KITAJIMA Koichi Osaka University Graduate School of Medicine, Assistant Professor, 医学系研究科, 助手 (00314310)
HANAFUSA Toshiaki Osaka University Graduate School of Medicine, Lecturer, 医学系研究科, 講師 (60164886)
YAMASAKI Tomoyuki Osaka University Medical School, Assistant Professor, 医学部, 助手 (00303975)
浜口 朋也 大阪大学, 医学部・附属病院, 医員
HAMAGUCHI Tomoya Osaka University Hospital, Medical Staff
|Budget Amount *help
¥3,100,000 (Direct Cost : ¥3,100,000)
Fiscal Year 1999 : ¥1,400,000 (Direct Cost : ¥1,400,000)
Fiscal Year 1998 : ¥1,700,000 (Direct Cost : ¥1,700,000)
In the pancreatic beta cells, the mode of insulin secretion in response to glucose is known as biphasic. In the first phase of insulin secretion, glucokinase (GK) acts as a glucose sensor, regulating the glucose flux and ATP synthesis rate. But if we try to explain the biphasic profile of insulin secretion through the metabolic consideration, only the nonallosteric GK component is not sufficient for a biphasic mode of reaction.
There are three distinct isozymes in phosphofructokinase (PFK), the other key regulatory enzyme of glycolysis in the liver, as well as in the beta cell. We have shown that expression of all three isozymes of L (liver form), M (muscle form) and P (platelet, brain or fibroblast form) is seen in the pancreatic tissue. Among these, the major isozymes in the beta cells are L and M forms. Both isozymes of PFK are well-known typical allosteric enzymes. To note, the attitue of allosteric inhibition by ATP and citrate is more steep in PFK-M than in PFK-L. Activation by AD
P/AMP is stronger in PFK-M. The first stage of glucose entry to beta cells would be characterized by low ATP/ADP ratio and low citrate levels. Then PFK-M dominantly works the intermediate glycolytic step. When the steady state glucose uptake and phosphorylation are established and intracellular contents of ATP and citrate are increased, allosteric inhibition affects more on PFK-M and glycolysis will go on mainly by PFK-L. The latter phenomenon may responsible for characterizing the second phase of insulin secretory response in view from the metabolic consideration.
To evaluate and examine this hypothesis, we are trying to knockout the specific PFK isozyme in the beta cells.
We have established in this research process, the precise analytical methods applicable to living cells for the determination of intracellular calcium ion, proton and ATP concentrations by using the real-time cell-imaging analyzer, confocal laser microscopy, and luciferase overexpression system, respectively.
We are now establishing the cellular knockout of PFK isozymes in MIN6 cells by the stable transformation of antisense RNA expression system. Transient overexpression of antisense vector is also prepared by the adenoviral system. The latter is aimed to evaluate the hypothesis using the isolated islets and pancreas organ perfusion.
During this research period, establishment of the specific knockout system and the cytoanalytical system have been prepared. These will be used in the following study periods and the forthcoming results will give further rationale for establishing isozyme specific knockout mice to determine the whole body effect of beta cell PFK isozymes.