Keisuke Tachibana

Activation of peroxisome proliferator-activated receptor δ induces fatty acid β-oxidation in skeletal muscle and attenuates metabolic syndrome

In this study, we defined the role of peroxisome proliferator-activated receptor β/δ (PPARδ) in metabolic homeostasis by using subtype selective agonists. Analysis of rat L6 myotubes treated with the PPARδ subtype-selective agonist, GW501516, by the Affymetrix oligonucleotide microarrays revealed that PPARδ controls fatty acid oxidation by regulating genes involved in fatty acid transport, β-oxidation, and mitochondrial respiration. Similar PPARδ-mediated gene activation was observed in the skeletal muscle of GW501516-treated mice. Accordingly, GW501516 treatment induced fatty acid β-oxidation in L6 myotubes as well as in mouse skeletal muscles. Administration of GW501516 to mice fed a high-fat diet ameliorated diet-induced obesity and insulin resistance, an effect accompanied by enhanced metabolic rate and fatty acid β-oxidation, proliferation of mitochondria, and a marked reduction of lipid droplets in skeletal muscles. Despite a modest body weight change relative to vehicle-treated mice, GW501516 treatment also markedly improved diabetes as revealed by the decrease in plasma glucose and blood insulin levels in genetically obese ob/ob mice. These data suggest that PPARδ is pivotal to control the program for fatty acid oxidation in the skeletal muscle, thereby ameliorating obesity and insulin resistance through its activation in obese animals.

The Role of PPARs in Cancer.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that belong to the nuclear hormone receptor superfamily. PPARα is mainly expressed in the liver, where it activates fatty acid catabolism. PPARα activators have been used to treat dyslipidemia, causing a reduction in plasma triglyceride and elevation of high-density lipoprotein cholesterol. PPARδ is expressed ubiquitously and is implicated in fatty acid oxidation and keratinocyte differentiation. PPARδ activators have been proposed for the treatment of metabolic disease. PPARγ2 is expressed exclusively in adipose tissue and plays a pivotal role in adipocyte differentiation. PPARγ is involved in glucose metabolism through the improvement of insulin sensitivity and represents a potential therapeutic target of type 2 diabetes. Thus PPARs are molecular targets for the development of drugs treating metabolic syndrome. However, PPARs also play a role in the regulation of cancer cell growth. Here, we review the function of PPARs in tumor growth.

Sterol-mediated Regulation of Human Lipin 1 Gene Expression in Hepatoblastoma Cells

Lipin 1 plays a crucial role in lipid metabolism in adipose tissue, skeletal muscle, and liver. Its physiological role involves two cellular functions: regulation of phosphatidate phosphatase activity and regulation of fatty acid oxidation. In this study, we have demonstrated that lipin 1 gene (LPIN1) expression is regulated by cellular sterols, which are key regulators of lipid metabolism. We have also characterized the sterol-response element and nuclear factor Y-binding sites in the human LPIN1 promoter. Using a luciferase assay, electrophoretic mobility shift assay, and chromatin immunoprecipitation assay, we demonstrated that these elements are responsible for the transcription of LPIN1 gene, mediated by SREBP-1 (sterol regulatory element-binding protein 1) and nuclear factor Y. Furthermore, we investigated whether lipin 1 is involved in lipogenesis by transfection of LPIN1 small interfering RNA. We infer that sterol-mediated regulation of lipin 1 gene transcription modulates triglyceride accumulation. This modulation involves changes in the activity of phosphatidate phosphatase.

Fenofibrate suppresses growth of the human hepatocellular carcinoma cell via PPARα-independent mechanisms.

Fenofibrate, a peroxisome proliferator-activated receptor (PPAR) α agonist, is a hypolipidemic drug. Although several studies have explored the fenofibrate-induced antiproliferative effect in cultured human cells, it is not clear which role PPARα plays in this antiproliferative effect. Therefore, we investigated the antiproliferative mechanism of fenofibrate in Huh7 (human hepatoma cell line). Cell viability was measured by the WST-8 assay and cell proliferation was assessed using the BrdU incorporation assay. The cell cycle was analyzed by flow cytometry. The cyclins, tumor suppressor proteins and regulators of the AKT signaling pathway were analyzed by immunoblotting. Using flow cytometry, we showed that fenofibrate blocks entry into the S phase of the cell cycle. We certified that this G1 arrest is caused by the reduction of cyclin A and E2F1 and the accumulation of the cyclin-dependent kinase inhibitor p27. Interestingly, the antiproliferative effect of fenofibrate was not affected by the PPARα antagonist (GW6471) or by PPARα-specific siRNA. These results suggest that fenofibrate suppresses Huh7 cell growth through a PPARα independent mechanism. Furthermore, we showed that treatment of Huh7 cells with fenofibrate leads to suppression of AKT phosphorylation. We also found for the first time that fenofibrate increased the C-terminal modulator protein (CTMP), which inhibits AKT phosphorylation. Our data suggest that fenofibrate inhibits the proliferation of Huh7 cells by blocking Akt activation, and that CTMP is one of the key players for this antiproliferative property of fenofibrate in Huh7 cells.

Identification of human low-density lipoprotein receptor as a novel target gene regulated by liver X receptor alpha

Liver X receptor alpha (LXRα) is a member of the nuclear receptor superfamily that is activated by oxysterols, and plays a pivotal role in regulating the metabolism, transport and uptake of cholesterol. Here, we demonstrate that LXRα also regulates the low‐density lipoprotein receptor (LDLR) gene, which mediates the endocytic uptake of LDL cholesterol in the liver. An LXR agonist induced the expression of LDLR in cultured hepatoblastoma cells. Moreover, the LDLR promoter contained an LXR response element that was recognized by LXRα/RXRα (retinoid X receptor alpha) heterodimers in hepatoblastoma cells. These results suggest a novel pathway whereby LXRα might modulate cholesterol metabolism.

