iapp, and Npy.
2) Treatment with BRD7389 caused α-cells to take on a clustered morphology resembling β-cells and to express low levels of insulin protein.
3) Profiling and biochemical assays identified BRD7389 as an inhibitor of RSK kinases, and knockdown experiments supported a role for RSK kin
Set7/9 is a lysine methyltransferase that interacts with the transcription factor Pdx1. This study found that:
1) Set7/9 methylates two lysine residues (Lys-123 and Lys-131) on Pdx1. Methylation only occurred with full-length Pdx1, indicating structural requirements.
2) Lys-131 methylation by Set7/9 augments Pdx1's transcriptional activity in luciferase reporter assays.
3) Mice with beta-cell specific deletion of Set7/9 (SetΔβ mice) showed glucose intolerance and impaired insulin secretion from islets, similar to Pdx1-deficient mice. Target genes
Insulin resistance occurs when cells become less responsive to insulin. It can be caused by genetic or diet-induced factors, with obesity being the most common cause. Insulin resistance is associated with inflammation, oxidative stress, and lipid accumulation in tissues like muscle and fat cells. Disrupted gut microbiota may also contribute by impacting energy harvest and metabolite production. Regulators of insulin sensitivity include PPARγ, AMPK, mTOR, and sirtuins, which control pathways involved in glucose and fatty acid metabolism. Gene expression analysis using PCR arrays can help study mechanisms of insulin resistance.
Estradiol Partially Recapitulates Murine Pituitary Cell Cycle Response to Pre...Michael M
This document discusses pituitary lactotroph hyperplasia induced by pregnancy or estradiol (E2) administration in mice. The key findings are:
1. Mouse pituitary mass and the proliferation markers PCNA and Ki-67 increased during pregnancy and with long-term E2 treatment, indicating hyperplasia.
2. The cell cycle inhibitors p15INK4b and p21Cip1 were up-regulated in the pituitary during pregnancy and E2 administration, coinciding with increased proliferation.
3. E2 administration partially mimicked the pituitary cell cycle expression changes seen during pregnancy, increasing proliferation while also inducing the antiproliferative factors p15INK4b and p21Cip1.
The document describes a study investigating the molecular mechanisms by which an extract from the brown seaweed Fucus vesiculosus (Fv1) mediates cell cycle inhibition and cell death in pancreatic cancer cells. Gene expression profiling found that Fv1 treatment upregulated the cell cycle inhibitor p57 and downregulated genes involved in cell cycle progression. This suggests Fv1 induces cell cycle arrest. Fv1 also altered the morphology of treated cells and inhibited their viability and proliferation in a caspase-independent manner. Proliferation was found to be a prerequisite for Fv1's effectiveness.
Neuronal inactivation of PGC-1α in mice leads to protection from diet-induced obesity and degenerative brain lesions. Researchers generated mice with PGC-1α knocked out specifically in neurons (BαKO mice) using CaMKIIα-Cre. BαKO mice had normal metabolic responses in peripheral tissues but were hypermetabolic, resistant to obesity, and had improved metabolic profiles like whole-body PGC-1α knockouts. BαKO mice also developed striatal lesions and impaired hypothalamic regulation of genes controlling energy balance. This demonstrates neuronal PGC-1α regulates whole-body energy balance and is necessary for neuronal health.
This study found that Sfrp5, which encodes secreted frizzled-related protein 5, is downregulated in the pancreatic islets of rats fed a cafeteria diet (obese model) and in pancreatic islets of human obese patients. The researchers demonstrated that silencing Sfrp5 increases beta cell proliferation in rats, which correlates with activation of the Wnt signaling pathway and increased proliferation markers. They also showed that expression of Sfrp5 in beta cells is modulated by IGF binding protein 3 secreted from visceral adipose tissue. Together, these findings reveal that Sfrp5 and the Wnt signaling pathway play an important role in regulating beta cell proliferation during the expansion of beta cell mass that occurs with
The document discusses research into the regulation and role of the transcription factor REST in neuronal differentiation of SH-SY5Y human neuroblastoma cells exposed to IGF-I and PMA. The key findings are: 1) IGF-I and PMA increase nuclear REST levels early in treatment but REST declines with longer exposure as neurite outgrowth increases, 2) Downregulating REST with antisense oligonucleotides enhances the effects of IGF-I and PMA on neuronal markers and neurite growth, 3) REST degradation involves the ubiquitin-proteasome system and contributes to later stage neuronal differentiation. The document also analyzes the O-glycosylation pattern of REST protein in these cells.
The document describes research investigating how the protein amyloid-beta (Aβ) may amplify itself in Alzheimer's disease. It finds that Aβ inhibits the enzyme ADAM10, reducing cleavage of amyloid precursor protein (APP) and allowing more amyloidogenic processing and Aβ production. It also finds that the protein netrin-1 can disrupt this process, reducing Aβ levels both in vitro and in mouse models. Testing netrin-1 and netrin-mimetic peptides as potential Alzheimer's therapeutics is discussed.
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Effectiveness of Resveratrol on Metastasis: A Reviewiosrphr_editor
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
Effectiveness of Resveratrol on Metastasis: A Review
GW8510 manuscript
1. GW8510 Increases Insulin Expression in Pancreatic Alpha
Cells through Activation of p53 Transcriptional Activity
Dina Fomina-Yadlin1,2
, Stefan Kubicek3
, Amedeo Vetere1
, Kaihui Hu He1
, Stuart L. Schreiber1,4,5
,
Bridget K. Wagner1
*
1 Chemical Biology Program, the Broad Institute of Harvard and MIT, Cambridge, Massachusetts, United States of America, 2 Department of Molecular and Cellular
Biology, Harvard University, Cambridge, Massachusetts, United States of America, 3 Research Centre for Molecular Medicine of the Austrian Academy of Sciences, Vienna,
Austria, 4 Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, United States of America, 5 Howard Hughes Medical Institute,
the Broad Institute of Harvard and MIT, Cambridge, Massachusetts, United States of America
Abstract
Background: Expression of insulin in terminally differentiated non-beta cell types in the pancreas could be important to
treating type-1 diabetes. Previous findings led us to hypothesize involvement of kinase inhibition in induction of insulin
expression in pancreatic alpha cells.
