Enzymes of carbohydrate and fat metabolism in anti-insulin serum diabetes; inactivation by free fatty acids and the protective effect of cellular protein

Abstract

The possibility that free fatty acids (FFA) and their CoN esters may directly inhibit the activity of enzymes of glycolysis and lipogenesis was studied in liver and adipose tissue of acutely diabetic rats. Despite a marked elevation in tissue FFA, the activity of glucose-6phosphate dehydrogenase, phospbofructokinase, pyruvate kinase, u-glycerophosphate dehydrogenase, aldolase, citrate synthetase, and several other enzymes was not affected 3 or 6 h after anti-insulin serum injection. The activities of hepatic and adipose tissue acetyl-Coi carboxylase, hepatic glucokinase and adipose tissue hexokinase were decreased. The tissue FFA levels were compared to linoleate concentrations required for halfmaximal inhibition (Ki) of several enzymes in tissue fractions after in vitro contact of 30 rain at 37 ~ C. Linoleate irreversibly inactivated the enzymes to a varying degree but its effect was dependent on the protein content of the system. The Ki of linoleate increased linearly with cellular protein concentration; when extrapolated to the protein level in the intact cell it became unphysiologieally high, markedly exceeding the liver or adipose tissue FFA concentration of acutely diabetic rats. The Ki linoleate and the actual FFA concentrations were particularly discrepant in the cytoplasmic compartment, comprising most of the investigated enzymes, hut only a small proportion of tissue FFA, as determined by measurements of intracellular FFi distribution. Similar discrepancy was found for the interaction of pMmityl-CoA with glucose-6-phosphate dehydrogenase and aeetyl-CoA carboxylase. Thus, the selective decrease in the activity of glueokinase and acetyl-CoA carboxylase in acute diabetes was attributed to factors other than FFA or their CoN esters. The nature of FFA-cnzyme interaction, causing enzyme inactivation rather than specific inhibition is discussed, concluding that FFA do not act as direct homeostatic modifiers of enzymes regulating carbohydrate and fat metabolism.

Long-chain acyl-CoA esters inhibit phosphorylation of AMP-activated protein kinase at threonine-172 by LKB1/STRAD/MO25

“…An earlier study demonstrated that the LCACE-mediated inhibitory effect decreased with increasing homogenate concentration, suggesting that unphysiologically high LCACE concentrations would be required to exert a physiologically relevant inhibition (47). Although total LCACE concentrations in tissues may range from 5 to 160 M, free LCACE concentration has been estimated to be Ͻ200 nM, although the actual free LCACE concentration for any tissue is not known (9).…”

Specific, reversible inactivation of phosphofructokinase by fructose-1,6-bisphosphatase. Involvement of adenosine 5'-triphosphate, oleate, and 3-phosphoglycerate

“…Evidence from in vivo or liver perfusion studies indicates that there is not a sufficient variation in tissue concentrations of ATP, ADP, or AMP to bring about significant alterations in the two enzyme activities (Clark et al, 1974;Tarnowski and Seemann, 1967;Exton and Park, 1969;Burch et al, 1970;Hems et al, 1966). Under conditions that stimulate gluconeogenesis (starvation, glucagon administration, and diabetes), the known rise in the concentration of 3-P-glycerate (Exton and Park, 1969) and possibly free fatty acids (Shafrir and Ruderman, 1974) would favor increased FDPase activity and, in the presence of the high intracellular levels of ATP (>3 µ /g wet weight), decreased PFK activity. Refeeding or a lowered glucagon level would result in a drop in 3-P-glycerate concentrations that would allow inactivation of FDPase by ATP and increased activity of PFK.…”