Naravat Poungvarin

Glucose-dependent transcriptional regulation by an evolutionarily conserved glucose-sensing module

We report here a novel mechanism for glucose-mediated activation of carbohydrate response element binding protein (ChREBP), a basic helix-loop-helix/leucine zipper (bHLH/ZIP) transcription factor of Mondo family that binds to carbohydrate response element in the promoter of some glucose-regulated genes and activates their expression upon glucose stimulation. Structure-function analysis of ChREBP in a highly glucose-sensitive system using GAL4-ChREBP fusion constructs revealed a glucose-sensing module (GSM) that mediates glucose responsiveness of ChREBP. GSM is conserved among Mondo family members; MondoA, a mammalian paralog of unknown function, and the GSM region of a Drosophila homolog were also found to be glucose responsive. GSM is composed of a low-glucose inhibitory domain (LID) and a glucose-response activation conserved element (GRACE). We have identified a new mechanism accounting for glucose responsiveness of ChREBP that involves specific inhibition of the transactivation activity of GRACE by LID under low glucose concentration and reversal of this inhibition by glucose in an orientation-sensitive manner. The intramolecular inhibition and its release by glucose is a regulatory mechanism that is independent of changes of subcellular localization or DNA binding activity, events that also appear to be involved in glucose responsiveness. This evolutionally conserved mechanism may play an essential role in glucose-responsive gene regulation.

Carbohydrate response element-binding protein (ChREBP) plays a pivotal role in beta cell glucotoxicity

Aims/hypothesisThis study was aimed at the elucidation of the pathogenesis of glucotoxicity, i.e. the mechanism whereby hyperglycaemia damages pancreatic beta cells. The identification of pathways in the process may help identify targets for beta cell-protective therapy. Carbohydrate response element-binding protein (ChREBP), a transcription factor that regulates the expression of multiple hyperglycaemia-induced genes, is produced in abundance in pancreatic beta cells. We hypothesise that ChREBP plays a pivotal role in mediating beta cell glucotoxicity.MethodsWe assessed the role of ChREBP in glucotoxicity in 832/13 beta cells, isolated mouse islets and human pancreas tissue sections using multiple complementary approaches under control and high-glucose-challenge conditions as well as in adeno-associated virus-induced beta cell-specific overexpression of Chrebp (also known as Mlxipl) in mice.ResultsUnder both in vitro and in vivo conditions, ChREBP activates downstream target genes, including fatty acid synthase and thioredoxin-interacting protein, leading to lipid accumulation, increased oxidative stress, reduced insulin gene transcription/secretion and enhanced caspase activity and apoptosis, processes that collectively define glucotoxicity. Immunoreactive ChREBP is enriched in the nucleuses of beta cells in pancreatic tissue sections from diabetic individuals compared with non-diabetic individuals. Finally, we demonstrate that induced beta cell-specific Chrebp overexpression is sufficient to phenocopy the glucotoxicity manifestations of hyperglycaemia in mice in vivo.Conclusions/interpretationThese data indicate that ChREBP is a key transcription factor that mediates many of the hyperglycaemia-induced activations in a gene expression programme that underlies beta cell glucotoxicity at the molecular, cellular and whole animal levels.

Glucose-Mediated Transactivation of Carbohydrate Response Element-Binding Protein Requires Cooperative Actions from Mondo Conserved Regions and Essential Trans-Acting Factor 14-3-3

Carbohydrate response element-binding protein (ChREBP) is a basic helix-loop-helix/leucine zipper transcription factor that binds to the carbohydrate response element in the promoter of certain lipogenic and glycolytic genes. High glucose can activate ChREBP by releasing an intramolecular inhibition within the glucose-sensing module (GSM) that occurs in low glucose. We report here that the glucose response of GSM is mediated by cooperation between five conserved submodules known as Mondo conserved regions (MCRs) I through V within GSM. Deletion of individual MCRs leads to complete (for MCR II, III, and IV) or partial (MCR I) loss of glucose response of ChREBP. MCR IV is necessary and sufficient for inhibiting the transcriptional activity of ChREBP under low glucose. The roles of MCR II and III in glucose response of ChREBP are independent of and distinct from their function in controlling subcellular localization. We further demonstrate that, instead of inhibiting ChREBP activity as would be predicted from its cytoplasmic retentive function, 14-3-3 binding with MCR III is essential for the glucose responsiveness of ChREBP. The interaction between 14-3-3 and ChREBP is constitutive, indicating a permissive role of 14-3-3 in the glucose response of ChREBP. We further uncovered an unconventional 14-3-3 binding motif (residues 116-135) lacking phosphor-serine/threonine within MCR III, a predicted alpha-helix highly conserved in all Mondo proteins. We conclude that individual subdomains in the GSM (MCR I through V) play diverse but crucial roles in cooperation with essential trans-acting cofactors such as 14-3-3 proteins to mediate the glucose response of ChREBP.