Methodology/Principal Findings: Alpha (aTC1.6) cells and human islets were treated with GW8510 and other small-
molecule inhibitors for up to 5 days. Alpha cells were assessed for gene- and protein-expression levels, cell-cycle status,
promoter occupancy status by chromatin immunoprecipitation (ChIP), and p53-dependent transcriptional activity. GW8510,
a putative CDK2 inhibitor, up-regulated insulin expression in mouse alpha cells and enhanced insulin secretion in
dissociated human islets. Gene-expression profiling and gene-set enrichment analysis of GW8510-treated alpha cells
suggested up-regulation of the p53 pathway. Accordingly, the compound increased p53 transcriptional activity and
expression levels of p53 transcriptional targets. A predicted p53 response element in the promoter region of the mouse Ins2
gene was verified by chromatin immunoprecipitation (ChIP). Further, inhibition of Jun N-terminal kinase (JNK) and p38
kinase activities suppressed insulin induction by GW8510.
Conclusions/Significance: The induction of Ins2 by GW8510 occurred through p53 in a JNK- and p38-dependent manner.
These results implicate p53 activity in modulation of Ins2 expression levels in pancreatic alpha cells, and point to a potential
approach toward using small molecules to generate insulin in an alternative cell type.
Citation: Fomina-Yadlin D, Kubicek S, Vetere A, He KH, Schreiber SL, et al. (2012) GW8510 Increases Insulin Expression in Pancreatic Alpha Cells through
Activation of p53 Transcriptional Activity. PLoS ONE 7(1): e28808. doi:10.1371/journal.pone.0028808
Editor: Kathrin Maedler, University of Bremen, Germany
Received July 27, 2011; Accepted November 15, 2011; Published January 5, 2012
Copyright: ß 2012 Fomina-Yadlin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was funded by the Juvenile Diabetes Research Foundation (S.L.S., B.K.W.), the NIGMS (National Institute of General Medical Sciences)
(GM38627 to S.L.S.) and a Type 1 Diabetes Pathfinder Award (DP2-DK083048, NIH-NIDDK, to B.K.W.). S.K. acknowledges support by the Ernst Schering Research
Foundation and the European Union FP7 Marie Curie program (PIOF-GA-2008-221135). D.F.-Y. acknowledges support from MCO training grant at Harvard
University. S.L.S. is an Investigator at the Howard Hughes Medical Institute. The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: bwagner@broadinstitute.org
Introduction
Autoimmune attack on pancreatic beta cells in type-1 diabetes
results in insulin deficiency and an inability to maintain glucose
homeostasis [1]. Inducing the production of insulin in other cell
types has the potential to assuage diabetes pathogenesis.
Pancreatic alpha cells are attractive candidates because of their
secretory nature, their developmental proximity to beta cells, and
their location within the islet of Langerhans [2]. Further,
conversion of alpha cells to functional beta cells has already been
demonstrated in mice by ectopic expression of a single
transcription factor, PAX4, in the developing pancreas [3].
Therefore, we hypothesized that small molecule-mediated stimu-
lation of insulin expression in alpha cells is a necessary initial step
for insulin production that does not require viral delivery [4] of
master-regulatory transcription factors, and could lead to an
alternative therapeutic strategy for type-1 diabetes.
Insulin expression is largely restricted to pancreatic beta cells,
but there are low levels of expression in extra-pancreatic tissues,
such as the brain [5,6] and the thymus [7]. Temporal and tissue-
specific regulation of the insulin gene demonstrates complexity
across species [8]. The cis-regulatory 400-base pair region flanking
the transcriptional start site (TSS) is highly regulated, controlled by
both beta cell-specific transcriptional regulators and general
transcription factors with widespread tissue distribution [8]. To
date, most pancreatic endocrine cell research has focused on
rodent cell lines [9]. In contrast to humans, rodents have a two-
gene insulin system, with Ins2 similar to the human insulin gene,
and Ins1 the result of a duplication-transposition of a partially
processed Ins2 mRNA product that lost the second intron [10,11].
Here, we focused on modulation of Ins2 transcription in mouse
alpha cells.
Previously, we reported a high-content screen to identify small-
molecule inducers of insulin expression in alpha cells, and the
PLoS ONE | www.plosone.org 1 January 2012 | Volume 7 | Issue 1 | e28808
2. discovery of a putative kinase inhibitor [12]. A more focused
exploration of the effects of other kinase inhibitors on insulin
expression in alpha cells led us to discover that GW8510, a
compound annotated as a CDK2 inhibitor [13], also up-regulates
Ins2 expression. Through further characterization of GW8510’s
effects on alpha cells, we demonstrate involvement of the p53 signal
transduction pathway in the modulation of Ins2 expression levels.
P53 has transcription-factor activity and performs most of its
biological functions through direct regulation of downstream
transcriptional targets [14]. It functions by binding to specific
DNA sequences containing p53 response elements, which results in
either activation or repression of promoter activity of target genes
[15,16]. Integration of upstream signals leads to a variety of cellular
responses to p53 activation, ranging from cell-cycle arrest, to
differentiation, to apoptosis [17,18]. Because of its functional
diversity and its importance in cell-fate decisions, p53 levels and
activity are tightly regulated through positive and negative feedback
[19]. The best characterized negative feedback loop involves an E3
ubiquitin ligase, MDM2 [20], which is up-regulated by increased
p53 levels or transcriptional activity [21]. Many signal-transduction
pathways converge on p53, but result in differential regulation of
downstream targets [22]. Thus, we sought to examine the
mechanism by which GW8510 activates the p53 pathway and up-
regulates Ins2 expression. We determined that p53 binds to the Ins2
promoter in alpha cells, and that GW8510 increases Ins2 expression
by up-regulating p53 transcriptional activity in a JNK- and p38-
dependent manner. These results suggest that modulating these
pathways with small molecules could be part of a feasible strategy
for generating insulin in an alternative cell type.
