ABSTRACT
Tumor necrosis factor-alpha (TNF alpha) is involved in the physiological and biological abnormalities found in two opposite metabolic situations: cachexia and obesity. In an attempt to identify novel genes and proteins that could mediate the effects of TNFalpha on adipocyte metabolism and development, we have used a differential display technique comparing 3T3-L1 cells exposed or not to the cytokine. We have isolated a novel adipose cDNA encoding a TNF alpha-inducible 470-amino acid protein termed TIARP, with six putative transmembrane regions flanked by a large amino-terminal and a short carboxyl-terminal domain, a structure reminiscent of channel and transporter proteins. Commitment into the differentiation process is required for cytokine responsiveness. The differentiation process per se is accompanied by a sharp emergence of TIARP mRNA transcripts, in parallel with the expression of the protein at the plasma membrane. Transcripts are present at high levels in white and brown adipose tissues, and are also detectable in liver, kidney, heart, and skeletal muscle. Whereas the biological function of TIARP is presently unknown, its pattern of expression during adipose conversion and in response to TNF alpha exposure as a transmembrane protein mainly located at the cell surface suggest that TIARP might participate in adipocyte development and metabolism and mediate some TNF alpha effects on the fat cell as a channel or a transporter.
Subject(s)
Adipocytes/metabolism , Adipose Tissue/metabolism , Membrane Proteins/chemistry , Membrane Proteins/physiology , Tumor Necrosis Factor-alpha/metabolism , 3T3 Cells , Amino Acid Sequence , Amino Acids/chemistry , Animals , Blotting, Northern , Blotting, Western , Cell Differentiation , Cell Line , Cell Membrane/chemistry , Cell Membrane/metabolism , DNA, Complementary/metabolism , Dose-Response Relationship, Drug , Gene Expression Profiling , Gene Library , Immunohistochemistry , Mice , Mice, Inbred C3H , Microscopy, Fluorescence , Molecular Sequence Data , Obesity/metabolism , Protein Binding , Protein Biosynthesis , Protein Structure, Tertiary , RNA, Messenger/metabolism , Sequence Homology, Amino Acid , Time Factors , Tissue Distribution , Transcription, GeneticABSTRACT
We have used a mRNA differential display technique to identify new genes involved in the reprogramming of gene expression during the adipose conversion of mouse 3T3 preadipocyte cell lines. We report here on the identification and cloning of a novel adipose-specific cDNA encoding a predicted membrane protein of 413 amino acids. The level of the corresponding 3.2-kilobase mRNA is tremendously increased during 3T3-L1 and 3T3-F442A differentiation into adipocytes. A single, very abundant 3.2-kilobase transcript is also found in inguinal and epididymal white adipose tissues and in interscapular brown adipose tissue but not in any other tissues examined. Its expression in adipose tissue is under tight nutritional regulation. The level of this novel 3.2-kilobase transcript becomes virtually nondetectable during fasting but is dramatically increased when fasted mice are refed a high carbohydrate diet. Based on its adipose specificity and dietary regulation, the novel gene product has been designated adiponutrin. The expression of adiponutrin mRNA is also 50-fold elevated in genetically obese fa/fa rats, indicating a link between adiponutrin and obesity. Western blot and confocal imagery analyses with epitope-tagged protein transiently expressed in 3T3-L1 adipocytes, and COS cells show that adiponutrin strictly localizes to membranes and is absent from the cytosol. Sequence analysis reveals homologies with several other members of related eukaryotic proteins, including a human paralog, which has been recently described in vesicular transport mechanisms. This leads us to suggest that adiponutrin could be involved in vesicular targeting and protein transport restricted to the adipocyte function.
Subject(s)
Adipose Tissue/metabolism , Cell Membrane/metabolism , Diet , Membrane Proteins/biosynthesis , Membrane Proteins/genetics , Obesity/genetics , RNA, Messenger/metabolism , 3T3 Cells , Adipocytes/metabolism , Amino Acid Sequence , Amino Acids/chemistry , Animals , Base Sequence , Blotting, Northern , Blotting, Western , COS Cells , Cell Lineage , Cells, Cultured , Cloning, Molecular , DNA, Complementary/metabolism , Epitopes/chemistry , Gene Expression Profiling , Gene Expression Regulation , Glucose/pharmacology , Membrane Proteins/chemistry , Mice , Microscopy, Confocal , Molecular Sequence Data , Plasmids/metabolism , Polymerase Chain Reaction , Protein Structure, Tertiary , Rats , Rats, Zucker , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Subcellular Fractions/metabolism , Tissue Distribution , TransfectionABSTRACT
3T3-F442A adipocytes, which express major beta 3-adrenergic receptors (beta 3-AR) (90%) and minor beta 1-AR (< 10%) and beta 2-AR (< 1%) populations, were used to investigate regulation by n-butyric acid of beta-AR subtype expression. Following butyrate treatment, EC50 values of beta 1- and beta 2-selective agonists, dobutamine and fenoterol, were decreased, whereas that of the beta 3-selective agonist BRL37344 was increased. Direct binding and competition of (-)-[125I]iodocyanopindolol binding by selective beta 1- and beta 2-AR antagonists, CGP20712A and ICI118551, and by the beta 3-AR agonist, BRL37344, revealed that both beta 1- and beta 2-AR were increased in butyrate-treated adipocytes, whereas beta 3-AR almost totally disappeared. In control adipocytes, beta 1-, beta 2-, and beta 3-AR transcripts (quantitated by a polymerase chain reaction assay) represented 6.5, 0.5, and 93% of total beta-AR mRNA, respectively. In butyrate-exposed cells, proportions of beta-AR proteins and mRNAs were, respectively, 87 and 94% for beta 1 and 9 and 1% for beta 2-AR. beta 3-ARs were barely detectable in binding assays and accounted for 4.5% of beta-AR transcripts. Variations of beta-AR protein and mRNA levels were accompanied by parallel changes in the transcription rates of the corresponding genes. The differential regulation of the three beta-ARs by n-butyric acid, a dietary factor produced from colonic fermentation, may have significant nutritional and energetic consequences.