Genome-Wide Analysis of ChREBP Binding Sites on Male Mouse Liver and White Adipose Chromatin

Glucose is an essential nutrient that directly regulates the expression of numerous genes in liver and adipose tissue. The carbohydrate response element-binding protein (ChREBP) links glucose as a signaling molecule to multiple glucose-dependent transcriptional regulatory pathways, particularly genes involved in glycolytic and lipogenic processes. In this study, we used chromatin immunoprecipitation followed by next-generation sequencing to identify specific ChREBP binding targets in liver and white adipose tissue. We found a large number of ChREBP binding sites, which are attributable to 5825 genes in the liver, 2418 genes in white adipose tissue, and 5919 genes in both tissues. The majority of these target genes were involved in known metabolic processes. Pathways in insulin signaling, the adherens junction, and cancers were among the top 5 pathways in both tissues. Motif analysis revealed a consensus sequence CAYGYGnnnnnCRCRTG that was commonly shared by ChREBP binding sites. Putative ChREBP binding sequences were enriched on promoters of genes involved in insulin signaling pathway, insulin resistance, and tumorigenesis.

ChREBP Regulates Itself and Metabolic Genes Implicated in Lipid Accumulation in β–Cell Line

Carbohydrate response element binding protein (ChREBP) is an important transcription factor that regulates a variety of glucose-responsive genes in hepatocytes. To date, only two natural isoforms, Chrebpα and Chrebpβ, have been identified. Although ChREBP is known to be expressed in pancreatic β cells, most of the glucose-responsive genes have never been verified as ChREBP targets in this organ. We aimed to explore the impact of ChREBP expression on regulating genes linked to accumulation of lipid droplets, a typical feature of β-cell glucotoxicity. We assessed gene expression in 832/13 cells overexpressing constitutively active ChREBP (caChREBP), truncated ChREBP with nearly identical amino acid sequence to Chrebpβ, or dominant negative ChREBP (dnChREBP). Among multiple ChREBP-controlled genes, ChREBP was sufficient and necessary for regulation of Eno1, Pklr, Mdh1, Me1, Pdha1, Acly, Acaca, Fasn, Elovl6, Gpd1, Cpt1a, Rgs16, Mid1ip1,Txnip, and Chrebpβ. Expression of Chrebpα and Srebp1c were not changed by caChREBP or dnChREBP. We identified functional ChREBP binding sequences that were located on the promoters of Chrebpβ and Rgs16. We also showed that Rgs16 overexpression lead to increased considerable amounts of lipids in 832/13 cells. This phenotype was accompanied by reduction of Cpt1a expression and slight induction of Fasn and Pklr gene in these cells. In summary, we conclude that Chrebpβ modulates its own expression, not that of Chrebpα; it also regulates the expression of several metabolic genes in β-cells without affecting SREBP-1c dependent regulation. We also demonstrate that Rgs16 is one of the ChREBP-controlled genes that potentiate accumulation of lipid droplets in β-cells.

FABP4-Cre Mediated Expression of Constitutively Active ChREBP Protects Against Obesity, Fatty Liver, and Insulin Resistance

Carbohydrate response element binding protein (ChREBP) regulates cellular glucose and lipid homeostasis. Although ChREBP is highly expressed in many key metabolic tissues, the role of ChREBP in most of those tissues and the consequent effects on whole-body glucose and lipid metabolism are not well understood. Therefore, we generated a transgenic mouse that overexpresses a constitutively active ChREBP isoform under the control of the fatty acid binding protein 4-Cre-driven promoter (FaChOX). Weight gain was blunted in male, but not female, FaChOX mice when placed on either a normal chow diet or an obesogenic Western diet. Respiratory exchange ratios were increased in Western diet-fed FaChOX mice, indicating a shift in whole-body substrate use favoring carbohydrate metabolism. Western diet-fed FaChOX mice showed improved insulin sensitivity and glucose tolerance in comparison with controls. Hepatic triglyceride content was reduced in Western diet-fed FaChOX mice in comparison with controls, suggesting protection from fatty liver. Epididymal adipose tissue exhibited differential expression of genes involved in differentiation, browning, metabolism, lipid homeostasis, and inflammation between Western diet-fed FaChOX mice and controls. Our findings support a role for ChREBP in modulating adipocyte differentiation and adipose tissue metabolism and inflammation as well as consequent risks for obesity and insulin resistance.