Results
Using high-content screening, we previously identified a
compound, BRD7389, which induced insulin expression in alpha
cells, and inhibited a wide variety of kinases biochemically [12].
To determine whether selective kinase inhibition could also induce
insulin expression, we treated the mouse pancreatic alpha cell line,
aTC1 clone 6 (aTC1.6), with the putative CDK2 inhibitor
GW8510 for five days, and observed a dose-dependent induction
of Ins2 expression following treatment (Figure 1A). We observed
similar effects when we used aTC1 clone 9 cells, which express
more insulin basally (Figure S1). Transcript levels of glucagon, the
endocrine hormone normally expressed in alpha cells, were
unaffected by the compound. Expression of Pdx1, a beta cell-
specific transcription factor capable of directly activating the Ins2
promoter [23], was significantly induced at 1.65 mM and higher.
An examination of the time course of gene expression revealed
that GW8510 induced Ins2 gene expression to its maximum after
48 hours, while Pdx1 was only up-regulated about two-fold after
96 hours (Figure 1B).
Because BRD7389 treatment enhanced insulin secretion in
dissociated human islet cells [12], we decided to explore the effects
of GW8510 on the same process. Five-day treatment with
GW8510 increased basal insulin secretion at 1.67 mM glucose
as well as glucose-stimulated insulin secretion at 16.7 mM
(Figure 1C). Interestingly, treatment with low levels of staurospor-
ine, a potent broad-range kinase inhibitor [24], also enhanced
insulin secretion in this system. Examination of the total cell
number and the numbers of alpha and beta cells, quantified by
immunofluorescence analysis, revealed that GW8510 treatment
decreased the total number of cells, but did not cause a significant
decline in beta cell numbers (Figure 1D, E). Interestingly, the
number of alpha cells seems to be increased following treatment
with both 3.3 mM GW8510 and staurosporine (Figure 1D, E).
Pancreatic islets are made up of a heterogeneous population of
cells [2], and it is difficult to pinpoint a compound’s effect on a
particular cell type, even in the dissociated islet system. Therefore,
we decided to focus on elucidating the mechanism of GW8510-
induced insulin expression in mouse alpha cells.
Since the induction of Ins2 gene expression precedes Pdx1 in this
case, the initial increase in Ins2 expression is likely to be induced by
a mechanism not involving Pdx1. Furthermore, knock-down of
CDK2 (Figure S2A) only marginally increased Ins2 expression,
while the use of other known CDK2 inhibitors (Figure S2B) had
no effect on Ins2 expression, suggesting that mechanisms other
than CDK2 inhibition are likely to be responsible for up-
regulation of Ins2 by GW8510. Thus, in order to determine a
potential mechanism of insulin induction, we treated mouse alpha
cells with 3.3 mM GW8510 or 0.1% DMSO for five days, and
performed gene-expression profiling of nearly 14,000 transcripts
(see Methods). Following GW8510 treatment, 364 genes were up-
regulated and 347 genes were down-regulated by at least two-fold
over the matched vehicle controls (Figure S3). Gene-set enrich-
ment analysis (GSEA) [25] revealed that gene sets containing p53-
responsive genes were significantly enriched after GW8510
treatment (Table S1). Specifically, microarray measurements
showed that the p53 transcriptional targets Cdkn1a, Mdm2, and
Ccng1 were up-regulated by compound treatment (Figure 2A).
Microarray-detected transcriptional changes in cell cycle-specific
genes and direct p53 transcriptional targets were confirmed on the
same samples by quantitative real-time RT-PCR (Figure 2B).
Seeing as p53-responsive genes were induced following
compound treatment, we sought to determine whether p53
transcriptional activity itself was enhanced. Using a p53-luciferase
reporter-gene assay, we found that 24-hour treatment with 3.3 mM
GW8510 increased reporter activity approximately seven-fold over
DMSO-treated controls (Figure 2C). Quantitative real-time RT-
PCR measurements confirmed that the transcript levels of Cdkn1a,
Mdm2, and Ccng1 were also significantly up-regulated following
treatment with 1.65 mM GW8510 (Figure 2D). In particular,
Cdkn1a and Ccng1 were strongly induced after both three- and five-
day treatments. Mdm2 transcript was only slightly affected by
three-day treatment, but showed significant up-regulation after
five days with 1.65 mM GW8510. This GW8510 concentration
was used in all further studies, because we could detect a full effect
on Ins2 induction without the toxicities observed at 3.3 mM.
Next, we explored downstream effects of p53 activation by
GW8510 on protein levels in alpha cells. The protein product of
Cdkn1a, p21, was increased after as little as eight hours, and
elevated more than ten-fold over basal levels at 48 hours
(Figure 3A, B). Cyclin G protein production was also increased,
but the induction was slower and reached only a two-fold increase
over basal levels by 48 hours of treatment. As with other cyclins,
cyclin G is an unstable protein that is quickly degraded [26], but
we could detect up-regulation of both the full-length protein and
the degradation product. Consistent with our previous assessment
of early Mdm2 expression, MDM2 protein levels were unaffected
after 48 hours of treatment with GW8510.
