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Lipogenic enzyme activities and mRNAS in rat adipose tissue during weaning: Role of the diet
CONTRIBUTION OF MOLECULAR BIOLOGY TO STUDIES ON MECHANISMS BROWN ADIPOCYTES.
OF THERMOGENESIS
IN
D. Hicouler, Centre de Hecherche sur la Nutrition. Centre National de la Recherche Scientifique. 9 rue Jules Hetzel. 92 190 Meudon. France. Thermogcnesis in brown adipose tissue results from uncoupled respiration related to a high proton conductance of the inner miiochondrial membrane. This unique proton translocation is due to a characteristic membraneous component, the Uncoupling Protein or UCP. of which the activity is triggered by free fatty acids and inhibited by purine nucleotides. Following acute interaction of norepinephrine with B-adrenoceptors of brown adipocytes. the metabolic cascade results in UCP activation and heat production. In other respects. chronic stimulation of brown adipocytes by norepinephrine induces cell hyperplasia. mitochondrlogenesis and synthesis of various components including UCP. This trophic response is also controlled by T3. We have developed molecular studies on UCP to investigate both the activity of UCP itself and the regulation of UCP gene expression. cDNAs for rat and bovine UCP were isolated. Sequencing of rat UCP cDNA confirmed previous cell-free synthesis experiments showing that synthesized UCP has no N-terminal targeting extension.UCP is made of six hydrophobic segments interpersed with hydrophilic loops. UCP is partially homologous to other ubiquitous mitochondrial carriers such as the ADP/ATP and the phosphate carriers. Diagon analysis identified a domain related to an ADP binding site of the ADP/ATp carrier. In order to understand the structure of UCP and residues or domains involved in proton translocation or regulation by nucleotides. experiments of expression of UCP in E. coli. yeasts and Xenopus oocytes have been undertaken. Hat and human UCP genes have been isolated. The complete exonic/intronic sequence of rat UCP gene has been determined as well as the organization of human gene. A long 4.5 kb fragment of the 5’ upstream region flanking the start site of transcription has been sequenced and shown to contain Nuclease sensitive sites as well as possible regulatory elements. Analysis of promotor and nuclear factors is under progress. Moreover the human gene of UCP has been localiaed in chromosome 4 in q31. Northern blot experiments indicated that UCP mHNA could be strongly induced by cold exposure, B-agonist drug dosing or refeeding of animals after starvation. Genomic probes were used to detect UCP mRNA in human samples. Northern blot experiments in developing calves raised the question of a possible transformation of thermogenic brown adipose tissue into white adipose tissue.
OF PHOSPHATE ACTIVATED GLUTAHINASE ACTIVITY AND mRNAIN PERIPORTAL AND PERIVENOUSHEPATOCYTES. M. Watford and E.M. Smith, Divisions of Nutritional and Biological Sciences, Savage Hall, Cornell University, Ithaca, New York 14853 USA.
DISTRIBUTION
Glutamine is considered an important substrate for hepatic urea synthesis and liver possesses a unique isozyme of phosphate activated glutaminase. Rat liver also has the capacity for glutamine synthesis (glutamine synthetase). These two enzymes appear to be localized in different parts of the liver acinus. Glutamine synthetase (activity, protein, and mRNA) has been shown to be present in a discrete perivenous population of hepatocytes (1.2) and there is functional evidence for a predominantly periportal location of glutaminase (3). We have determined the zonation of hepatic glutaminase in periportal and perivenous hepatocytes isolated by the digitonin perfusion technique. In periportal cells the specific activity of phosphate dependent glutaminase is 2-fold higher than in perivenous cells. Similarly, when Northern blots of RNA isolated from these cells were probed with a cDNA to hepatic glutaminase the mean relative abundance of the mRNAwas 2.6fold higher in samples from periportal cells. We have also confirmed the exclusively perivenous location of glutamine synthetase. These results suggest that phosphate activated glutaminase is predominantly expressed in the periportal zone of the liver acinus. This location is in accord with roles for hepatic glutaminase in providing substrate for the urea cycle and possible involvement in an intercellular glutamine cycle (3). 1. 2. 3.
