Distinct functional roles of the two terminal halves of eukaryotic phosphofructokinase.

Eukaryotic PFK (phosphofructokinase), a key regulatory enzyme in glycolysis, has homologous N- and C-terminal domains thought to result from duplication, fusion and divergence of an ancestral prokaryotic gene. It has been suggested that both the active site and the Fru-2,6-P2 (fructose 2,6-bisphosphate) allosteric site are formed by opposing N- and C-termini of subunits orientated antiparallel in a dimer. In contrast, we show in the present study that in fact the N-terminal halves form the active site, since expression of the N-terminal half of the enzymes from Dictyostelium discoideum and human muscle in PFK-deficient yeast restored growth on glucose.

Isolation of novel animal cell lines defective in glycerolipid biosynthesis reveals mutations in glucose-6-phosphate isomerase.

Glycerolipids are structural components for membranes and serve in energy storage. We describe here the use of a photodynamic selection technique to generate a population of Chinese hamster ovary cells that display a global deficiency in glycerolipid biosynthesis. One isolate from this population, GroD1, displayed a profound reduction in the synthesis of phosphatidylcholine, phosphatidylethanolamine, and triglycerides but presented high levels of phosphatidic acid and normal levels of phosphatidylinositol synthesis.

Mice deficient in phosphofructokinase-M have greatly decreased fat stores.

Synthesis of triacylglycerol requires the glucose-derived glycerol component, and glucose uptake has been viewed as the rate-limiting step in glucose metabolism in adipocytes. Furthermore, adipose tissue contains all three isoforms of the glycolytic enzyme phosphofructokinase (PFK). We here report that mice deficient in the muscle isoform PFK-M have greatly reduced fat stores.

Tissue-dependent loss of phosphofructokinase-M in mice with interrupted activity of the distal promoter: impairment in insulin secretion.

Phosphofructokinase is a key enzyme of glycolysis that exists as homo- and heterotetramers of three subunit isoforms: muscle, liver, and C type. Mice with a disrupting tag inserted near the distal promoter of the phosphofructokinase-M gene showed tissue-dependent differences in loss of that isoform: 99% in brain and 95-98% in islets, but only 50-75% in skeletal muscle and little if any loss in heart. This correlated with the continued presence of proximal transcripts specifically in muscle tissues.

Ca2+, NAD(P)H and membrane potential changes in pancreatic beta-cells by methyl succinate: comparison with glucose.

The present study was undertaken to determine the main metabolic secretory signals generated by the mitochondrial substrate MeS (methyl succinate) compared with glucose in mouse and rat islets and to understand the differences. Glycolysis and mitochondrial metabolism both have key roles in the stimulation of insulin secretion by glucose. Both fuels elicited comparable oscillatory patterns of Ca2+ and changes in plasma and mitochondrial membrane potential in rat islet cells and clonal pancreatic beta-cells (INS-1).