Cellular p53 activity is regulated at the post-translational level
by combinations of modifications, such as phosphorylation and
acetylation. For example, Ser392 phosphorylation has been
reported to increase p53 DNA-binding capacity and transcrip-
tional activity [27,28]. Furthermore, p53 acetylation promotes p53
stability and accumulation [29]. Since we observed an increase in
p53 activity after treatment with GW8510, we decide to examine
p53 post-translational modification status. We observed that two-
to five-day treatment with 1.65 mM GW8510 increased Ser392
phosphorylation in alpha cells, indicating a higher transactivation
p53 Activates Mouse Insulin Promoter
PLoS ONE | www.plosone.org 2 January 2012 | Volume 7 | Issue 1 | e28808
3. capacity of p53 following compound treatment (Figure 3C,D). We
also detected an up-regulation of K379 acetylation (Figure 3C, D),
which is also consistent with increased p53 activity. MDM2, a
direct p53 transcriptional target, is an E3 ubiquitin ligase that
regulates p53 stability by targeting it for degradation through a
negative feedback mechanism [20]. Akt-mediated phosphorylation
Figure 1. Effects of GW8510 treatment on mouse alpha cells and dissociated human islet cells. Pancreatic gene expression was measured
by quantitative real-time RT-PCR (qPCR) following a (A) 5-day dose-response and (B) time-course with 1.65 mM GW8510. (C) Insulin secretion
measurements in dissociated human islets following 5-day compound treatment at indicated concentrations. (D) Immunofluorescence analysis
quantification of total cell numbers, measured by nuclear count, and numbers of alpha and beta cells, measured by glucagon and insulin staining,
respectively, following compound treatment. (E) Representative images shown. All data represent the mean6SD of at least three experiments;
*p,0.05, **p,0.01 and ***p,0.001.
doi:10.1371/journal.pone.0028808.g001
p53 Activates Mouse Insulin Promoter
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4. of MDM2 on Ser166 increases its nuclear localization and
interaction with p300, which in turn, enhances p53 ubiquitination
and degradation [30,31]. Treatment with GW8510 caused a
decrease in Ser166 phosphorylation (Figure 3C, D), presumably
preventing p53 ubiquitination and enhancing p53 stability. Hence,
the post-translational modification states of p53 and MDM2 are
consistent with enhanced cellular p53 activity, and, therefore, with
previous results indicating up-regulation of p53 transcriptional
targets following compound treatment.
Because GW8510 is reported to inhibit CDK2 [13] and has
been shown here to enhance p53 activity, we examined the cell-
cycle profile of alpha cells following compound treatment. CDK2
inhibition leads to an arrest in G1/S, while p53 over-expression
and activation can induce either a G1/S or a G2/M cell-cycle
arrest [32]. In light of our results showing elevated p21, cyclin G,
and MDM2 levels, we anticipated that compound treatment
would produce a cell-cycle phenotype. Indeed, FACS analysis
following three-day treatment with GW8510 showed an enrich-
Figure 2. Involvement of the p53 pathway and quantification of p53 transcriptional activity and expression levels of p53 target
genes following GW8510 treatment of alpha cells. (A) Heat-map display of one of the enriched gene sets (INGA_p53_TARGETS) in gene-
expression profiling of alpha cells treated with 3.3 mM GW8510 for five days. Relative expression values in three biological replicates are plotted by
color. Red, high expression levels, blue, low expression levels. (B) Reproducibility of gene-expression changes following GW8510 treatment, measured
by microarray and qPCR. (C) Cellular p53 activity measured using a dual-luciferase reporter system. Activity of the firefly luciferase p53-reporter
construct was normalized to constitutively active co-transfected Renilla luciferase, and to positive and negative controls. (D) qPCR measurement of
transcript levels of direct p53 targets following 3- and 5-day treatments with 1.65 mM GW8510. Data represent the mean6SD of at least three
experiments; **p,0.01 and ***p,0.001.
doi:10.1371/journal.pone.0028808.g002
p53 Activates Mouse Insulin Promoter
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5. ment of the G2/M population, from 17 to 33% (Figure 4A, B).
This result suggested a G2/M arrest following induction of p53
activity, as opposed to G1/S arrest, which should result in
enrichment of the G0/G1 population. We observed a decrease in
the number of mitotic nuclei following treatment with GW8510
(Figure S4A), suggesting a delayed entry into mitosis. In contrast,
the DNA-damaging agents doxorubicin and etoposide caused an
increase in the number of mitotic nuclei (Figure S4A), consistent
with previous observations that DNA damage delays exit from
mitosis [33]. We also observed that GW8510 had no effect on
phosphorylation of either ATR (Figure S4B) or ATM (Figure S5)
protein kinases, which are activated following DNA damage [34].
In contrast, doxorubicin and etoposide increased ATR phosphor-
ylation (Figure S4B) and activated the ATM-CHK2-p53 pathway
(Figure S5). Accordingly, these DNA damaging agents did not
induce insulin expression in alpha cells (Figure S6). We sought
further evidence of a G2/M arrest by immunofluorescence
analysis for Ser10 phosphorylation of histone H3, a marker of
mitosis [35]. GW8510 treatment decreased the proportion of
mitotic cells in a concentration-dependent manner (Figure 4C, D).
The increase in the G2/M population, and the decrease in the
number M-phase cells, indicate enrichment of G2, as would
happen following a G2/M arrest.
Since p53 is a transcription factor, the trans-activation capacity
of which seems to be enhanced following treatment with GW8510,
we sought to determine whether p53 could directly trans-activate
Ins2. We mined a database for genome-scale computational
discovery of conserved regulatory elements, cisRED, in the mouse
genome, which contains conserved sequence motifs in promoters
of about 17,500 genes [36]. Interestingly, cisRED analysis
predicted a p53 response element in the promoter region of the
Ins2 gene, with a discovery p-value of 0.02 (Figure 5A, Table S2).
This analysis also predicted known response elements in p53 target
genes, such as Cdkn1a and Ccng1, with discovery p-values of 0.06
and 0.004, respectively. It should also be noted that no such
response element is predicted for Ins1, and no Ins1 up-regulation
was, in fact, detected by microarray or real time RT-PCR (data
not shown).
