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Tu-P7:4 A new human adipocyte cell system to study energy metabolism
185
Tuesday, June 20, 2006: Poster Session P7
BASIC SCIENCE
(lst PART)
CHRONIC CENTRAL O V E R E X P R E S S I O N OF LEPTIN ELEVATES B L O O D PRESSURE DESPITE E X T R E M E HYPOLEPTINEMIA
adipose tissue. These genes seems to have general importance in the development of obesity, because these high expressions were observed in both of the obese strains, SHR-SP(fa+/+) and ZF.
~
N. Turner 1'2, M.K. Matheny 2 , P.J. Scaxpace 2 . 1GRECC, VA Medical Center;
Gainesville, USA: 2Dept. Pharmacology, Univ. of Florida, Gainesville, USA Objective: Central infusion of leptin elevates mean arterial blood pressure (MAP), even in obese models that axe resistant to the metabolic actions of leptin. In our model, chronic overexpression of hypothalamic leptin induces leptin resistance in the absence of obesity. We investigated if MAP is increased after prolonged elevated central leptin despite resistance to the metabolic actions of leptin. Methods: Recombinate adeno-associated virus encoding leptin (rAAVleptin) or control were administered (icy) to lean F344xBN male rats. Food intake and body weight was assessed daily and MAP by tail cuff was assessed at day 80. Results: rAAV-leptin reduced food intake by 30% by day 5, which gradually returned to control level by day 80. Body weight diminished by 50g, whereas control rats gained weight. We previously determined that such rats axe unresponsive to exogenous central leptin, and hence leptin resistance. At day 80, serum leptin was extremely low (7.99-4-.72 vs. 0.39-4-.08 ng/ml). Previous studies indicated that after 80 days, the rats gradually gain weight despite unabated leptin transgene expression in the hypothalamus. At day 80, MAP was elevated in rAAV-leptin rats (148.5-4-5.3 vs. 126.9+2.2mmHg, P<.001). Conclusions: These data indicate that chronically elevated central leptin increases blood pressure in a model of leptin resistance where peripheral leptin levels are extremely low. These data suggest that only prolonged elevated central leptin is necessary to elevate blood pressure and that this pressor response is not subject to same attenuation observed for metabolic responses. Funding: VA Research, NIH AG026159.
~
ALTERED G E N E E X P R E S S I O N IN A B D O M I N A L A D I P O S E TISSUE OF A N O V E L A N I M A L M O D E L FOR T H E METABOLIC S Y N D R O M E
K. TaJaira 1, T. Ueno 1, N. Fukuda 1 , H. Takagi 1 , M. Mitsumata 2, A. Tsunemi 1 , K. Ikeda 3 , J. Yamamoto 3 , K. Matsumoto 1 , Y. Yamori 4 .
1Department of Medicine, Nihon Universi~ School of Medicine, Itabashi, Tokyo, Japan: 2Department of Pathology, Nihon Universi~ School of Medicine, Itabashi, Tokyo, Japan: 3School of Human Em,irolmlental Science, Mukogawa Women's University, Nishinomiya,Hyogo, Japan: 4WHO Collaborating Center For Researeh On Primatw Prevention of Cardiovascular Diseases, Sakyo-Ku, Kyoto, Japan Objective: Changes in the balance of gene expression in abdominal adipose tissue is thought to play major roles in the determination of phenotypes of the metabolic syndrome. We have established a new congenic rat strain carrying fa mutation of Zucker fatty rat (ZF) in a SHR-SP background (SHR-SP(fa+/fa+)) as an animal model of the metabolic syndrome. In this study, we analyzed gene expression in abdominal adipose tissue in this obese congenic rats. Methods: SHR-SP(fa+/fa+), showed higher blood pressure, plasma lipids and steady-state plasma glucose levels and obesity compared with those in SHR-SP. LDL particle size evaluated by fast-phase liquid chromatography was smaller in the SHR-SP(fa+/+) strain compared with that in SHR-SP. Gene expressions in abdominal adipose tissue were analyzed by Gene Chip and RTPCR method. Results: Significantly, highly expressed genes in abdominal adipose tissue in obese SHR-SP(fa+/+) analysed by Gene Chip compared with those in Wister Kyoto strain were as follows, glutamate receptor, neurotransmitter-induced early gene 4, heat shock protein 70, calcium channel alpha-1 subunit. These genes were also highly expressed in ZF strain and these results were confirmed by RT-PCR method. Conclusion: By the Gene Chip analysis and RT-PCR method, several interesting genes have been found out as highly expressed genes in abdominal
