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Specific elevation of transcript levels of particular protein subtypes induced in brown adipose tissue by cold exposure

Faculty of Pharmaceutical Sciences, University of Tokushima, Shomachi-1, Tokushima 770-8505, Japan
Biochimica et Biophysica Acta (Impact Factor: 4.66). 05/2000; 1457(3):263-272. DOI: 10.1016/S0005-2728(00)00107-9

ABSTRACT To understand the difference in metabolic flow in rat brown adipose tissue (BAT) from that in white adipose tissue (WAT) at the molecular level, we examined the steady-state transcript levels of 39 proteins in both adipose tissues with and without cold exposure by Northern blot analysis. In addition to the transcript levels of uncoupling protein isoforms, those of proteins involved in the transport and catabolism of fatty acids and glucose in BAT were elevated by cold exposure, suggesting the stimulation of utilization of fatty acids and glucose as fuels in BAT. As to these changes, the muscle-type subtypes were remarkable; and therefore, they were suggested to be responsible for the cold exposure-induced acceleration of energy expenditure in BAT. Furthermore, of the isoforms of β-adrenergic receptor (β-AR) and CCAAT enhancer binding protein (C/EBP), transcript levels of β1-AR and C/EBPβ in BAT were increased by the cold exposure. Possible roles of these proteins in energy metabolism in BAT were discussed.

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Available from: Takiko Daikoku, Jul 30, 2015
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    • "In most previous reports concerning cold exposure, the period of exposure was short, being several hours to weeks (Puigserver et al. 1998; Guardiola-Diaz et al. 1999; Daikoku et al. 2000; Yu et al. 2002; Goetzman et al. 2005). The expression levels of many proteins including PPARa ⁄ c dizzily changed during several weeks of cold exposure (Guardiola-Diaz et al. 1999). "
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    ABSTRACT: Peroxisome proliferator-activated receptor alpha (PPARalpha) is a member of the nuclear receptor family, regulating fatty acid degradation in many organs. Two-dimensional SDS-PAGE of brown adipose tissue (BAT) from PPARalpha-null mice produced a higher-density spot. Proteomic analysis indicated that the protein was pyruvate dehydrogenase beta (PDHbeta). To observe PDHbeta regulation in BAT, the organ was stimulated by long-term cold exposure, and the activities of associated enzymes were investigated. Histological and biochemical analyses of BAT showed a significant decrease in the triglyceride content in wild-type mice and some degree of decrease in PPARalpha-null mice on cold exposure. Analyses of molecules related to glucose metabolism showed that the expression of PDHbeta is under PPARalpha-specific regulation, and that glucose degradation ability may decrease on cold exposure. In contrast, analyses of molecules related to fatty acid metabolism showed that numerous PPARalpha/gamma target molecules are induced on cold exposure, and that fatty acid degradation ability in wild-type mice is markedly enhanced and also increases to same degree in PPARalpha-null mice on cold exposure. Thus, this study proposes novel and multiple roles of PPARalpha in BAT.
    Genes to Cells 12/2009; 15(2):91-100. DOI:10.1111/j.1365-2443.2009.01368.x · 2.86 Impact Factor
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    • "Under these conditions, up-regulation of UCP1 is well known to enable effective thermogenesis in BAT. As mentioned above, our previous studies indicated that certain genes encoding metabolic proteins, especially their subtypes known to be significantly expressed in heart/skeletal muscle, are up-regulated in the BAT of cold-exposed animals [7] [8]. Essentially the same conclusion as ours was also obtained by Adams et al. [9]. "
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    ABSTRACT: To identify genes whose expression in brown adipose tissue (BAT) is up- or down-regulated in cold-exposed rats, we performed microarray analysis of RNA samples prepared from the BAT of cold-exposed rats and of rats kept at room temperature. Previously reported elevations of transcript levels of uncoupling protein (UCP1), type II iodothyronine deiodinase (DIO2), and type III adenylate cyclase (AC3) in the BAT of cold-exposed rats over those in that of rats maintained at room temperature were confirmed. In addition to these changes, remarkable elevations of the transcript levels of several genes that seemed to be associated with the processes of cell-cycle regulation and DNA replication were detected in the BAT of cold-exposed rats, possibly reflecting the significant proliferation of brown adipocytes in response to cold exposure. Up-regulation of the gene encoding sarcomeric mitochondrial type creatine kinase in the BAT of cold-exposed rats was also detected by microarray analysis, but subsequent Northern analysis revealed that the expression of not only the sarcomeric mitochondrial type enzyme, but also that of 2 other subtypes, i.e., cytoplasmic brain type and cytoplasmic muscle type, was elevated in the BAT of cold-exposed rats. Microarray analysis also revealed a significant expression of myoglobin in BAT and its elevation in the BAT of cold-exposed rats, and this result was supported by calibrated Northern analysis. On the contrary, several genes such as regulator of G-protein signaling 2 and IMP dehydrogenase 1 were down-regulated in the BAT of cold-exposed rats. The physiological meaning of these changes accompanying cold exposure was discussed.