Subject(s)
Adipocytes/metabolism , Butyrates/pharmacology , Gene Expression Regulation/drug effects , Receptors, Adrenergic, beta-1/biosynthesis , Receptors, Adrenergic, beta-2/biosynthesis , Receptors, Adrenergic, beta/biosynthesis , 3T3 Cells , Adenylyl Cyclase Inhibitors , Adipocytes/drug effects , Adrenergic beta-1 Receptor Antagonists , Adrenergic beta-2 Receptor Antagonists , Animals , Base Sequence , Butyric Acid , DNA Primers , DNA, Complementary/biosynthesis , Ethanolamines/pharmacology , Imidazoles/pharmacology , Iodocyanopindolol , Isoproterenol/pharmacology , Mice , Molecular Sequence Data , Oligonucleotides, Antisense , Pindolol/analogs & derivatives , Pindolol/metabolism , Polymerase Chain Reaction , Propanolamines/pharmacology , Receptors, Adrenergic, beta/drug effects , Receptors, Adrenergic, beta/metabolism , Receptors, Adrenergic, beta-1/metabolism , Receptors, Adrenergic, beta-2/metabolism , Receptors, Adrenergic, beta-3 , Transcription, Genetic/drug effectsABSTRACT
When confluent 3T3-F442A cell cultures (Day 0) were grown for 3 days in fetal calf serum-supplemented medium containing isobutyl methyl xanthine and dexamethasone (induction phase) and then shifted to serum-free hormone-defined medium (expression phase), they spontaneously exhibited a sharp rise in lipoprotein lipase activity (LPL); at the peak (Day 7) the LPL activity was about 25 times higher than in control cultures and was further enhanced by insulin. Although this expression of LPL activity was spontaneous, the emergence of glycerophosphate dehydrogenase (G3PDH) activity was completely dependent upon insulin as well as upon the expression of the differentiated phenotype. In committed cells, insulin elicited sustained DNA synthesis associated with limited cell proliferation. The addition of retinoic acid during the phase of expression inhibited insulin-dependent terminal differentiation (i.e., the emergence of G3PDH activity and acquisition of the differentiated phenotype). In addition, retinoic acid counteracted the stimulating effect of insulin upon LPL activity, but affected neither the mitotic process nor the spontaneous emergence of LPL activity. When added during the phase of induction, it prevented the overall process of adipogenic differentiation. Thus, the use of retinoic acid can indicate independent control of the mitogenic and lipogenic effects of insulin following commitment to adipogenic differentiation.
Subject(s)
Adipose Tissue/drug effects , Tretinoin/pharmacology , 1-Methyl-3-isobutylxanthine/pharmacology , Adipose Tissue/cytology , Cell Differentiation/drug effects , Cell Division/drug effects , Cell Line , DNA/biosynthesis , Dexamethasone/pharmacology , Glycerolphosphate Dehydrogenase/metabolism , Insulin/pharmacology , Lipoprotein Lipase/metabolismABSTRACT
Differentiation of 3T3-F442A adipocytes, monitored by accumulation of neutral lipid and by using the sensitive marker glycerophosphate dehydrogenase, is inhibited by incubation of confluent 3T3-F442A fibroblasts in medium containing retinoic acid or dexamethasone. When added together, dexamethasone (0.25 microM) potentiates about 50-fold the inhibitory effect of retinoic acid (10 microM). Insulin cannot counteract the retinoic acid blockade; however, it can overcome the inhibition of differentiation elicited by dexamethasone. These differential effects of insulin are used for characterizing the adipose conversion cycle. We describe cell culture conditions where terminal differentiation of 3T3-F442A preadipocytes is achieved by low, physiological levels of insulin. They include the switch from a high-serum medium containing isobutyl methyl xanthine and dexamethasone to a serum-free, hormone-supplemented medium. The data reported establish the existence of two successive states for commitment to adipogenic differentiation: a first commitment point (CA) to differentiation which requires serum adipogenic factors, and a second commitment point (CH) controlled by lipogenic hormones, namely insulin, after which terminal maturation can resume. We demonstrate that retinoic acid can prevent and interrupt differentiation by blocking the cells within the early differentiation phase.