SEX- A N D SITE-SPECIFIC G E N E EXPRESSION IN T H E A D I P O S E TISSUE OF OB/OB MICE
S. Shiozaki 1 , T. Chiba 1 , K. Kokame 2, T. Miyata 2 , M. Ai 1 , E. Kaneko 1 , M. Yoshida I , K. Shimokado I . 1Tokyo Medical attd Dental Universi~
Graduate School, Tokyo, Japan: 2National Cardiovascular Center Researeh Institute, Osaka, Japan Objectives: Estrogen is likely involved in sex differences in adipose tissue distribution, but it is not cleax whether estrogen has site-specific effects. Methods: Gene-expression patterns in adipose tissue of ob/ob mice (10 weeks) of both sexes and of male mice injected with estradiol (51~g/animal, twice) were analyzed with an Affymetrix Genechip. Expression patterns were confirmed by real-time PCR for selected genes. Results: A m o n g the 12,488 genes in the array, about 6000 were expressed in adipose tissue. There were significant differences in gene-expression patterns between the subcutaneous and visceral adipose tissue, and between male and female mice. Estradiol injection in male mice changed the expression of some genes toward a female pattern, while the expression of other genes was not affected or changed in the opposite direction. Conclusions: Estradiol changed the gene-expression pattern in a sitespecific manner, and therefore may control the development of the sexdependent distribution of adipose tissue. Funding: Special Coordination Funds from the Ministry of Education, Science, Technology, and Culture of Japan.
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A N E W H U M A N A D I P O C Y T E CELL S Y S T E M TO STUDY E N E R G Y M E T A B O L I S M
J. Prawitt 1 , A. Niemeier 1 , M. Kassem 2, U. Beisiegel 1 , J. Heeren 1 . IlBMII: Molecular Cell Biology, Universi~ Medical Center Hamburg-Eppendotf Hamburg, Germany: 2Depat'tment of Endocrinology, Universi~ Hospital Odense, Odense, Denn~trk Objective: Only very few adequate cell models are available to study human adipocyte metabolism. The aim of this project is to establish a human adipocyte cell culture system which will allow us to analyse lipid metabolism with regard to insulin responsiveness. Methods: We have generated this cell model by differentiation of an immortalised human mesenchymal stem cell line (Simonsen et al 2002). Adipocyte morphology was verified by Oil Red O staining and the detection of adipocyte markers by RT-PCR, quantitative PCR and Western Blot. The effect of insulin on the cells was tested by analysis of distinct phosphorylated signaling proteins and uptake of radioactively labeled glucose. Results: After 14 to 21 days of differentiation the cells show an advanced accumulation of monoloculax lipid droplets consisting predominantly of triglycerides. These adipocytes express a typical pattern of adipocyte markers and proteins of lipid metabolism on mRNA and protein level (e.g. A D D - l , PPARgamma, LRP1, ApoE). First quantitative PCR data reveal a m a x i m u m in expression of the insulin-dependent Glut4 around day 14 while the expression of Glutl increases subsequently during differentiation. In response to insulin, phosphorylation of Akt was observed while no insulin-dependent uptake of glucose was measured at day 21. Conclusion: We established a human adipocyte cell system which exhibits characteristics of mature human adipocytes. The insulin-mediated phosphorylation of Akt is not reflected in insulin-dependent uptake of glucose which might be explained by the expression pattern of the glucose receptors. Funding: Government Hamburg
XIV bztetTtational Symposium on Atherosclerosis, Rome, Italy, June 18-22, 2006
Tuesday, June 20, 2006: Poster Session P7
BASIC SCIENCE
(lst PART)
CHRONIC CENTRAL O V E R E X P R E S S I O N OF LEPTIN ELEVATES B L O O D PRESSURE DESPITE E X T R E M E HYPOLEPTINEMIA
adipose tissue. These genes seems to have general importance in the development of obesity, because these high expressions were observed in both of the obese strains, SHR-SP(fa+/+) and ZF.
