Kiyoto Motojima

Inhibition of RXR and PPARγ ameliorates diet-induced obesity and type 2 diabetes

PPARgamma is a ligand-activated transcription factor and functions as a heterodimer with a retinoid X receptor (RXR). Supraphysiological activation of PPARgamma by thiazolidinediones can reduce insulin resistance and hyperglycemia in type 2 diabetes, but these drugs can also cause weight gain. Quite unexpectedly, a moderate reduction of PPARgamma activity observed in heterozygous PPARgamma-deficient mice or the Pro12Ala polymorphism in human PPARgamma, has been shown to prevent insulin resistance and obesity induced by a high-fat diet. In this study, we investigated whether functional antagonism toward PPARgamma/RXR could be used to treat obesity and type 2 diabetes. We show herein that an RXR antagonist and a PPARgamma antagonist decrease triglyceride (TG) content in white adipose tissue, skeletal muscle, and liver. These inhibitors potentiated leptins effects and increased fatty acid combustion and energy dissipation, thereby ameliorating HF diet-induced obesity and insulin resistance. Paradoxically, treatment of heterozygous PPARgamma-deficient mice with an RXR antagonist or a PPARgamma antagonist depletes white adipose tissue and markedly decreases leptin levels and energy dissipation, which increases TG content in skeletal muscle and the liver, thereby leading to the re-emergence of insulin resistance. Our data suggested that appropriate functional antagonism of PPARgamma/RXR may be a logical approach to protection against obesity and related diseases such as type 2 diabetes.

Nrf2 regulates the alternative first exons of CD36 in macrophages through specific antioxidant response elements

We previously demonstrated that Nrf2 regulates oxidized LDL-mediated CD36 expression in macrophages. The current study aimed to determine the mechanism of Nrf2-mediated macrophage CD36 induction. Treatment with the Nrf2 activator diethylmaleate, but not PPARgamma specific ligands, caused marked upregulation of CD36 in mouse macrophage RAW264.7 cells. Similarly, Nrf2 activators induced CD36 expression in bone marrow-derived macrophages in a Nrf2-dependent manner. Induced expression of the three alternative first exons of mouse CD36, deemed 1A, 1B, and 1C, occurred upon Nrf2 activation with exon1A mainly contributing to the CD36 expression. Four antioxidant response elements (AREs) lie within close proximity to these three exons, and chromatin immunoprecipitation assays demonstrated that two AREs upstream of exon1A, the distal 1A-ARE1, and the proximal 1A-ARE2, were Nrf2-responsive. Luciferase reporter assays conclusively demonstrated that 1A-ARE2 is the critical regulatory element for the Nrf2-mediated gene expression. Thus Nrf2 directly regulates CD36 gene expression by binding to 1A-ARE2.

Identification of ERRα as a specific partner of PGC‐1α for the activation of PDK4 gene expression in muscle

Pyruvate dehydrogenase kinaseu20034 (PDK4) is a key regulatory enzyme involved in switching the energy source from glucose to fatty acids in response to physiological conditions. Transcription of the PDK4 gene is activated by fasting or by the administration of a PPARα ligand in a tissue‐specific manner. Here, we show that the two mechanisms are independent, and that ERRα is directly involved in PPARα‐independent transcriptional activation of the PDK4 gene with PGC‐1α as a specific partner. This conclusion is based on the following evidence. First, detailed mutation analyses of the cloned PDK4 gene promoter sequence identified a possible ERRα‐binding motif as the PGC‐1α responsive element. Second, overexpression of ERRα by cotransfection enhanced, and the knockout of it by shRNAs diminished, PGC‐1α‐dependent activation. Third, specific binding of ERRα to the identified PGC‐1α responsive sequence was confirmed by the electrophoresis mobility shift assay. Finally, cell‐type‐specific responsiveness to PGC‐1α was observed and this could be explained by differences in the expression levels of ERRα, however, ectopic expression of ERRα in poorly responsive cells did not restore PGC‐1α responsiveness, indicating that ERRα is necessary, but not sufficient for the response.

Hydrophobic statins induce autophagy in cultured human rhabdomyosarcoma cells.