Highly sensitive EGFR mutation detection by specific amplification of mutant alleles.

Mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) gene predict benefit from tyrosine kinase inhibitors in patients suffering from non-small-cell lung cancer. In this study, we developed a fast, simple, cost-effective and highly sensitive assay for detection of five clinically important EGFR mutations in exon 19 (2235_2249del and 2236_2250del), exon 20 (C2369T) and exon 21 (T2573G and c.2573_2574 TG > GT). We designed EGFR mutation detection assays by combining allele-specific PCR amplification with the detection of SYBR Green I fluorescence, and optimized PCR conditions to specifically amplify mutant alleles. These one-step assays were able to detect the mutations at levels as low as 1.5 mutant copies in a DNA sample. Commercially available probe-based allele-specific PCR exhibited relatively poor performance when detecting very low copies of mutated DNA, especially in exon 19 and 20. Our assays offered dramatically less reagent cost than that of the commercial kit and generated results in less than 90 min after DNA extraction. These protocols can also be applied to conventional thermal cyclers followed by gel electrophoresis detection.

Thai herbal antipyretic 22 formula (APF22) inhibits UVA-mediated melanogenesis through activation of Nrf2-regulated antioxidant defense: Polyherbal formula inhibits UVA-induced melanogenesis via Nrf2

Thai herbal antipyretic 22 formula (APF22), a polyherbal formula, has been traditionally used to treat dermatologic problems including hyperpigmentation. Exposure of the skin to ultraviolet A (UVA) causes abnormal melanin production induced by photooxidative stress. This study thus aimed to investigate the protective effects of APF22 extracts and phenolic compounds, ferulic acid (FA), and gallic acid (GA; used as positive control and reference compounds), on melanogenesis through modulation of nuclear factor E2‐related factor 2 (Nrf2) signaling and antioxidant defenses in mouse melanoma (B16F10) cells exposed to UVA. Our results revealed that the APF22 extracts, FA, and GA reduced melanin synthesis as well as activity and protein levels of tyrosinase in UVA‐irradiated B16F10 cells. Moreover, APF22 extracts and both FA and GA were able to activate Nrf2‐antioxidant response element signaling and promote antioxidant defenses including glutathione, catalase, glutathione peroxidase, and the glutathione‐S‐transferase at both mRNA and enzyme activity levels in irradiated cells. In conclusion, APF22 extracts suppressed UVA‐mediated melanogenesis in B16F10 cells possibly via redox mechanisms involving activation of Nrf2 signaling and upregulation of antioxidant defenses. Moreover, pharmacological action of the APF22 extracts may be attributed to the phenolic compounds, FA, and GA, probably serving as the APF22s active compounds.

Development of ultra-short PCR assay to reveal BRAF V600 mutation status in Thai colorectal cancer tissues

The protein kinase BRAF is one of the key players in regulating cellular responses to extracellular signals. Somatic mutations of the BRAF gene, causing constitutive activation of BRAF, have been found in various types of human cancers such as malignant melanoma, and colorectal cancer. BRAF V600E and V600K, most commonly observed mutations in these cancers, may predict response to targeted therapies. Many techniques suffer from a lack of diagnostic sensitivity in mutation analysis in clinical samples with a low cancer cell percentage or poor-quality fragmented DNA. Here we present allele-specific real-time PCR assay for amplifying 35- to 45-base target sequences in BRAF gene. Forward primer designed for BRAF V600E detection is capable of recognizing both types of BRAF V600E mutation, i.e. V600E1 (c.1799T>A) and V600E2 (c.1799_1800delTGinsAA), as well as complex tandem mutation caused by nucleotide changes in codons 600 and 601. We utilized this assay to analyze Thai formalin-fixed paraffin-embedded tissues. Forty-eight percent of 178 Thai colorectal cancer tissues has KRAS mutation detected by highly sensitive commercial assays. Although these DNA samples contain low overall yield of amplifiable DNA, our newly-developed assay successfully revealed BRAF V600 mutations in 6 of 93 formalin-fixed paraffin-embedded colorectal cancer tissues which KRAS mutation was not detected. Ultra-short PCR assay with forward mutation-specific primers is potentially useful to detect BRAF V600 mutations in highly fragmented DNA specimens from cancer patients.