Yong-Ho Ahn

Up-regulation of Acetyl-CoA Carboxylase α and Fatty Acid Synthase by Human Epidermal Growth Factor Receptor 2 at the Translational Level in Breast Cancer Cells

Expression of the HER2 oncogene is increased in ∼30% of human breast carcinomas and is closely correlated with the expression of fatty acid synthase (FASN). In the present study, we determined the mechanism by which FASN and acetyl-CoA carboxylase α (ACCα) could be induced by HER2 overexpression. SK-BR-3 and BT-474 cells, breast cancer cells that overexpress HER2, expressed higher levels of FASN and ACCα compared with MCF-7 and MDA-MB-231 breast cancer cells in which HER2 expression is low. The induction of FASN and ACCα in BT474 cells were not mediated by the activation of SREBP-1. Exogenous HER2 expression in MDA-MB-231 cells induced the expression of FASN and ACCα, and the HER2-mediated increase in ACCα and FASN was inhibited by both LY294002, a phosphatidylinositol 3-kinase inhibitor, and rapamycin, a mammalian target of rapamycin (mTOR) inhibitor. In addition, the activation of mTOR by the overexpression of RHEB in MDA-MB-231 cells increased the synthetic rates of both FASN and ACCα. On the other hand, FASN and ACCα were reduced in BT-474 cells by a blockade of the mTOR signaling pathway. These changes observed in their protein levels were not accompanied by changes in their mRNA levels. The 5′- and 3′-untranslated regions of both FASN and ACCα mRNAs were involved in selective translational induction that was mediated by mTOR signal transduction. These results strongly suggest that the major mechanism of HER2-mediated induction of FASN and ACCα in the breast cancer cells used in this study is translational regulation primarily through the mTOR signaling pathway.

Lipin1 is a key factor for the maturation and maintenance of adipocytes in the regulatory network with CCAAT/enhancer-binding protein alpha and peroxisome proliferator-activated receptor gamma 2.

Lipin1 expression was induced at a late stage of differentiation of 3T3-L1 preadipocytes and maintained at high levels in mature adipocytes. Knockdown of expression of lipin1 by small interfering RNA in 3T3-L1 preadipocytes almost completely inhibited differentiation into adipocytes, whereas overexpression of lipin1 accelerated adipocyte differentiation, demonstrating that lipin1 is required for adipocyte differentiation. In mature adipocytes, transfection of lipin1-small interfering RNA decreased the expression of adipocyte functional genes, indicating the involvement of lipin1 in the maintenance of adipocyte function. Lipin1 increases the transcription-activating function of peroxisome proliferator-activated receptor γ2 (PPARγ2) via direct physical interaction, whereas lipin1 did not affect the function of other adipocyte-related transcription factors such as C/EBPα, liver X-activated receptor α, or sterol regulatory element binding protein 1c. In mature adipocytes, lipin1 was specifically recruited to the PPARγ-response elements of the phosphoenolpyruvate carboxykinase gene, an adipocyte-specific gene. C/EBPα up-regulates lipin1 transcription by directly binding to the lipin1 promoter. Based on the existence of a positive feedback loop between C/EBPα and PPARγ2, we propose that lipin1 functions as an amplifier of the network between these factors, resulting in the maintenance of high levels of the specific gene expression that are required for adipogenesis and mature adipocyte functions.

Integrated Expression Profiling and Genome-Wide Analysis of ChREBP Targets Reveals the Dual Role for ChREBP in Glucose-Regulated Gene Expression

The carbohydrate response element binding protein (ChREBP), a basic helix-loop-helix/leucine zipper transcription factor, plays a critical role in the control of lipogenesis in the liver. To identify the direct targets of ChREBP on a genome-wide scale and provide more insight into the mechanism by which ChREBP regulates glucose-responsive gene expression, we performed chromatin immunoprecipitation-sequencing and gene expression analysis. We identified 1153 ChREBP binding sites and 783 target genes using the chromatin from HepG2, a human hepatocellular carcinoma cell line. A motif search revealed a refined consensus sequence (CABGTG-nnCnG-nGnSTG) to better represent critical elements of a functional ChREBP binding sequence. Gene ontology analysis shows that ChREBP target genes are particularly associated with lipid, fatty acid and steroid metabolism. In addition, other functional gene clusters related to transport, development and cell motility are significantly enriched. Gene set enrichment analysis reveals that ChREBP target genes are highly correlated with genes regulated by high glucose, providing a functional relevance to the genome-wide binding study. Furthermore, we have demonstrated that ChREBP may function as a transcriptional repressor as well as an activator.