    Biochimica et Biophysica Acta 02/2008; 1777(1):104-12. DOI:10.1016/j.bbabio.2007.10.014 · 4.66 Impact Factor
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    ABSTRACT: Im ersten Teil der vorliegenden Arbeit wurde eine mögliche Funktion der mitochondrialen Transportproteine UCP2, UCP3 und UCPx als Protonentransporter bzw. Entkopplerproteine untersucht. Für diese Analysen wurde ein Testsystem etabliert, mit dem ein möglicher Einfluss der Entkopplerproteine auf die Atmung von HEK293-Zellen nach transienter Expression nachgewiesen werden konnte. Da UCP1 das bislang einzige funktionell charakterisierte Mitglied der Entkopplerproteinfamilie ist, wurde das Messverfahren zunächst mit UCP1 transfizierten HEK293-Zellen validiert. Die Expression und Inkorporation von UCP1 und UCP3 in die Mitochondrien von HEK293-Zellen wurde mittels Western-Blots bestätigt. Die Sauerstoffverbrauchsmessungen haben gezeigt, dass die UCP1 transfizierte Zellen nach Inhibition der ATP-Synthase durch Oligomycin eine geringere Kopplung der Atmung zeigten, als die Kontrollzellen. Nach Palmitatzugabe erhöhte sich die Atmungsrate der UCP1-Zellen signifikant. Diese fettsäureabhängige Aktivierung bestätigte die Expression von funktionsfähigem UCP1. Bei den Kontrollen erfolgte keine Steigerung der Atmung nach Fettsäurezugabe. Zellen die mit UCP2, UCP3 und UCPx transfizierten wurden, zeigten weder eine erhöhte entkoppelte Atmung in Anwesenheit von Oligomycin, noch eine durch Fettsäuren induzierte Entkopplung der Atmung. Eine mit UCP1 vergleichbare Entkopplerfunktion von UCP2, UCP3 und UCPx konnte damit nicht bestätigt werden. Des Weiteren wurde ein vermuteter regulatorischer bzw. inhibitorischer Einfluss des mitochondrialen Folat-Carriers SOUP auf die UCP vermittelte Entkopplung, nach Koexpression mit UCPx und UCP1 in HEK293-Zellen geprüft. Die Koexpression von SOUP mit UCPx oder UCP1 hatte allerdings keinen Einfluss auf die mitochondriale Atmung der Zellen bzw. auf die UCP1 vermittelte Entkopplung. Im zweiten Teil dieser Arbeit wurde die mögliche Bedeutung von UCP3 als Regulator des mitochondrialen Fettsäurestoffwechsels in Dsungarischen Zwerghamstern untersucht, die aufgrund einer bislang unbekannten Mutation ein gewebespezifisches UCP3 Defizit im braunen Fettgewebe besitzen. Für die Analyse wurden Wildtyphamster und Mutanten sieben Tage kälteexponiert, 48 h gefastet oder unter Kontrollbedingungen bei 23°C mit ad libitum Futter gehalten. Von Wildtypen und Mutanten wurde die Genexpression von UCP3, sowie von Schlüsselenzymen des Fettsäurestoffwechsels analysiert, um Hinweise auf eine mögliche Störung des Fettsäurestoffwechsels bei den Mutanten zu erhalten. Analysiert wurde die mRNA-Expression der mitochondrialen Thioesterase-I (MTE-I) und der Carnitin-Palmitoyltransferase-I (CPT-I). Im braunen Fettgewebe von Mutanten konnte weder die UCP3 mRNA, noch das UCP3-Protein mittels Northern bzw Western Blots nachgewiesen werden. Im braunen Fettgewebe von Wildtypen waren keine Unterschiede im mRNA-Spiegel zwischen Kontrollen, gefasteten und kaltakklimatisierten Hamstern feststellbar. Der Proteingehalt bei kälteexponierten Wildtyptieren war allerdings 3-fach höher und bei den gefasteten Tieren tendenziell erniedrigt gegenüber den Kontrollen. Die Analyse mRNA-Expression der MTE-I in Wildtyphamstern und Mutanten hat gezeigt, dass offenbar keine gekoppelte Regulation der Expression der MTE-I und UCP3 im braunen Fettgewebe erfolgt, womit offensichtlich keine direkte funktionelle Kopplung zwischen beiden Proteinen besteht. Es konnte jedoch feststellt werden, dass die Regulation von UCP3 und der MTE-I offenbar abhängig vom Fettsäurestoffwechsel im Gewebe erfolgt. Die mRNA-Expression der CPT-I mRNA im braunen Fettgewebe der Wildtypen und Mutanten war vergleichbar. Demzufolge gab es keine Anzeichen für eine veränderte Regulation oder eine Inhibition des Fettsäureimports in die Mitochondrien der Mutanten. Die Fettsäureoxidationskapazität des braunen Fettgewebes von gefasteten, kaltakklimatisierten und Kontroll-Wildtyphamstern und Mutanten wurde in Gewebeproben in vitro anhand der CO2-Produktion mit Oleat als Substrat ermittelt. Zudem wurde die Fettsäureoxidationskapazität von isolierten Braunfettmitochondrien aus kälteexponierten Wildtyphamstern und Mutanten durch Messung des O2-Verbrauchs in Anwesenheit von Palmitoyl-Carnitin als Substrat bestimmt. Diese Messungen haben ergeben, dass die mitochondriale Fettsäureoxidationskapazität des braunen Fettgewebes offensichtlich nicht durch das Fehlen von UCP3 beeinträchtigt