Statins are widely used to treat hypercholesterolemia, but they are associated with muscle-related adverse events, by as yet, inadequately resolved mechanisms. In this study, we report that statins induced autophagy in cultured human rhabdomyosarcoma A204 cells. Potency differed widely among the statins: cerivastatin induced autophagy at 0.1muM, simvastatin at 10muM but none was induced by pravastatin. Addition of mevalonate, but not cholesterol, blocked induction of autophagy by cerivastatin, suggesting that this induction is dependent on modulation of isoprenoid metabolic pathways. The statin-induced autophagy was not observed in other types of cells, such as human hepatoma HepG2 or embryonic kidney HEK293 cells. Muscle-specific abortive induction of autophagy by hydrophobic statins is a possible mechanism for statin-induced muscle-related side effects.

Identification and characterization of the ER/lipid droplet-targeting sequence in 17β-hydroxysteroid dehydrogenase type 11

17beta-Hydroxysteroid dehydrogenase type 11 (17betaHSD11) is mostly localized on the endoplasmic reticulum (ER) membrane under normal conditions and redistributes to lipid droplets (LDs) when the formation of LDs is induced. In this study, confocal microscopy analyses of the subcellular localization of the mutated 17betaHSD11 proteins in cells with or without LDs revealed that both an N-terminal hydrophobic sequence and an adjacent sequence that has a weak homology with the PAT motif are independently necessary and both parts together (28 amino acid residues in total) are sufficient for the dual localization of 17betaHSD11. Mutation analyses suggest that the PAT-like motif in 17betaHSD11 will not be functionally similar to the canonical PAT motif. Hsp60 was identified as a possibly interacting protein with the PAT-like motif, and biochemical and microscopic analyses suggest that Hsp60 may be partly, but not necessarily involved in recognition of the PAT-like part of the targeting sequence of 17betaHSD11.

Hydrophobic statins induce autophagy and cell death in human rhabdomyosarcoma cells by depleting geranylgeranyl diphosphate.

Statins are the most common type of medicine used to treat hypercholesterolemia; however, they are associated with a low incidence of myotoxicity such as myopathy and rhabdomyolysis. The mechanisms for the adverse effects remain to be fully elucidated for safer chronic use and drug development. The results of our earlier work suggested that hydrophobic statins induce autophagy in cultured human rhabdomyosarcoma A204 cells. In this study, we first confirmed the statin-induced autophagy by assessing other criteria, including induced expression of the autophagy-related genes, enhanced protein degradation of autophagy marker protein p62 and electron microscopic observation of induced formation of autophagosome. We next demonstrated that the extent of inhibition of HMG-CoA reductase in the cell is parallel with the ability of a statin to induce autophagy. Thus, the primary activity of statins causes autophagy in A204 cells. Considering the mechanism for the induction, we showed that statins induce autophagy by depleting cellular levels of geranylgeranyl diphosphate (GGPP) mostly through an unknown pathway that does not involve two major small G proteins, Rheb and Ras. Finally, we demonstrated that the ability of statins to induce autophagy parallels their toxicity to A204 cells and that both can be suppressed by GGPP.

Maturation of the Extracellular Matrix and Cell Adhesion Molecules in Layered Co-cultures of HepG2 and Endothelial Cells

We previously reported that using thermo-responsive culture surfaces, a layered co-culture was achieved by placing an endothelial cell sheet onto a layer of human hepatoma cell line HepG2 in order to develop a culture model that mimics hepatic lobules. In the layered co-culture cells, the expression levels of liver-specific genes gradually increased. A cross-sectional view of the layered co-culture cells showed that the thickness of the layer slowly increased after layering, as did extracellular matrix (ECM) deposition around HepG2 cells. In this study, we report that the molecular compositions of ECM and cell-adhesion molecules changed in the layered co-culture cells. Gene expression of integrin alpha4 and decorin gradually increased after layering, and the time-course pattern of these genes was correlated with that of liver-specific genes. Moreover, the layered co-culture system has the ability to assemble a branching network of fibronectin fibrils. These results suggest that a vastly different extracellular environment in layered co-culture cells may induce an increase in liver-specific functions.