Gebhardt, R. and Mecke, D. (1983) EMBO J. 2, 567-570 Gebhardt, R., Ebert, A. and Bauer, G. (1988) Febs. Lett. 241, 89-93 Haussinger, D., Gerok, W. and Sies, H. (1984) Trends Biochem. Sci. 9. 300-302
OF THERMOGENESIS
IN
D. Hicouler, Centre de Hecherche sur la Nutrition. Centre National de la Recherche Scientifique. 9 rue Jules Hetzel. 92 190 Meudon. France. Thermogcnesis in brown adipose tissue results from uncoupled respiration related to a high proton conductance of the inner miiochondrial membrane. This unique proton translocation is due to a characteristic membraneous component, the Uncoupling Protein or UCP. of which the activity is triggered by free fatty acids and inhibited by purine nucleotides. Following acute interaction of norepinephrine with B-adrenoceptors of brown adipocytes. the metabolic cascade results in UCP activation and heat production. In other respects. chronic stimulation of brown adipocytes by norepinephrine induces cell hyperplasia. mitochondrlogenesis and synthesis of various components including UCP. This trophic response is also controlled by T3. We have developed molecular studies on UCP to investigate both the activity of UCP itself and the regulation of UCP gene expression. cDNAs for rat and bovine UCP were isolated. Sequencing of rat UCP cDNA confirmed previous cell-free synthesis experiments showing that synthesized UCP has no N-terminal targeting extension.UCP is made of six hydrophobic segments interpersed with hydrophilic loops. UCP is partially homologous to other ubiquitous mitochondrial carriers such as the ADP/ATP and the phosphate carriers. Diagon analysis identified a domain related to an ADP binding site of the ADP/ATp carrier. In order to understand the structure of UCP and residues or domains involved in proton translocation or regulation by nucleotides. experiments of expression of UCP in E. coli. yeasts and Xenopus oocytes have been undertaken. Hat and human UCP genes have been isolated. The complete exonic/intronic sequence of rat UCP gene has been determined as well as the organization of human gene. A long 4.5 kb fragment of the 5’ upstream region flanking the start site of transcription has been sequenced and shown to contain Nuclease sensitive sites as well as possible regulatory elements. Analysis of promotor and nuclear factors is under progress. Moreover the human gene of UCP has been localiaed in chromosome 4 in q31. Northern blot experiments indicated that UCP mHNA could be strongly induced by cold exposure, B-agonist drug dosing or refeeding of animals after starvation. Genomic probes were used to detect UCP mRNA in human samples. Northern blot experiments in developing calves raised the question of a possible transformation of thermogenic brown adipose tissue into white adipose tissue.
OF PHOSPHATE ACTIVATED GLUTAHINASE ACTIVITY AND mRNAIN PERIPORTAL AND PERIVENOUSHEPATOCYTES. M. Watford and E.M. Smith, Divisions of Nutritional and Biological Sciences, Savage Hall, Cornell University, Ithaca, New York 14853 USA.
DISTRIBUTION
Glutamine is considered an important substrate for hepatic urea synthesis and liver possesses a unique isozyme of phosphate activated glutaminase. Rat liver also has the capacity for glutamine synthesis (glutamine synthetase). These two enzymes appear to be localized in different parts of the liver acinus. Glutamine synthetase (activity, protein, and mRNA) has been shown to be present in a discrete perivenous population of hepatocytes (1.2) and there is functional evidence for a predominantly periportal location of glutaminase (3). We have determined the zonation of hepatic glutaminase in periportal and perivenous hepatocytes isolated by the digitonin perfusion technique. In periportal cells the specific activity of phosphate dependent glutaminase is 2-fold higher than in perivenous cells. Similarly, when Northern blots of RNA isolated from these cells were probed with a cDNA to hepatic glutaminase the mean relative abundance of the mRNAwas 2.6fold higher in samples from periportal cells. We have also confirmed the exclusively perivenous location of glutamine synthetase. These results suggest that phosphate activated glutaminase is predominantly expressed in the periportal zone of the liver acinus. This location is in accord with roles for hepatic glutaminase in providing substrate for the urea cycle and possible involvement in an intercellular glutamine cycle (3). 1. 2. 3.
Gebhardt, R. and Mecke, D. (1983) EMBO J. 2, 567-570 Gebhardt, R., Ebert, A. and Bauer, G. (1988) Febs. Lett. 241, 89-93 Haussinger, D., Gerok, W. and Sies, H. (1984) Trends Biochem. Sci. 9. 300-302