~
N. Turner 1'2, M.K. Matheny 2 , P.J. Scaxpace 2 . 1GRECC, VA Medical Center;
Gainesville, USA: 2Dept. Pharmacology, Univ. of Florida, Gainesville, USA Objective: Central infusion of leptin elevates mean arterial blood pressure (MAP), even in obese models that axe resistant to the metabolic actions of leptin. In our model, chronic overexpression of hypothalamic leptin induces leptin resistance in the absence of obesity. We investigated if MAP is increased after prolonged elevated central leptin despite resistance to the metabolic actions of leptin. Methods: Recombinate adeno-associated virus encoding leptin (rAAVleptin) or control were administered (icy) to lean F344xBN male rats. Food intake and body weight was assessed daily and MAP by tail cuff was assessed at day 80. Results: rAAV-leptin reduced food intake by 30% by day 5, which gradually returned to control level by day 80. Body weight diminished by 50g, whereas control rats gained weight. We previously determined that such rats axe unresponsive to exogenous central leptin, and hence leptin resistance. At day 80, serum leptin was extremely low (7.99-4-.72 vs. 0.39-4-.08 ng/ml). Previous studies indicated that after 80 days, the rats gradually gain weight despite unabated leptin transgene expression in the hypothalamus. At day 80, MAP was elevated in rAAV-leptin rats (148.5-4-5.3 vs. 126.9+2.2mmHg, P<.001). Conclusions: These data indicate that chronically elevated central leptin increases blood pressure in a model of leptin resistance where peripheral leptin levels are extremely low. These data suggest that only prolonged elevated central leptin is necessary to elevate blood pressure and that this pressor response is not subject to same attenuation observed for metabolic responses. Funding: VA Research, NIH AG026159.
~
ALTERED G E N E E X P R E S S I O N IN A B D O M I N A L A D I P O S E TISSUE OF A N O V E L A N I M A L M O D E L FOR T H E METABOLIC S Y N D R O M E
K. TaJaira 1, T. Ueno 1, N. Fukuda 1 , H. Takagi 1 , M. Mitsumata 2, A. Tsunemi 1 , K. Ikeda 3 , J. Yamamoto 3 , K. Matsumoto 1 , Y. Yamori 4 .
1Department of Medicine, Nihon Universi~ School of Medicine, Itabashi, Tokyo, Japan: 2Department of Pathology, Nihon Universi~ School of Medicine, Itabashi, Tokyo, Japan: 3School of Human Em,irolmlental Science, Mukogawa Women's University, Nishinomiya,Hyogo, Japan: 4WHO Collaborating Center For Researeh On Primatw Prevention of Cardiovascular Diseases, Sakyo-Ku, Kyoto, Japan Objective: Changes in the balance of gene expression in abdominal adipose tissue is thought to play major roles in the determination of phenotypes of the metabolic syndrome. We have established a new congenic rat strain carrying fa mutation of Zucker fatty rat (ZF) in a SHR-SP background (SHR-SP(fa+/fa+)) as an animal model of the metabolic syndrome. In this study, we analyzed gene expression in abdominal adipose tissue in this obese congenic rats. Methods: SHR-SP(fa+/fa+), showed higher blood pressure, plasma lipids and steady-state plasma glucose levels and obesity compared with those in SHR-SP. LDL particle size evaluated by fast-phase liquid chromatography was smaller in the SHR-SP(fa+/+) strain compared with that in SHR-SP. Gene expressions in abdominal adipose tissue were analyzed by Gene Chip and RTPCR method. Results: Significantly, highly expressed genes in abdominal adipose tissue in obese SHR-SP(fa+/+) analysed by Gene Chip compared with those in Wister Kyoto strain were as follows, glutamate receptor, neurotransmitter-induced early gene 4, heat shock protein 70, calcium channel alpha-1 subunit. These genes were also highly expressed in ZF strain and these results were confirmed by RT-PCR method. Conclusion: By the Gene Chip analysis and RT-PCR method, several interesting genes have been found out as highly expressed genes in abdominal
