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1.
J Biol Chem ; 286(35): 30561-30570, 2011 Sep 02.
Article in English | MEDLINE | ID: mdl-21757760

ABSTRACT

The protein deacetylase, sirtuin 1 (SIRT1), is a proposed master regulator of exercise-induced mitochondrial biogenesis in skeletal muscle, primarily via its ability to deacetylate and activate peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α). To investigate regulation of mitochondrial biogenesis by SIRT1 in vivo, we generated mice lacking SIRT1 deacetylase activity in skeletal muscle (mKO). We hypothesized that deacetylation of PGC-1α and mitochondrial biogenesis in sedentary mice and after endurance exercise would be impaired in mKO mice. Skeletal muscle contractile characteristics were determined in extensor digitorum longus muscle ex vivo. Mitochondrial biogenesis was assessed after 20 days of voluntary wheel running by measuring electron transport chain protein content, enzyme activity, and mitochondrial DNA expression. PGC-1α expression, nuclear localization, acetylation, and interacting protein association were determined following an acute bout of treadmill exercise (AEX) using co-immunoprecipitation and immunoblotting. Contrary to our hypothesis, skeletal muscle endurance, electron transport chain activity, and voluntary wheel running-induced mitochondrial biogenesis were not impaired in mKO versus wild-type (WT) mice. Moreover, PGC-1α expression, nuclear translocation, activity, and deacetylation after AEX were similar in mKO versus WT mice. Alternatively, we made the novel observation that deacetylation of PGC-1α after AEX occurs in parallel with reduced nuclear abundance of the acetyltransferase, general control of amino-acid synthesis 5 (GCN5), as well as reduced association between GCN5 and nuclear PGC-1α. These findings demonstrate that SIRT1 deacetylase activity is not required for exercise-induced deacetylation of PGC-1α or mitochondrial biogenesis in skeletal muscle and suggest that changes in GCN5 acetyltransferase activity may be an important regulator of PGC-1α activity after exercise.


Subject(s)
Group III Histone Deacetylases/chemistry , Mitochondria/metabolism , Sirtuin 1/chemistry , Trans-Activators/metabolism , p300-CBP Transcription Factors/metabolism , AMP-Activated Protein Kinases/metabolism , Animals , Cell Nucleus/metabolism , DNA/metabolism , Mice , Mice, Knockout , Models, Biological , Muscle, Skeletal/metabolism , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha , Physical Conditioning, Animal , Time Factors , Transcription Factors
2.
Reprod Toxicol ; 32(1): 85-92, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21679764

ABSTRACT

Resveratrol (trans-3,5,4'-trihydroxystilbene), a polyphenol found in red wine, has multiple beneficial activities that are similar to caloric restriction. In this study, we analyzed the effect of resveratrol on the gonadotropin genes, follicle-stimulating hormone (FSHß) and luteinizing hormone (LHß) in LßT2 immortalized mouse gonadotrope cells. Resveratrol specifically inhibited activin-induced FSHß mRNA and protein expression, and reduced activin-stimulated Smad2/3 phosphorylation. Knockdown of SirT1 gene expression or SirT1 inhibition did not block repression of FSHß expression or suppression of Smad2/3 phosphorylation, but did increase p53 acetylation. Taken together, our results suggest that resveratrol down-regulates Smad2/3 phosphorylation and suppresses FSHß expression via a SirT1-independent pathway.


Subject(s)
Antioxidants/pharmacology , Follicle Stimulating Hormone, beta Subunit/genetics , Gene Expression/drug effects , Gonadotrophs/drug effects , Smad2 Protein/metabolism , Smad3 Protein/metabolism , Stilbenes/pharmacology , Animals , Cell Line , Follicle Stimulating Hormone, beta Subunit/metabolism , Gene Expression Profiling , Gonadotrophs/metabolism , Mice , Oligonucleotide Array Sequence Analysis , Phosphorylation/drug effects , RNA, Messenger/metabolism , Resveratrol , Sirtuin 1/genetics , Sirtuin 1/metabolism
3.
Nephron Physiol ; 104(1): p56-60, 2006.
Article in English | MEDLINE | ID: mdl-16733371

