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GRK 1482 Jahrbuch 2011-2014

Publications [1] Rumessen JJ, Gudmand-Høyer E. Absorption capacity of fructose in healthy adults. Comparison with sucrose and its constituent monosaccharides. Gut. 1986; 27:1161-8. [2] Wasserman D, Hoekstra JH, Tolia V, Taylor CJ, Kirschner BS, Takeda J, Bell GI, Taub R, Rand EB. Molecular ana- lysis of the fructose transporter gene (GLUT5) in isola- ted fructose malabsorption. J Clin Invest. 1996; 98:2398- 402. [4] Barone S, Fussell SL, Singh AK, Lucas F, Xu J, Kim C, Wu X, Yu Y, Amlal H, Seidler U, Zuo J, Soleimani M. Sl c2a5 (Glut5) is essential for the absorption of fructose in the intestine and generation of fructose-induced hyper- tension. J Biol Chem. 2009; 284:5056-66. [4] Manolescu A, Witkowska K, Kinnaird A, Cessford T, Cheeseman C. Facilitated hexose transporters: new per- spectives on form and function. Physiology. 2007, 22:234-40. [5] Debosch BJ, Chi M, Moley KH. Glucose transporter 8 (GLUT8) regulates enterocyte fructose transport and global mammalian fructose utilization. Endocrinology. 2012, 153:4181-91. PhD FELLOWS GRK Progress Report 2011-2014 | Page 51 Aim To unveil the molecular basis of fructose malabsorption, we will perform genetic analysis and functional characterisation of in- testinal fructose transporters. Furthermore, the beneficial effect of equimolar glucose administration will be studied. Methods and Results We will analyse at least 100 patients with fructose malabsorpti- on and 100 subjects with normal fructose breath test by a can- didate gene approach. Additionally, we will recruit at least 300 phenotypically well characterised controls. In these individuals, we will perform fructose and lactose breath test, and in selec- ted cases lactulose or glucose breath test. Since GLUT5 and GLUT7 are strong candidates for fructose malabsorption we will analyse the coding, intronic and pro- moter regions. Moreover, the coding regions of other intestinal fructose transporters will be sequenced. The uptake of 14C-Fructose in GLUT7-injected Xenopus lae- vis oocytes and not-injected oocytes was measured. The ma- ximum fructose uptake in GLUT7-injected oocytes was twice as high as in not-injected oocytes. Because this effect was not as high as desired and not always reproducible we decided to search for another expression system. Since CHO cells show a low fructose uptake because of lacking GLUT5, we transiently transfected these cells with GLUT7 and measured uptake of 14C-fructose. We observed only small and non-reproducible effects. Therefore, we will use retroviral transduction to create NIH-3T3 cells that stably express GLUT7 and GLUT7 variants to compare 14C-fructose uptake. We analysed the coding regions of GLUT5 and GLUT7 in 36 pa- tients with fructose malabsorption, 34 controls with negative fructose breath test and in 60 (GLUT5)/ 250 (GLUT7) population based controls (with unknown fructose absorption state). We found only 1 amino acid changing variant in GLUT5 (p.T14M) in 1/36 patients but not in control subjects. In GLUT7, we detected 5 non-synonymous variants. The variant p.R224C was found in 3/36 patients, 0/34 controls and 3/250 population controls. Noteworthy, the arginine at codon 224 is conserved in all hu- man GLUTs as well as in GLUT7 of different species. The co- ding sequence of GLUT9 was analysed in 40 patients and 30 controls, the 5’UTR additionally in 194 population controls. The 5’UTR variant 4:10042100 G>A was found in 7/40 patients, 1/30 controls and 10/194 population controls. Outlook Further studies in larger patient cohorts will reveal if GLUT5 pro- moter or GLUT7 promoter/coding variants play a role in fruc- tose malabsorption. In addition, we will investigate if genetic alterations in other intestinal fructose transporters of the GLUT family are involved in the pathogenesis of fructose malabsorp- tion. To investigate the functional consequences of the variants found, we will characterise fructose transport activity of mu- tants in stably transfected NIH-3T3 fibroblasts. Hypothetical structure of GLUT7. Amino acid changes found so far are designa- ted. The in vitro amino acid exchange p.302V studied by Manolescu et al. [4] is also depicted Supervisors Prof. Dr. Heiko Witt I TUM I Paediatric Nutritional Medicine Prof. Dr. Hannelore Daniel I TUM | Physiology of Human Nutrition Start of project: September 2011 Academic background: Studies of Nutrition at University of Hohenheim