PHOSPHOFRUCTOKINASE ISOZYMES IN PANCREATIC-ISLETS AND CLONAL BETA-CELLS (INS-1)

Normal insulin secretion is oscillatory in vivo, and the oscillations are impaired in type II diabetes, We and others have shown oscillation s in insulin secretion from isolated perifused islets stimulated with glucose, and in this study we show oscillations in insulin secretion f rom the glucose-sensitive clonal beta-cell line INS-1, We have propose d that the oscillatory insulin secretion may be caused by spontaneous oscillations of glycolysis and the ATP:ADP ratio in the beta-cell, ana logous to those seen in glycolyzing muscle extracts, The mechanism of the latter involves autocatalytic activation of the key regulatory enz yme, phosphofructokinase (PFK), by its product fructose 1,6-bisphospha te (F16BP), However, of the three PFK subunit isoforms (M- [muscle], L - [liver], and C-type, predominant in fibroblasts), only M-type is act ivated by micromolar F16BP at near-physiological conditions, We theref ore studied PFK isoforms in the beta-cell, Western analysis of PFK sub units in isolated rat islets and INS-1 cells showed the presence of M- type, as well as C-type and perhaps lesser amounts of L-type, Kinetic studies of PFK activity in INS-I cell extracts showed strong activatio n by micromolar concentrations of F16BP at near-physiological concentr ations of ATP (several millimolar) and AMP and fructose 6-phosphate (m icromolar), indicative of the M-type isoform, Activation by submicromo lar concentrations of fructose 2,6-bisphosphate (F26BP) and potent inh ibition by citrate were also observed, The F16BP-stimulatable activity was about one-half of the F26BP-stimulatable activity, These experime nts demonstrate that beta-cells contain tire M-type isoform of PFK wit h the requisite regulatory properties for generating glycolytic oscill ations that may be the basis of oscillatory insulin secretion.