Regulation of glucose transporter type 4 isoform gene expression in muscle and adipocytes

The gene expression of glucose transporter type 4 isoform (GLUT4) is known to be controlled by metabolic, nutritional, or hormonal status. Understanding the molecular mechanisms governing GLUT4 gene expression is critical, because glucose disposal in the body depends on the activities of GLUT4 in the muscle and adipocytes. The GLUT4 activities are regulated by a variety of mechanisms. One of them is transcriptional regulation. GLUT4 gene expression is regulated by a variety of transcriptional factors in muscle and adipose tissue. These data are accumulating regarding the transcriptional factors regulating GLUT4 gene expression. These include MyoD, MEF2A, GEF, TNF‐α, TR‐1α, KLF15, SREBP‐1c, C/EBP‐α, O/E‐1, free fatty acids, PAPRγ, LXRα, NF‐1, etc. These factors are involved in the positive or negative regulation of GLUT4 gene expression. In addition, there is a complex interplay between these factors in transactivating GLUT4 promoter activity. Understanding the mechanisms controlling GLUT4 gene transcription in these tissues will greatly promote the potential therapeutic drug development for obesity and T2DM. IUBMB Life, 59: 134‐145, 2007

Interrelationship between Liver X Receptor α, Sterol Regulatory Element-binding Protein-1c, Peroxisome Proliferator-activated Receptor γ, and Small Heterodimer Partner in the Transcriptional Regulation of Glucokinase Gene Expression in Liver

Liver glucokinase (LGK) plays an essential role in controlling blood glucose levels and maintaining cellular metabolic functions. Expression of LGK is induced mainly regulated by insulin through sterol regulatory element-binding protein-1c (SREBP-1c) as a mediator. Since LGK expression is known to be decreased in the liver of liver X receptor (LXR) knockout mice, we have investigated whether LGK might be directly activated by LXRα. Furthermore, we have studied interrelationship between transcription factors that control gene expression of LGK. In the current studies, we demonstrated that LXRα increased LGK expression in primary hepatocytes and that there is a functional LXR response element in the LGK gene promoter as shown by electrophoretic mobility shift and chromatin precipitation assay. In addition, our studies demonstrate that LXRα and insulin activation of the LGK gene promoter occurs through a multifaceted indirect mechanism. LXRα increases SREBP-1c expression and then insulin stimulates the processing of the membrane-bound precursor SREBP-1c protein, and it activates LGK expression through SREBP sites in its promoter. LXRα also activates the LGK promoter by increasing the transcriptional activity and induction of peroxisome proliferator-activated receptor (PPAR)-γ, which also stimulates LGK expression through a peroxisome proliferator-responsive element. This activation is tempered through a negative mechanism, where a small heterodimer partner (SHP) decreases LGK gene expression by inhibiting the transcriptional activity of LXRα and PPARγ by directly interacting with their common heterodimer partner RXRα. From these data, we propose a mechanism for LXRα in controlling the gene expression of LGK that involves activation through SREBP-1c and PPARγ and inhibition through SHP.

Regulation of GLUT4 gene expression by SREBP-1c in adipocytes

Expression of the GLUT4 (glucose transporter type 4 isoform) gene in adipocytes is subject to hormonal or metabolic control. In the present study, we have characterized an adipose tissue transcription factor that is influenced by fasting/refeeding regimens and insulin. Northern blotting showed that refeeding increased GLUT4 mRNA levels for 24 h in adipose tissue. Consistent with an increased GLUT4 gene expression, the mRNA levels of SREBP (sterol-regulatory-element-binding protein)-1c in adipose tissue were also increased by refeeding. In streptozotocin-induced diabetic rats, insulin treatment increased the mRNA levels of GLUT4 in adipose tissue. Serial deletion, luciferase reporter assays and electrophoretic mobility-shift assay studies indicated that the putative sterol response element is located in the region between bases -109 and -100 of the human GLUT4 promoter. Transduction of the SREBP-1c dominant negative form to differentiated 3T3-L1 adipocytes caused a reduction in the mRNA levels of GLUT4, suggesting that SREBP-1c mediates the transcription of GLUT4. In vivo chromatin immunoprecipitation revealed that refeeding increased the binding of SREBP-1 to the putative sterol-response element in the GLUT4. Furthermore, treating streptozotocin-induced diabetic rats with insulin restored SREBP-1 binding. In addition, we have identified an Sp1 binding site adjacent to the functional sterol-response element in the GLUT4 promoter. The Sp1 site appears to play an additive role in SREBP-1c mediated GLUT4 gene upregulation. These results suggest that upregulation of GLUT4 gene transcription might be directly mediated by SREBP-1c in adipose tissue.