Regulated expression by PPARα and unique localization of 17β‐hydroxysteroid dehydrogenase type 11 protein in mouse intestine and liver

17β‐Hydroxysteroid dehydrogenase typeu200311 (17β‐HSD11) is a member of the short‐chain dehydrogenase/reductase family involved in the activation and inactivation of sex steroid hormones. We recently identified 17β‐HSD11 as a gene that is efficiently regulated by peroxisome proliferator‐activated receptor‐α PPARα in the intestine and the liver [Motojima K (2004) Eur J Biochem271, 4141–4146]. In this study, we characterized 17β‐HSD11 at the protein level to obtain information about its physiologic role in the intestine and liver. For this purpose, specific antibodies against 17β‐HSD11 were obtained. Western blotting analysis showed that administration of a peroxisome proliferator‐activated receptor‐α agonist induced 17β‐HSD11 protein in the jejunum but not in the colon, and to a much higher extent than in the liver of mice. A subcellular localization study using Chinese hamster ovary cells and green fluorescent protein‐tagged 17β‐HSD11 showed that it was mostly localized in the endoplasmic reticulum under normal conditions, whereas it was concentrated on lipid droplets when they were induced. A pulse‐chase experiment suggested that 17β‐HSD11 was redistributed to the lipid droplets via the endoplasmic reticulum. Immunohistochemical analysis using tissue sections showed that 17β‐HSD11 was induced mostly in intestinal epithelia and hepatocytes, with heterogeneous localization both in the cytoplasm and in vesicular structures. A subcellular fractionation study of liver homogenates confirmed that 17β‐HSD11 was localized mostly in the endoplasmic reticulum when mice were fed a normal diet, but was distributed in both the endoplasmic reticulum and the lipid droplets of which formation was induced by feeding a diet containing a proliferator‐activated receptor‐α agonist. Taken together, these data indicate that 17β‐HSD11 localizes both in the endoplasmic reticulum and in lipid droplets, depending on physiologic conditions, and that lipid droplet 17β‐HSD11 is not merely an endoplasmic reticulum contaminant or a nonphysiologically associated protein in the cultured cells, but a bona fide protein component of the membranes of both intracellular compartments.

Up-Regulation of Drug-Metabolizing Enzyme Genes in Layered Co-Culture of a Human Liver Cell Line and Endothelial Cells

Primary human hepatocytes are used extensively to study drug-metabolizing enzymes such as the cytochrome P450 (CYP) enzymes. However, the activities of these enzymes decrease rapidly during culture. In the present study, using a thermo-responsive culture dish, layered co-culture was achieved by placing a bovine pulmonary artery endothelial cell (BPAEC) sheet onto the human hepatoma cell line HepG2. In the BPAEC/HepG2 layered co-culture system, real-time polymerase chain reaction analysis showed that the expression levels of various CYP enzymes were more than 10 times greater 21 days after layering than with a HepG2 monolayer. The expression levels of CYP1B1, CYP2C9, CYP2E1, and CYP3A4 were up-regulated in a time-dependent manner, gradually increasing from day 10 after layering, and continuing to increase until at least day 21. The gene expression levels of the various CYP enzymes were almost identical to that of human liver. These results suggest that our layered co-culture system enhances the function of HepG2 cells and that our BPAEC/HepG2 layered co-culture system can serve as a useful model for the in vitro evaluation of CYP regulation.

17β-Hydroxysteroid dehydrogenase type 13 is a liver-specific lipid droplet-associated protein

17beta-Hydroxysteroid dehydrogenase (17betaHSD) type 13 is identified as a new lipid droplet-associated protein. 17betaHSD type 13 has an N-terminal sequence similar to that of 17betaHSD type 11, and both sequences function as an endoplasmic reticulum and lipid droplet-targeting signal. Localization of native 17betaHSD type 13 on the lipid droplets was confirmed by subcellular fractionation and Western blotting. In contrast to 17betaHSD type 11, however, expression of 17betaHSD type 13 is largely restricted to the liver and is not enhanced by peroxisome proliferator-activated receptor alpha and its ligand. Instead the expression level of 17betaHSD type 13 in the receptor-null mice was increased several-fold. 17betaHSD type 13 may have a distinct physiological role as a lipid droplet-associated protein in the liver.