ABSTRACT

During embryonic development, the structures of the nephron from the glomerulus to distal tubule derive from the metanephric mesenchyme. The mesenchymal cells change their cell type and produce highly organized epithelia under the influence of signals from the ureteric bud. The morphological sequence of this conversion includes the formation of a corona of mesenchymal cells surrounding the tips of the ureteric bud, followed by the development of a pre-tubular aggregate, which evolves into preliminary forms of the segmented nephron. Currently, these stages are largely based on histomorphologic criteria and expression of marker molecules. However, to dissect the effects of inductive signals from the ureteric bud in more detail, a sophisticated readout of stages in the conversion process is required, based on the onset of epithelial polarity and the occurrence of vectorial transport. In this review, we discuss some of the new approaches in establishing the staging of the conversion process.


Subject(s)
Embryonic Induction , Epithelial Cells/cytology , Gene Expression Regulation, Developmental , Kidney/embryology , Mesoderm/cytology , Animals , Cadherins/genetics , Cadherins/metabolism , Epithelial Cells/metabolism , Gene Expression Profiling , Humans , Interleukin-6/metabolism , Kidney/cytology , Kidney/metabolism , Leukemia Inhibitory Factor , Mesoderm/metabolism , Nephrons/cytology , Nephrons/embryology , Nephrons/metabolism , Receptors, Cytokine/metabolism , Wnt Proteins/metabolism
4.
J Biol Chem ; 280(34): 30143-9, 2005 Aug 26.
Article in English | MEDLINE | ID: mdl-15994332

ABSTRACT

Although many organic anion transport protein (Oatp) family members have PDZ consensus binding sites at their C termini, the functional significance is unknown. In the present study, we utilized rat Oatp1a1 (NM_017111) as a prototypical member of this family to examine the mechanism governing its subcellular trafficking. A peptide corresponding to the C-terminal 16 amino acids of rat Oatp1a1 was used to affinity-isolate interacting proteins from rat liver cytosol. Protein mass fingerprinting identified PDZK1 as the major interacting protein. This was confirmed by immunoprecipitation of an Oatp1a1-PDZK1 complex from cotransfected 293T cells as well as from native rat liver membrane extracts. Oatp1a1 bound predominantly to the first and third PDZ binding domains of PDZK1, whereas the high density lipoprotein receptor, scavenger receptor B type I binds to the first domain. Although it is possible that PDZK1 forms a complex with these two integral membrane proteins, this did not occur, suggesting that as yet undescribed factors lead to selectivity in the interaction of these protein ligands with PDZK1. Oatp1a1 protein expression was near normal in PDZK1 knock-out mouse liver. However, it was located predominantly in intracellular structures, in contrast to its normal basolateral plasma membrane distribution. Plasma disappearance of the Oatp1a1 ligand [35S]sulfobromophthalein was correspondingly delayed in knock-out mice. These studies show a critical role for oligomerization of Oatp1a1 with PDZK1 for its proper subcellular localization and function. Because its ability to transport substances into the cell requires surface expression, this must be considered in any assessment of physiologic function.


Subject(s)
Hepatocytes/cytology , Membrane Proteins/metabolism , Neoplasm Proteins/metabolism , Organic Anion Transporters/metabolism , Amino Acid Sequence , Animals , Cell Line , Cell Membrane/metabolism , DNA, Complementary/metabolism , Gene Expression Regulation , Hepatocytes/metabolism , Humans , Immunoprecipitation , Ligands , Liver/metabolism , Mass Spectrometry , Membrane Proteins/chemistry , Mice , Mice, Knockout , Microscopy, Fluorescence , Molecular Sequence Data , Neoplasm Proteins/chemistry , Peptides/chemistry , Protein Binding , Protein Structure, Tertiary , Rats , Sulfobromophthalein/chemistry , Transfection
5.
J Biol Chem ; 280(24): 23390-6, 2005 Jun 17.
Article in English | MEDLINE | ID: mdl-15837786