wird. Auch die Messung des Fettsäurestoffwechsels in isolierten Mitochondrien von Wildtypen und Mutanten hat keine eindeutigen Hinweise auf eine Störung der mitochondrialen beta-Oxidation durch das UCP3 Defizit ergeben. Zusammenfassend kann man sagen, dass anhand der in dieser Arbeit durchgeführten Genexpressionsstudien und in vitro Messungen des Fettsäurestoffwechsels, kein direkter funktioneller Zusammenhang oder ein regulativer Einfluss von UCP3 auf den Fettsäurestoffwechsel bestätigt werden kann. In the first part of the present work a possible function of UCP2, UCP3 and UCPx, as proton transporters or uncoupling proteins were analysed. For the analysis a new assay was set up, to show a possible influence of transient expressed uncoupling proteins on mitochondrial respiration of HEK293 cells. Because UCP1 is up to now the only funcional characterized memeber of the protein family, the assay was tested with UCP1 transfected HEK293 cells. The expression and incorporation of UCP1 and UCP3 into the mitochondria were proved by western blot analysis. The measurement of O2 consumption showed less coupling of respiration in UCP1 transfected cells compared to control cells after inhibition of the ATP synthase with oligomycin. After addition of the fatty acid palmitate the respiration of UCP1 transfected cells were significantly increased, whereas control cells showed no increase of respiration in presence of fatty acids. By this the expression of functional UCP1 has been confirmed. UCP2, UCP3 and UCPx transfected cells neither showed an increased of uncoupled respiration in presence of oligomycin nor could be activated by addition of palmitate. A with UCP1 comparable uncoupling function of UCP2, UCP3 and UCPx could not be confirmed. Additionally a possible regulative or inhibitory influence of the mitochondrial folate transporter SOUP on UCP induced uncoupling were investigated after coexpression of SOUP with either UCPx or UCP1. Coexpression of SOUP and UCPx did not effect the mitochondrial respiration. Also the coexpression of SOUP and UCP1 did not influence UCP1 induced uncoupled respiration. In the second part of the present work a possible regulative function of UCP3 in fatty acid metabolism was analysed in hamsters which show a tissue specific lack of UCP3 in brown adipose tissue due to an unknown mutation. For the analysis wildtype and mutant hamsters were fasted for 48 h, exposed to 5°C for seven days or kept at 23°C with food ad libitum (control group). Geneexpression of UCP3 and keyenzymes of fatty acid metabolism were analysed in wildtype and mutant hamsters to confirm a possible impairement of fatty acid metabolism due to the lack of UCP3. Beyond UCP3 the mRNA expression of the mitochondrial thioesterase I (MTE-I) and the carnitine palmitoyl transferase I (CPT-I) were analysed. In brown adipose tissue of mutant hamsters no UCP3 mRNA or protein were detectable by northern and western blot analysis. In wildtype hamsters the UCP3 mRNA expression in brown adipose tissue were similar in fasted, cold exposed and control animals. The UCP3 protein content in cold exposed hamsters showed a 3-fold increase, in fasted animals protein content were slightly deacreased compared to controls. Analysis of MTE-I mRNA expression in wildtypes and mutants revealed no coupled regulation of MTE-I and UCP3 expression in brown adipose tissue. Therefore both proteins are not functionally coupled, but their expression in this tissue depends obviously on fatty acid metabolism. Expression of CPT-I mRNA in brown adipose tissue of wildtype hamsters and mutants was similar. Therefore there seemst to be no impairement of regulation or an inhibition of fatty acid import into mitochondria of mutant hamsters. The capacity of fatty acid oxidation in brown adipose tissue of fasted, cold exposed or control wildtype and mutant hamsters were compared by measuring the CO2 production of tissue samples in vitro with oleate as substrate. Additionally β-oxidation were analysed by measuring the O2 consumption of isolated mitochondria of cold exposed animals of both phenotypes with palmitoyl carnitine as substrate. All measurements revealed no impairement of fatty acid oxidation due to the lack of UCP3 neither in tissue samples nor in isolated mitochondria. In conclusion, according to gene expression studies and in vitro measurements of fatty acid oxidation, no direct functional relation or regulative influence of UCP3 in fatty acid metabolism could be confirmed.
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