Uptake of glucose in mammalian cells occurs by facilitated diffusion by glucose transporters. Conversion of glucose to glucose-6-phosphate (G-6-P) catalyzed by hexokinases plays a critical role by maintaining downhill gradient necessary for glucose entry into cells. The major focus of the proposal is on hexoskinase II (HKII) which, like HKI and HKIII is a 100 kD isoform that is inhibited by physiologic concentrations of G-6-P. HKII is selectively found in adipose tissue, skeletal muscle and heart, tissues in which insulin stimulates glucose uptake and utilization. Studies have shown that insulin increase HKII activity in these tissues. The recent cloning of the HKII cDNA and gene make it possible to study structure and regulation of the gene. The ratio of glucose transporters and HKs in a cell can be manipulated so that relative contributions of each glucose uptake and metabolism can be studied.
Aim 1 will establish whether glucagon (cAMP), glucocorticoids and insulin modulate HKII gene transcription. If so, the respective hormone-response elements will be mapped. Transgenic mice will be used to determine the promoter elements responsible for the unique tissue distribution of HKII.
Aim 2 will use specific mutations to locate the glucose, ATP and G-6-P binding domains. The HKII, the N- and C-halves will be expressed in E. coli and compared for function with HKI and gulucokinae (GK). Whether catalytic and regulatory domains co-exist in each half of HKII or separate halves will be determined. The ultimate goal is to provide a crystallographic analysis.
Aim 3 is to define the kinetics of expressed HKs, and their interactions with selected transporter isoforms in frog oocytes. This approach will determine how HKs control metabolic flux and whether compartmentation is involved in the process.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
1R01DK046867-01
Application #
3248217
Study Section
Endocrinology Study Section (END)
Project Start
1993-09-30
Project End
1997-08-31
Budget Start
1993-09-30
Budget End
1994-08-31
Support Year
1
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Vanderbilt University Medical Center
Department
Type
Schools of Medicine
DUNS #
004413456
City
Nashville
State
TN
Country
United States
Zip Code
37212
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Whitesell, Richard R; Ardehali, Hossein; Printz, Richard L et al. (2003) Control of glucose phosphorylation in L6 myotubes by compartmentalization, hexokinase, and glucose transport. Biochem J 370:47-56
Perriott, L M; Kono, T; Whitesell, R R et al. (2001) Glucose uptake and metabolism by cultured human skeletal muscle cells: rate-limiting steps. Am J Physiol Endocrinol Metab 281:E72-80
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Vogt, C; Ardehali, H; Iozzo, P et al. (2000) Regulation of hexokinase II expression in human skeletal muscle in vivo. Metabolism 49:814-8
Halseth, A E; O'Doherty, R M; Printz, R L et al. (2000) Role of Ca(2+) fluctuations in L6 myotubes in the regulation of the hexokinase II gene. J Appl Physiol 88:669-73
Ardehali, H; Printz, R L; Whitesell, R R et al. (1999) Functional interaction between the N- and C-terminal halves of human hexokinase II. J Biol Chem 274:15986-9
Yamada, K; Printz, R L; Osawa, H et al. (1999) Human ZHX1: cloning, chromosomal location, and interaction with transcription factor NF-Y. Biochem Biophys Res Commun 261:614-21
Yamada, K; Osawa, H; Granner, D K (1999) Identification of proteins that interact with NF-YA. FEBS Lett 460:41-5
Printz, R L; Osawa, H; Ardehali, H et al. (1997) Hexokinase II gene: structure, regulation and promoter organization. Biochem Soc Trans 25:107-12

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