Design and evaluation of locked nucleic acid-based splice-switching oligonucleotides in vitro

Antisense-mediated modulation of pre-mRNA splicing is an attractive therapeutic strategy for genetic diseases. Currently, there are few examples of modulation of pre-mRNA splicing using locked nucleic acid (LNA) antisense oligonucleotides, and, in particular, no systematic study has addressed the optimal design of LNA-based splice-switching oligonucleotides (LNA SSOs). Here, we designed a series of LNA SSOs complementary to the human dystrophin exon 58 sequence and evaluated their ability to induce exon skipping in vitro using reverse transcription-polymerase chain reaction. We demonstrated that the number of LNAs in the SSO sequence and the melting temperature of the SSOs play important roles in inducing exon skipping and seem to be key factors for designing efficient LNA SSOs. LNA SSO length was an important determinant of activity: a 13-mer with six LNA modifications had the highest efficacy, and a 7-mer was the minimal length required to induce exon skipping. Evaluation of exon skipping activity using mismatched LNA/DNA mixmers revealed that 9-mer LNA SSO allowed a better mismatch discrimination. LNA SSOs also induced exon skipping of endogenous human dystrophin in primary human skeletal muscle cells. Taken together, our findings indicate that LNA SSOs are powerful tools for modulating pre-mRNA splicing.

Sterol-regulatory-element-binding protein 2 and nuclear factor Y control human farnesyl diphosphate synthase expression and affect cell proliferation in hepatoblastoma cells

FDPS (farnesyl diphosphate synthase) catalyses the formation of farnesyl diphosphate, a key intermediate in the synthesis of cholesterol and isoprenylated cellular metabolites. FDPS is also the molecular target of nitrogen-containing bisphosphonates, which are used as bone-antiresorptive drugs in various disorders. In the present study, we characterized the sterol-response element and NF-Y (nuclear factor Y)-binding site in the human FDPS promoter. Using a luciferase assay, electrophoretic mobility-shift assay and chromatin immunoprecipitation assay, we demonstrated that these elements are responsible for the transcription of the FDPS gene, and that its transcriptional activation is mediated by SREBP-2 (sterol-regulatory-element-binding protein 2) and NF-Y. We also investigated whether sterol-mediated FDPS expression is involved in the cell proliferation induced by zoledronic acid, an FDPS inhibitor. We show that the SREBP-2- and NF-Y-mediated regulation of FDPS gene transcription modulates cell proliferation. These results suggest that SREBP-2 and NF-Y are required to trigger cell proliferation through the induction of FDPS expression and that the pharmacological action of zoledronic acid is involved in this pathway.

Regulation of the human PDZK1 expression by peroxisome proliferator-activated receptor alpha.

Although PDZK1 is a well‐known adaptor protein, the mechanisms for its role in transcriptional regulation are largely unknown. The peroxisome proliferator‐activated receptor alpha (PPARα) is a ligand‐activated transcription factor that plays an important role in the regulation of lipid homeostasis. Previously, we established a tetracycline‐regulated human cell line that can be induced to express PPARα and identified candidate target genes, one of which was PDZK1. In this study, we cloned and characterized the promoter region of the human pdzk1 gene and determined the PPAR response element. Finally, we demonstrate that endogenous PPARα regulates PDZK1 expression.

Analysis of the subcellular localization of the human histone methyltransferase SETDB1.

SET domain, bifurcated 1 (SETDB1) is a histone methyltransferase that methylates lysine 9 on histone H3. Although it is important to know the localization of proteins to elucidate their physiological function, little is known of the subcellular localization of human SETDB1. In the present study, to investigate the subcellular localization of hSETDB1, we established a human cell line constitutively expressing enhanced green fluorescent protein fused to hSETDB1. We then generated a monoclonal antibody against the hSETDB1 protein. Expression of both exogenous and endogenous hSETDB1 was observed mainly in the cytoplasm of various human cell lines. Combined treatment with the nuclear export inhibitor leptomycin B and the proteasome inhibitor MG132 led to the accumulation of hSETDB1 in the nucleus. These findings suggest that hSETDB1, localized in the nucleus, might undergo degradation by the proteasome and be exported to the cytosol, resulting in its detection mainly in the cytosol.

PPARβ/δ activation of CD300a controls intestinal immunity

Macrophages are important for maintaining intestinal immune homeostasis. Here, we show that PPARβ/δ (peroxisome proliferator-activated receptor β/δ) directly regulates CD300a in macrophages that express the immunoreceptor tyrosine based-inhibitory motif (ITIM)-containing receptor. In mice lacking CD300a, high-fat diet (HFD) causes chronic intestinal inflammation with low numbers of intestinal lymph capillaries and dramatically expanded mesenteric lymph nodes. As a result, these mice exhibit triglyceride malabsorption and reduced body weight gain on HFD. Peritoneal macrophages from Cd300a−/− mice on HFD are classically M1 activated. Activation of toll-like receptor 4 (TLR4)/MyD88 signaling by lipopolysaccharide (LPS) results in prolonged IL-6 secretion in Cd300a−/− macrophages. Bone marrow transplantation confirmed that the phenotype originates from CD300a deficiency in leucocytes. These results identify CD300a-mediated inhibitory signaling in macrophages as a critical regulator of intestinal immune homeostasis.