Figure 3. GW8510 treatment effects on protein levels of p53 transcriptional targets and on post-translational modification status
of p53 and MDM2. (A) Western blot analysis and (B) quantification of protein levels of direct p53 targets following a two-day time-course with
1.65 mM GW8510. (C) Western blot analysis and (D) quantification of total protein and post-translational modification levels following 2–5 days of
treatment with 1.65 mM GW8510. Data represent the mean 6 SD of 3 biological replicates; *p,0.01, **p,0.01 and ***p,0.001.
doi:10.1371/journal.pone.0028808.g003
p53 Activates Mouse Insulin Promoter
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6. We verified the presence of p53-response elements in the Ins2
gene by using a covalent chromatin-capture protocol with
recombinant tagged p53 in mouse alpha cells. This method
employs covalent bond formation between the tag and the ligand
immobilized on beads, and has the advantage of being more
efficient and robust than conventional antibody-based ChIP. The
affinity tag in this system, haloalkane dehalogenase, is a derivative
of a bacterial hydrolase that allows covalent site-specific tethering
to resin (see Methods). Since the resin contains immobilized
synthetic ligands, the excess of free ligand can block the
interaction between the tagged protein and the resin. Thus,
addition of blocking ligand to half of the reaction allows
quantification of enrichment over the corresponding control
[37]. PCR analysis following p53 ChIP revealed the presence of
predicted response elements in promoter regions of known p53
transcriptional targets, Cdkn1a and Ccng1, as well as Ins2
(Figure 5B, C). Electrophoretic analysis and quantification of
PCR products demonstrated that 1.34% of total input DNA is
Figure 4. Cell-cycle effects of GW8510 treatment. (A) FACS-generated histograms of propidium iodide stained cells treated with either vehicle
control or GW8510 for 3 days. (B) Quantification of cell-cycle distributions from gated cellular populations in A expressed as percentage of the total
cellular population. Data represent the mean 6 SD of two biological replicates. (C) and (D) M-phase immunofluorescence analysis and quantification
using histone H3 phospho-Ser10 as a mitosis marker. Total cells were counted using Hoechst nuclear stain. Representative images are shown for
Hoechst, histone H3 phospho-Ser10, and overlay at indicated GW8510 concentrations. Values are expressed as fold over vehicle-treated controls.
Data represent the mean 6 SD of at least 3 biological replicates; ***p,0.001.
doi:10.1371/journal.pone.0028808.g004
p53 Activates Mouse Insulin Promoter
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7. pulled down by p53 at the Cdkn1a promoter and 0.15% at the
Ins2 promoter (Figure S7).
We then determined whether up-regulation of p53 transcrip-
tional activity was related to Ins2 induction by GW8510, and
whether modulation of p53 levels or activity would have an impact
on induction of Ins2 expression. P53 levels in alpha cells were
manipulated by overexpression or by siRNA-mediated knock-
down (Figure 6A). Knock-down of p53 decreased the induction of
Ins2 by GW8510 by 75%, while p53 overexpression resulted in a
three-fold increase in Ins2 induction following three-day treatment
with 1.65 mM GW8510 (Figure 6B). We also modulated p53
activity using chemical probes that either target p53 directly or act
upstream in the signal transduction pathways leading to p53
activation. Pifithrin-a, a reversible inhibitor of p53-mediated
apoptosis and p53-dependent transcription [38], suppressed
GW8510-induced Ins2 expression by 80% (Figure 6C). Pifithrin-
Figure 5. Evaluation of p53 response elements. (A) cisRED prediction of p53 response elements in promoter regions of indicated mouse genes.
Discovery p-value is plotted against experimentally determined fold change in gene expression. Selected genes are highlighted. (B) ChIP-PCR analysis
of predicted p53-response elements in promoter regions of Ins2, Cdkn1a and Ccng1. Cells were either untransfected (‘‘control’’) or transfected with
recombinant tagged p53 (‘‘p53-HaloTag’’), and p53-bound DNA immunoprecipitated. PCR was then performed on Ins2, Cdkn1a, or Ccng1 regions
containing predicted p53-response elements. The presence of blocking ligand helps determine the specificity of the interaction. (C) Fold enrichment
of promoter binding is calculated over the corresponding blocking ligand control in the p53-HaloTag condition.
doi:10.1371/journal.pone.0028808.g005
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8. m, which inhibits p53 mitochondrial signalling pathway without
having an effect on p53 transcriptional trans-activation capacity
[39], had no effect on induction of Ins2 by GW8510 (Figure 6C).
We examined pathways upstream of p53, and observed that both
SP600125, a JNK1/2/3 inhibitor [40], and SB202190, a p38
kinase inhibitor [41], almost entirely suppressed Ins2 induction by
GW8510 (Figure 6C). In contrast, the ERK inhibitor PD-09859
[42] reduced GW8510-induced expression by only 20%
(Figure 6C). We confirmed that siRNA-mediated silencing of
JNK and p38 also reduced GW8510-induced insulin expression
(Figure S8). This chemical epistasis analysis indicates that
GW8510 does not likely act at the level of p53 directly, but rather
upstream of p53, in signalling pathways involving JNK and p38
(Figure 6D).
Discussion
Small molecule-mediated alterations in cell state are important
in diseases of cellular deficiency such as type-1 diabetes. An
increase in insulin expression in other pancreatic cell types could
be a valuable part of a strategy to increase beta-cell mass. We
report induction of insulin expression in murine pancreatic alpha
cells with GW8510, a small molecule annotated as a CDK2
inhibitor. We previously described a putative kinase inhibitor,
BRD7389, able to modulate insulin levels in mouse alpha cells
[12], but have found that GW8510 more strongly increases Ins2
mRNA levels. Interestingly, both compounds enhanced insulin
secretion in dissociated human islets. We show that in the donor
tested, treatment with GW8510 and a general kinase inhibitor,
staurosporine, potentiated both basal and the glucose-stimulated
insulin secretion. In addition, a previous report indicates that
wortmannin, a phosphatidylinositol 3-kinase inhibitor, augmented
insulin secretion at 15 mM glucose [43]. Currently available
methodology does not enable us to distinguish between the effects
on insulin release from beta cells, which represent the majority of
the islet endocrine cell population, and potential contributions
from other islet cell types. However, these findings justify further
exploration of kinase inhibition on endocrine-cell composition of
pancreatic islets, generation of insulin in non-beta cell types, and
beta-cell function.