ABSTRACT

Fibrate drugs improve cardiovascular health by lowering plasma triglycerides, normalize low density lipoprotein levels, and raise high density lipoprotein (HDL) levels in patients with dyslipidemias. The HDL-raising effect of fibrates has been shown to be due in part to an increase in human apolipoprotein AI gene expression. However, it has recently been shown that fibrates can affect HDL metabolism in mouse by significantly decreasing hepatic levels of the HDL receptor scavenger receptor B-I (SR-BI) and the PDZ domain containing protein PDZK1. PDZK1 is essential for maintaining hepatic SR-BI levels. Therefore, decreased SR-BI might be secondary to decreased PDZK1, but the mechanism by which fibrates lower SR-BI has not been elucidated. Here we show that feeding PDZK1-deficient mice fenofibrate resulted in the near absence of SR-BI in liver, definitively demonstrating that the effect of fenofibrate on SR-BI is PDZK1-independent. Metabolic labeling experiments in primary hepatocytes from fenofibrate-fed mice demonstrated that fenofibrate enhanced the degradation of SR-BI in a post-endoplasmic reticulum compartment. Moreover, fenofibrate-induced degradation of SR-BI was independent of the proteasome, calpain protease, or the lysosome, and antioxidants did not inhibit fenofibrate-induced degradation of SR-BI. Using metabolic labeling coupled with cell surface biotinylation assays, fenofibrate did not inhibit SR-BI trafficking to the plasma membrane. Together, the data support a model in which fenofibrate enhances the degradation of SR-BI in a post-ER, post-plasma membrane compartment. The further elucidation of this novel degradation pathway may provide new insights into the physiological and pathophysiological regulation of hepatic SR-BI.


Subject(s)
Fenofibrate/pharmacology , Membrane Proteins/metabolism , Receptors, Lipoprotein/metabolism , Animals , Antioxidants/pharmacology , Biotinylation , Blotting, Western , Cell Membrane/metabolism , Down-Regulation , Endoplasmic Reticulum/metabolism , Exons , Hepatocytes/metabolism , Hypolipidemic Agents/pharmacology , Immunoprecipitation , Lipoproteins, HDL/metabolism , Liver/metabolism , Lysosomes/metabolism , Mice , Mice, Transgenic , Proteasome Endopeptidase Complex/metabolism , Protein Biosynthesis , Protein Structure, Tertiary , RNA Processing, Post-Transcriptional , Recombination, Genetic , Scavenger Receptors, Class B , Time Factors
6.
J Lipid Res ; 46(1): 86-92, 2005 Jan.
Article in English | MEDLINE | ID: mdl-15520449

ABSTRACT

ABCA7 is homologous to ABCA1 and has recently been shown in cell culture to bind apolipoprotein A-I (apoA-I) and to promote the efflux of phospholipids. However, it is not known if ABCA7 promotes lipid efflux in vivo. When expressed in HEK293 cells, both human and mouse ABCA7 promoted phospholipid efflux to apoA-I but no detectable cholesterol efflux. However, genetic knockdown of ABCA7 in mouse peritoneal macrophages did not affect phospholipid or cholesterol efflux to apoA-I. Moreover, in ABCA1-knockout macrophages, there was no detectable apoA-I-stimulated phospholipid efflux, inconsistent with a residual role of ABCA7. In contrast to plasma membrane localization of ABCA7 in transfected embryonic kidney cells, immunofluorescence microscopy of endogenous ABCA7 in macrophages showed a predominantly intracellular localization of the protein. Strikingly, immunofluorescence studies of adult mouse kidney revealed an apical brush border membrane localization of ABCA7 in the proximal tubule, suggesting that ABCA7 may come in contact with apoA-I in the glomerular filtrate. Although ABCA7 does not contribute to apolipoprotein-mediated lipid efflux in resting macrophages, its cell surface location in the kidney suggests that it could serve such a role in tissue microenvironments.


Subject(s)
ATP-Binding Cassette Transporters/metabolism , Apolipoprotein A-I/metabolism , Lipid Metabolism , Macrophages, Peritoneal/metabolism , ATP-Binding Cassette Transporters/genetics , Animals , Biological Transport , Fluorescent Antibody Technique , Kidney/chemistry , Kidney/cytology , Mice , Mice, Knockout , Nephrons/chemistry , Phospholipids/metabolism , Tissue Distribution , Transfection
7.
Proc Natl Acad Sci U S A ; 101(26): 9774-9, 2004 Jun 29.
Article in English | MEDLINE | ID: mdl-15210959

