Phosphofructokinase Isozymes in Pancreatic Islets and Clonal β-Cells (INS-1)

Abstract

Normal insulin secretion is oscillatory in vivo, and the oscillations are impaired in type II diabetes. We and others have shown oscillations in insulin secretion from isolated perifused islets stimulated with glucose, and in this study we show oscillations in insulin secretion from the glucose-sensitive clonal beta-cell line INS-1. We have proposed that the oscillatory insulin secretion may be caused by spontaneous oscillations of glycolysis and the ATP:ADP ratio in the beta-cell, analogous to those seen in glycolyzing muscle extracts. The mechanism of the latter involves autocatalytic activation of the key regulatory enzyme, phosphofructokinase (PFK), by its product fructose 1,6-bisphosphate (F16BP). However, of the three PFK subunit isoforms (M-[muscle], L-[liver], and C-type, predominant in fibroblasts), only M-type is activated by micromolar F16BP at near-physiological conditions. We therefore studied PFK isoforms in the beta-cell. Western analysis of PFK subunits 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-1 cell extracts showed strong activation by micromolar concentrations of F16BP at near-physiological concentrations of ATP (several millimolar) and AMP and fructose 6-phosphate (micromolar), indicative of the M-type isoform. Activation by submicromolar concentrations of fructose 2,6-bisphosphate (F26BP) and potent inhibition by citrate were also observed. The F16BP-stimulatable activity was about one-half of the F26BP-stimulatable activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolic Oscillations in β-Cells

“…The burst is terminated once the supply of high-energy phosphate donors is exhausted, allowing the ATP/ADP ratio to decay (83). Supporting the idea that glycolysis may oscillate is the presence of PFK-M, the PFK isoform with known oscillatory behavior, in islets (84). Interestingly, mutations in this enzyme are associated with impairment of oscillatory insulin release in humans (85).…”

Fatty acid metabolism and insulin secretion in pancreatic beta cells

“…These oscillations are driven by autocatalytic activation of the muscle isoform of the key glycolytic enzyme phosphofructokinase-1 (PFK-1) by its product fructose 1,6-bisphosphate resulting in large oscillations in the ATP:ADP ratio. The muscle isoform of this enzyme, PFK-M, has recently been shown to be the dominant PFK activity in beta cells [61].…”

Deficiency of phosphofructo-1-kinase/muscle subtype in humans impairs insulin secretion and causes insulin resistance.

“…Concerning insulin secretion of the pancreatic ␤ cell (17), a coincidence between reduced PFK1 activity and impaired insulin secretion has been described in vitro (18,19), but was explained by primary phosphate depletion and consequently impaired secretion in the particular experimental setup used. Recently, Yaney and coworkers (20) demonstrated the presence of all three isotypes of PFK1 in rat-derived ␤ cell lines and islets at the protein level. In an extensive investigation of the complex regulation of the enzyme, negative by both ATP and citrate, and autocatalytically positive by AMP-dependent activation by fructose-1,6-bisphosphate, they concluded that PFK1-M contributed most of the activity under physiological intracellular conditions.…”