We then explored the mechanism of GW8510-induced insulin
expression in mouse alpha cells. The canonical regulator of insulin
expression, Pdx1, was not implicated in the initial burst of Ins2
induction. In an attempt to identify the underlying mechanism of
Figure 6. Effects of manipulation of p53 levels and activity on induction of Ins2 by GW8510 treatments. (A) Experimental knockdown
and over-expression of p53 in alpha cells and (B) its effects on Ins2 induction by 3-day treatment with 1.65 mM GW8510. (C) Co-treatment with small
molecule inhibitors of p53 and upstream targets in the p53 signalling pathway and their effect on Ins2 induction following treatment with GW8510.
(D) Proposed model for GW8510-mediated induction of insulin expression via activation of p53 transcriptional activity. All data represent the
mean6SD of at least three experiments; *p,0.05, **p,0.01 and ***p,0.001.
doi:10.1371/journal.pone.0028808.g006
p53 Activates Mouse Insulin Promoter
PLoS ONE | www.plosone.org 8 January 2012 | Volume 7 | Issue 1 | e28808
9. action, we examined the effects of GW8510 on the alpha-cell
transcriptome, which revealed involvement of p53 pathway
activation in the observed phenotype. Furthermore, we validated
a cisRED-predicted p53 response element in the Ins2 promoter
region. Following identification of p53 as a direct transcriptional
regulator of Ins2 expression, we demonstrated that increasing p53
levels and activity enhances compound-mediated insulin induc-
tion, while decreasing p53 suppresses these effects.
P53 is present at low levels in normal tissues, including the
pancreas. However, a comparison of expression levels across
rodent pancreatic cell types revealed an enrichment of p53
expression in alpha and beta cells compared to intact pancreatic
islets or to whole pancreas preparations [44]. We observed high
p53 protein levels in the alpha cell line, aTC1.6 (Figure 2C),
indicating that modulating p53 activity in alpha cells could be a
feasible strategy to induce insulin expression. Furthermore,
treatment of NIH3T3 mouse embryonic fibroblasts with
GW8510 enhanced expression of the canonical p53 transcriptional
targets, e.g. Cdkn1a and Ccng1, but did not show an effect on Ins2
mRNA levels (Figure S9). Hence, the p53-dependent insulin
induction by GW8510, observed in pancreatic alpha cells, depends
on the phenotypic cellular context and does not occur in a non-
pancreatic cell type.
Consistent with previous observations in a variety of cell lines
[45], we found that p53 binds to response elements in promoters of
its target genes under basal conditions (Figure 5). DNA binding
itself, however, does not cause expression of p53 targets [45];
execution of the p53 transcriptional program depends on the
necessary set of post-translational modifications and interactions
with appropriate co-regulators. Increasing p53 levels by overex-
pression, or stimulating its activity with inhibitors of p53-MDM2
interaction, did not achieve the same effect (data not shown),
suggesting that intervention upstream of p53 is necessary for the
observed phenotype.
GW8510 does not appear to act at the level of p53 directly, but
acts upstream of p53 to target JNK- and p38-dependent signal-
transduction pathways. Chemical (Figure 6C) or genetic (Figure
S8) inhibition of either JNK or p38 activity interfered with
induction of Ins2 by GW8510. JNK and p38 kinases directly
phosphorylate and trans-activate p53 under stress conditions [46],
including genotoxic stress and DNA damage [47]. However,
treatment with the DNA damaging agents doxorubicin and
etoposide did not induce insulin expression in alpha cells (Figure
S6), suggesting that GW8510 does not function by inducing DNA
damage. The suppression of GW8510’s effects by JNK and p38
kinase inhibitors indicates that GW8510 acts upstream of these
two kinases, and that both signal-transduction pathways are
required to achieve the p53 post-translational modification
necessary for transcriptional activation.
Historically, p53-mediated transcription of target genes has
been thought to occur through sequence-specific binding of the
p53 tetramer to the consensus response element, composed of two
decamer half-sites separated by a 0–13 bp spacer region [48]. The
breadth of p53 transcriptional targets has been recently expanded
following identification of functional non-canonical response
elements, like the L and the K sites [49]. The predicted 11-bp
p53 half-site response element is located 1,267 bps upstream of
TSS for the mouse Ins2 gene, well outside the cis-regulatory
promoter-proximal region, which extends ,300 bp upstream and
100 bp downstream of the TSS [8]. The compact nature of the
promoter and the combinatorial complexity of transcription factor
regulatory elements allow tight positional and temporal control of
insulin expression in beta cells [8]. However, more distal
regulatory sequences may perhaps be utilized to induce ectopic
insulin expression in a non-beta cell type. Additionally, a distal
regulatory element may be in spatial proximity with the target
gene or other regulatory factors at the proximal promoter region
through looped chromatin conformation [50]. A detailed explo-
ration of looped genomic interactions at the insulin promoter may
yield further insights into regulation of insulin expression in
pancreatic beta cells and the potential for up-regulating insulin
expression in a non-beta cell type.
These results show that small-molecule activation of p53 in a
JNK- and p38-dependent manner can regulate Ins2 gene
expression in mouse alpha cells. The cisRED database also
predicts an identical response element located 1,444 bp upstream
of the rat Ins2 TSS (Table S5), indicating similarity in the
regulation of mouse and rat Ins2 genes. In contrast, there is .65%
identity in the main regulatory region of the insulin promoters of
humans and non-primate mammals (2300 to +1), and quickly
drops to ,40% identity in the first 600 bp upstream of the TSS
[8]. Nevertheless, the database also predicts a sequence ortholo-
gous to the p53 response element in the human insulin gene,
located 202 bp downstream of the TSS (Table S5). Evaluation of
this prediction will help determine whether modulation of p53
activity could also be used to alter insulin expression in human
endocrine cell types.