ABSTRACT

The mechanisms responsible for the inverse relationship between plasma high-density lipoprotein (HDL) levels and atherosclerotic cardiovascular disease are poorly understood. The ATP-binding cassette transporter A1 (ABCA1) mediates efflux of cellular cholesterol to lipid-poor apolipoproteins but not to HDL particles that constitute the bulk of plasma HDL. We show that two ABC transporters of unknown function, ABCG1 and ABCG4, mediate isotopic and net mass efflux of cellular cholesterol to HDL. In transfected 293 cells, ABCG1 and ABCG4 stimulate cholesterol efflux to both smaller (HDL-3) and larger (HDL-2) subclasses but not to lipid-poor apoA-I. Treatment of macrophages with an liver X receptor activator results in up-regulation of ABCG1 and increases cholesterol efflux to HDL. RNA interference reduced the expression of ABCG1 in liver X receptor-activated macrophages and caused a parallel decrease in cholesterol efflux to HDL. These studies indicate that ABCG1 and ABCG4 promote cholesterol efflux from cells to HDL. ABCG1 is highly expressed in macrophages and probably mediates cholesterol efflux from macrophage foam cells to the major HDL fractions, providing a mechanism to explain the relationship between HDL levels and atherosclerosis risk.


Subject(s)
ATP-Binding Cassette Transporters/metabolism , Cholesterol/metabolism , Lipoproteins, HDL/chemistry , Lipoproteins, HDL/metabolism , Lipoproteins/metabolism , ATP Binding Cassette Transporter 1 , ATP Binding Cassette Transporter, Subfamily G , ATP Binding Cassette Transporter, Subfamily G, Member 1 , ATP-Binding Cassette Transporters/genetics , Animals , Biological Transport , Cell Line , Cyclodextrins/metabolism , DNA-Binding Proteins , Gene Deletion , Humans , Lipoproteins/deficiency , Lipoproteins/genetics , Liver X Receptors , Macrophages/metabolism , Mice , Orphan Nuclear Receptors , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Receptors, Cytoplasmic and Nuclear/agonists , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Retinoic Acid/metabolism , Retinoid X Receptors , Transcription Factors/metabolism , Up-Regulation
8.
J Biol Chem ; 278(44): 42906-12, 2003 Oct 31.
Article in English | MEDLINE | ID: mdl-12917409

ABSTRACT

ATP-binding cassette transporter 1 (ABCA1), the defective transporter in Tangier disease, binds and promotes cellular cholesterol and phospholipid efflux to apolipoprotein I (apoA-I). Based on a high degree of sequence homology between ABCA1 and ABCA7, a transporter of unknown function, we investigated the possibility that ABCA7 might be involved in apolipoprotein binding and lipid efflux. Similarly to cells expressing ABCA1, HEK293 cells overexpressing ABCA7 showed specific binding and cross-linking of lipid-poor apoA-I. ABCA7 expression increased cellular phosphatidylcholine and sphingomyelin efflux to apoA-I in a manner similar to ABCA1 but had no effect on cholesterol efflux. Western analysis showed a high protein level of ABCA7 in mouse spleen, lung, adrenal, and brain but low expression in liver. In contrast to ABCA1, ABCA7 showed moderate basal mRNA and protein levels in macrophages and lymphocytes but no induction by liver X receptor activation. These studies show that ABCA7 has the ability to bind apolipoproteins and promote efflux of cellular phospholipids without cholesterol, and they suggest a possible role of ABCA7 in cellular phospholipid metabolism in peripheral tissues.


Subject(s)
ATP-Binding Cassette Transporters/physiology , Apolipoprotein A-I/metabolism , Cholesterol/metabolism , ATP-Binding Cassette Transporters/metabolism , Adrenal Glands/metabolism , Animals , Biological Transport , Blotting, Western , Brain/metabolism , CD4-Positive T-Lymphocytes/metabolism , Cell Line , Cells, Cultured , DNA-Binding Proteins , Electrophoresis, Polyacrylamide Gel , Genetic Vectors , Humans , Lipid Metabolism , Liver/metabolism , Liver X Receptors , Lung/metabolism , Lymphocytes/metabolism , Macrophages/metabolism , Mice , Microscopy, Confocal , Orphan Nuclear Receptors , Peritoneum/pathology , Phosphatidylcholines/metabolism , Phospholipids/metabolism , Plasmids/metabolism , Protein Binding , RNA, Messenger/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Sphingomyelins/metabolism , Spleen/metabolism , Tissue Distribution , Transfection
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