Peroxisome Proliferator-activated Receptor α Is Responsible for the Up-regulation of Hepatic Glucose-6-phosphatase Gene Expression in Fasting and db/db Mice

Glucose-6-phosphatase (G6Pase) is a key enzyme that is responsible for the production of glucose in the liver during fasting or in type 2 diabetes mellitus (T2DM). During fasting or in T2DM, peroxisome proliferator-activated receptor α (PPARα) is activated, which may contribute to increased hepatic glucose output. However, the mechanism by which PPARα up-regulates hepatic G6Pase gene expression in these states is not well understood. We evaluated the mechanism by which PPARα up-regulates hepatic G6Pase gene expression in fasting and T2DM states. In PPARα-null mice, both hepatic G6Pase and phosphoenolpyruvate carboxykinase levels were not increased in the fasting state. Moreover, treatment of primary cultured hepatocytes with Wy14,643 or fenofibrate increased the G6Pase mRNA level. In addition, we have localized and characterized a PPAR-responsive element in the promoter region of the G6Pase gene. Chromatin immunoprecipitation (ChIP) assay revealed that PPARα binding to the putative PPAR-responsive element of the G6Pase promoter was increased in fasted wild-type mice and db/db mice. These results indicate that PPARα is responsible for glucose production through the up-regulation of hepatic G6Pase gene expression during fasting or T2DM animal models.

Transcriptional Regulation of Glucose Sensors in Pancreatic β Cells and Liver

Derangement of glucose metabolism is a key feature of T2DM, with the liver and pancreatic β-cells playing a key role in glucose homeostasis. In the postprandial state, glucose is transported into hepatocytes and either metabolized to fatty acids or CO2, or stored as glycogen. Glucose also acts as a key signal in pancreatic β-cells for regulating insulin secretion. Because GLUT2 and GK expressed in liver and β-cells are responsible for sensing glucose levels in the blood, studies on the regulation of these biomolecules are important in understanding glucose homeostasis in vivo. These molecules are known to be regulated either transcriptionally or post-transcriptionally, and recent studies on the structure and function of promoters of these genes have revealed the involvement of various transcriptional factors in their regulation. Here, we review recent progress in elucidating the transcriptional regulation of glucose sensors in the liver and pancreatic β-cells and the relevance to T2DM.

Troglitazone activates p21Cip/WAF1 through the ERK pathway in HCT15 human colorectal cancer cells

In this study, we identified a new mechanism for the anti-proliferation of HCT15 colorectal cancer cells by troglitazone (TRO). Treating HCT15 cells with 20 microM of TRO transiently increased extracellular signal regulated kinase (ERK) activity within 15 min, and this subsequently induced p21Cip/WAF1 cell cycle regulator and localized in the nucleus. Raf-1 modification and MEK activation also occurred after TRO treatment, and Elk-1-dependent trans-reporter gene expression was concomitantly induced. The induction and nuclear localization of p21Cip/WAF1 by TRO were blocked by PD98059 pre-treatment, which suggested a role for the ERK pathway in p21Cip/WAF1 activation. TRO inhibited BrdU incorporation and no BrdU incorporation was observed in most p21Cip/WAF1-activated cells. Therefore, TRO regulates the proliferation of HCT15 cells at least partly by a mechanism involving the activation of p21Cip/WAF1.

Role of resveratrol in FOXO1-mediated gluconeogenic gene expression in the liver

During a state of fasting, the blood glucose level is maintained by hepatic gluconeogenesis. SIRT1 is an important metabolic regulator during nutrient deprivation and the liver-specific knockdown of SIRT1 resulted in decreased glucose production. We hypothesize that SIRT1 is responsible for the upregulation of insulin-suppressed gluconeogenic genes through the deacetylation of FOXO1. Treatment of primary cultured hepatocytes with resveratrol increased insulin-repressed PEPCK and G6Pase mRNA levels, which depend on SIRT1 activity. We found that the resveratrol treatment resulted in a decrease in the phosphorylation of Akt and FOXO1, which are independent of SIRT1 action. Fluorescence microscopy revealed that resveratrol caused the nuclear localization of FOXO1. In the nucleus, FOXO1 is deacetylated by SIRT1, which might make it more accessible to the IRE of the PEPCK and G6Pase promoter, causing an increase in their gene expression. Our results indicate that resveratrol upregulates the expression of gluconeogenic genes by attenuating insulin signaling and by deacetylating FOXO1, which are SIRT1-independent in the cytosol and SIRT1-dependent in the nucleus, respectively.