SEX- A N D SITE-SPECIFIC G E N E EXPRESSION IN T H E A D I P O S E TISSUE OF OB/OB MICE
S. Shiozaki 1 , T. Chiba 1 , K. Kokame 2, T. Miyata 2 , M. Ai 1 , E. Kaneko 1 , M. Yoshida I , K. Shimokado I . 1Tokyo Medical attd Dental Universi~
Graduate School, Tokyo, Japan: 2National Cardiovascular Center Researeh Institute, Osaka, Japan Objectives: Estrogen is likely involved in sex differences in adipose tissue distribution, but it is not cleax whether estrogen has site-specific effects. Methods: Gene-expression patterns in adipose tissue of ob/ob mice (10 weeks) of both sexes and of male mice injected with estradiol (51~g/animal, twice) were analyzed with an Affymetrix Genechip. Expression patterns were confirmed by real-time PCR for selected genes. Results: A m o n g the 12,488 genes in the array, about 6000 were expressed in adipose tissue. There were significant differences in gene-expression patterns between the subcutaneous and visceral adipose tissue, and between male and female mice. Estradiol injection in male mice changed the expression of some genes toward a female pattern, while the expression of other genes was not affected or changed in the opposite direction. Conclusions: Estradiol changed the gene-expression pattern in a sitespecific manner, and therefore may control the development of the sexdependent distribution of adipose tissue. Funding: Special Coordination Funds from the Ministry of Education, Science, Technology, and Culture of Japan.
~
A N E W H U M A N A D I P O C Y T E CELL S Y S T E M TO STUDY E N E R G Y M E T A B O L I S M
J. Prawitt 1 , A. Niemeier 1 , M. Kassem 2, U. Beisiegel 1 , J. Heeren 1 . IlBMII: Molecular Cell Biology, Universi~ Medical Center Hamburg-Eppendotf Hamburg, Germany: 2Depat'tment of Endocrinology, Universi~ Hospital Odense, Odense, Denn~trk Objective: Only very few adequate cell models are available to study human adipocyte metabolism. The aim of this project is to establish a human adipocyte cell culture system which will allow us to analyse lipid metabolism with regard to insulin responsiveness. Methods: We have generated this cell model by differentiation of an immortalised human mesenchymal stem cell line (Simonsen et al 2002). Adipocyte morphology was verified by Oil Red O staining and the detection of adipocyte markers by RT-PCR, quantitative PCR and Western Blot. The effect of insulin on the cells was tested by analysis of distinct phosphorylated signaling proteins and uptake of radioactively labeled glucose. Results: After 14 to 21 days of differentiation the cells show an advanced accumulation of monoloculax lipid droplets consisting predominantly of triglycerides. These adipocytes express a typical pattern of adipocyte markers and proteins of lipid metabolism on mRNA and protein level (e.g. A D D - l , PPARgamma, LRP1, ApoE). First quantitative PCR data reveal a m a x i m u m in expression of the insulin-dependent Glut4 around day 14 while the expression of Glutl increases subsequently during differentiation. In response to insulin, phosphorylation of Akt was observed while no insulin-dependent uptake of glucose was measured at day 21. Conclusion: We established a human adipocyte cell system which exhibits characteristics of mature human adipocytes. The insulin-mediated phosphorylation of Akt is not reflected in insulin-dependent uptake of glucose which might be explained by the expression pattern of the glucose receptors. Funding: Government Hamburg
XIV bztetTtational Symposium on Atherosclerosis, Rome, Italy, June 18-22, 2006