Materials and Methods
Reagents
All chemicals were obtained from Sigma Aldrich. Mouse
pancreatic alpha cell line aTC1 (clones 6 and 9) was purchased
from the American Type Culture Collection. Primers were bought
from Origene and Eurofins MWG Operon. Trp53, cdk2, p38 and
JNK siRNA constructs were purchased from Applied Biosciences,
mouse p53 vector was obtained from Origene (MC205636), and
p53-luciferase reporter constructs were bought from SABios-
ciences. Antibodies used in this study were purchased from Cell
Signaling for p53 (2524), p53 phospho-S392 (9281), p53 acetyl-
K379 (2570), mdm2 phospho-S116 (3521), and histone H3
phospho-Ser10 (9706), Santa Cruz for p21 (sc-6246), cyclin G1/
G2 (sc-851), and mdm2 (sc-56155), and Sigma for b-actin (A1978).
Fluorescently-labelled secondary antibodies were purchased from
Jackson ImmunoResearch, and poly-HRP conjugated antibodies
were purchased from Thermo Scientific Pierce.
Cell culture and compound treatments
aTC1 cells were grown in DMEM containing 1 g/L glucose,
supplemented with 10% FBS, 50 U/mL penicillin and 50 U/mL
streptomycin. Pancreatic islets from one donor (Age: 47 BMI: 23,
purity: 85%, viability: 99%) were dissociated and cultured as
previously described [12]. For compound treatments, cells were
plated in 6-well plates for Western blot and FACS analysis, 24-well
plates for gene-expression analysis and 96-well plates for
immunofluorescence analysis. Cells were allowed to adhere
overnight before addition of compound, and for 5-day treatments,
media was changed and new compound added on day 3. All
compound treatments were performed in 0.1% final DMSO
concentration.
Gene expression measurements
Following compound treatment, cells were lysed and RNA
isolated using the RNeasy Plus Mini kit (Qiagen) according to the
manufacturer’s protocol. 500 ng of RNA was reversed transcribed
using High Capacity RNA-to-cDNA Master Mix (Applied
Biosystems). Quantitative PCR was performed with SYBR Green
PCR Master Mix (Applied Biosystems) on an Applied Biosystems
p53 Activates Mouse Insulin Promoter
PLoS ONE | www.plosone.org 9 January 2012 | Volume 7 | Issue 1 | e28808
10. 7900HT real-time PCR machine using the primers listed in Table
S3. Microarray analysis was performed by the Broad Institute
Genetic Analysis Platform on 500 ng of total RNA using GeneChip
Mouse Genome 430A arrays from Affymetrix, measuring about
22,000 transcripts for approximately 14,000 genes. All data is
MIAME compliant, with the raw data deposited in Gene
Expression Omnibus (GEO), accession number GSE31102.
Western blot analysis
Cell extracts were generated by lysing cells in modified RIPA
buffer containing 1% NP-40, 0.1% sodium deoxycholate,
150 mM NaCl, 1 mM EDTA, 50 mM Tris, pH 7.5 supplemented
with protease inhibitors (Roche) and phosphatase inhibitors (1%
v/v cocktail 1, 0.5% v/v cocktail 2, 1% v/v cocktail 3, Sigma).
Protein concentrations were measured using BCA Protein Assay
Kit (Thermo Scientific Pierce), and 20 mg of each sample were run
on E-Page 48 gels (Invitrogen) and transferred to PVDF
membranes using an iBlot (Invitrogen). Membranes were probed
with 1:500 dilutions of primary antibodies from Cell Signaling,
1:100 dilutions of primary antibodies from Santa Cruz, and
1:1000 dilutions of secondary poly-HRP conjugated antibodies
(Thermo Scientific Pierce). Blots were imaged on an Image Station
4000MM PRO (Kodak/Carestream), and band intensities were
quantified using ImageJ software.
Immunofluorescence measurements
15,000 aTC1 cells per well were plated in 50 mL media in black
optical-bottom tissue culture-treated 96-well plates (Corning).
Following compound treatment, cells were fixed with 4%
paraformaldehyde for 20 minutes at room temperature. Cells
were permeabilized in PBS supplemented with 0.3% Triton X-100
for 20 minutes at room temperature and blocked with 3% BSA in
PBS supplemented with 0.1% Tween-20 (PBSTB3) for 30 min-
utes. Cells were then incubated in 1:250 dilution of primary
antibody in PBSTB3 overnight at 4uC. Following three washes
with PBS, cells were incubated in secondary antibody and 10 mg/
mL Hoechst 33342 in PBSTB3 for 1 h at room temperature in the
dark. Following three washes with PBS, cells were imaged using an
ImageXpress Micro automated microscope (Molecular Devices).
Image analysis and quantification was performed using the ‘‘Cell
Scoring’’ module of MetaXpress software (Molecular Devices).
Transfections
siRNA transfections were performed as outlined in the
Lipofectamine RNAiMax manufacturer’s protocol (Invitrogen).
30 pmol total siRNA was transfected per well of a 24-well plate,
using a combination of three siRNA constructs, 10 pmol each.
Lipofectamine 2000 was used for vector transfections according to
manufacturer’s protocol (Invitrogen). 0.8 mg of DNA was trans-
fected per well of a 24-well plate. Antibiotic-free DMEM
supplemented with 10% FBS was used for all transfections, and
was changed to the usual media 24 hours later. Indicated
compound treatments were started during the media change.
FACS analysis
Suspensions of aTC1 cells from a well of a 6-well plate (500 ml
in PBS) were fixed in 5 mL of cold ethanol and left at 4uC
overnight. Cells were washed twice and resuspended in 800 mL of
PBS containing 1% BSA. Cells were stained by addition of 50 mL
of 1 mg/mL propidium iodide solution (Invitrogen) and 100 mL
of 10 mg/mL RNase A solution (Sigma), and incubated at 37uC
for 30 minutes. Samples were analyzed on the BD LSRII flow
cytometer.
Reporter-gene assay
p53-luciferase constructs were resuspended in Opti-MEM and
reverse-transfected into aTC1 cells using SureFECT transfection
reagent according to manufacturer’s protocol (SABiosciences).
24 hours following transfection, media was changed to DMEM
containing 0.5% FBS and either 0.1% DMSO or the compound of
interest. Dual-GLO luciferase assay was performed 24 hours after
compound addition according to manufacturer’s protocol (Pro-
mega). Firefly and Renilla luminescence signal were read
consecutively on an EnVision Multilabel Plate Reader (PerkinEl-
mer). Reporter signal in each experimental condition was
normalized to the Renilla transfection control, and subsequently
to positive- and negative-control reporter wells.
Chromatin immunoprecipitation
Mouse p53 was amplified using the following primers:
Fw 59-AAAAGCGATCGCCACTGCCATGGAGGAGTCA-
CAGTC-39
Rv 59-AAAAGTTTAAACTCAGTCTGAGTCAGGCCCCA-
39
The insert was cloned into the pFN22K Halotag cmvd1 flexi
vector using PmeI/SgfI restriction sites. Transfections were
performed in 10-cm dishes overnight. Media was changed after
24 hours, and cells were fixed in 1% formaldehyde 48 hours after
transfection. Samples were lysed in HaloCHIP lysis buffer and
sonicated on ice using Branson Sonifier 250 Analog at 2.5 output
with 6 cycles of alternating 10 seconds on and 10 seconds off.
HaloCHIP system protocol (Promega) was performed on cell
lysates according to manufacturer’s instructions. Blocking ligand
was added to half the sample. Primers for PCR analysis of putative
p53 response elements are listed in Table S4.
Supporting Information
Figure S1 Effects of GW8510 on insulin expression in
alphaTC1, clone 9 cell line. Cells were treated for three days
with the indicated concentration of GW8510, and mRNA
collected for assessment of insulin expression by quantitative
PCR. Gene expression was normalized to actin expression. Data
represent the mean 6 SD of three biological replicates; * p,0.05.
(TIF)
Figure S2 Effects of reduction in cdk2 levels or activity
on insulin expression in mouse alpha cells. (A) siRNA-
mediated silencing of cdk2 induces insulin gene expression
approximately two-fold. Scrambled siRNA was used as a control.
(B) Ins2 gene expression changes following a 2-day treatment of
aTC1 cells with cdk2 inhibitors at indicated concentrations. Data
represent the mean 6 SD of three biological replicates.
(TIF)
Figure S3 Volcano plot of microarray measurements in
alpha cells following five-day treatment with 3.3 mM
GW8510. Fold change is calculated over matched DMSO
controls (n = 3) and plotted against the p-value. Genes down-
regulated and up-regulated following GW8510 treatment at least
2-fold with p,0.01 are counted.
(TIF)
Figure S4 Assessment of cell-cycle and ATR activation
following treatment with GW8510 and known DNA-
damaging agents. (A) Percent mitotic nuclei induced by the
indicated concentrations of each compound. (B) Phosphorylation
of ATR was assessed by immunofluorescence, with the nuclear
intensity of p-ATR staining, overlapping with Hoechst nuclear
dye, quantified using MetaXpress (Molecular Devices). Data
p53 Activates Mouse Insulin Promoter
PLoS ONE | www.plosone.org 10 January 2012 | Volume 7 | Issue 1 | e28808
11. represent the mean6SD of at least three experiments; **p,0.05,
**p,0.01 and ***p,0.001.
(TIF)
Figure S5 Assessment of induction of the ATM pathway
by GW8510. Nuclear intensities of (A) phosphorylated ATM, (B)
phosphorylated CHK2, and (C) phosphorylated p53 were assessed
by immunofluorescence and analysis using MetaXpress software
(Molecular Devices). Representative images are shown in (D).
Data represent the mean6SD of at least three experiments;
**p,0.05, **p,0.01 and ***p,0.001.
(TIF)
Figure S6 Ins2 gene expression changes following a 2-
day treatment of aTC1 cells with DNA-damaging agents.
Cells were treated with (A) doxorubicin, (B) etoposide, (C)
thymidine, and (D) ethidium bromide, at indicated concentrations.
Data represent the mean6SD of three biological replicates.
(TIF)
Figure S7 PCR analysis and quantification following
halo-tag p53 ChIP of predicted p53-response elements in
promoter regions of (A) a known p53 transcriptional
target, Cdkn1a, and (B) the novel target, Ins2. Percent
input at each predicted response element is calculated from the
standard input curve.
(TIF)
Figure S8 Knock-down of JNK and p38 inhibit induction
of Ins2 by GW8510. Alpha cells were transfected with the
indicated siRNAs for one day, followed by three-day treatment
with 1.65 mM GW8510. mRNA was collected for analysis of Ins2
gene expression by quantitative PCR, using actin as a normali-
zation control.
(TIF)
Figure S9 Effects of GW8510 on NIH3T3 cells. Gene
expression changes in NIH3T3 mouse embryonic fibroblasts
following a 3-day treatment of aTC1 cells GW8510 at indicated
concentrations. Data represent the mean6SD of three biological
replicates; *p,0.05, **p,0.01 and ***p,0.001.
(TIF)
Table S1 p53-pathway related GSEA gene sets found to
be enriched following 5-day treatment of alpha cells with
GW8510.
(DOC)
Table S2 CisRED prediction of p53 response elements
(group 200034) in promoter regions of selected genes
from the Mouse 4.0 database.
(DOC)
Table S3 Quantitative real-time PCR primers for
indicated mouse genes.
(DOC)
Table S4 Primers used for PCR analysis of putative p53
response elements predicted by CisRED.
(DOC)
Table S5 Species comparisons of orthologous sequenc-
es of the p53 response element predicted in mouse Ins2
promoter region by CisRED.
(DOC)
Acknowledgments
We thank the Genetic Analysis Platform at the Broad Institute for
performing microarray measurements, FAS Centre for Systems Biology at
Harvard University for help with FACS analysis, and Y. Yuan, J. Paulk,
and A. Shamji for technical assistance and discussion.
Author Contributions
Conceived and designed the experiments: DF-Y SK SLS BKW. Performed
the experiments: DF-Y SK AV KHH. Analyzed the data: DF-Y SK AV
KHH. Wrote the paper: DF-Y SK SLS BKW.
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p53 Activates Mouse Insulin Promoter
PLoS ONE | www.plosone.org 12 January 2012 | Volume 7 | Issue 1 | e28808