ABSTRACT
Non-alcoholic fatty liver disease (NAFLD) is associated with hepatic insulin resistance with the molecular basis of this association being not well understood. Here we studied the effect of hepatic triglyceride accumulation induced by postprandial triglyceride-rich lipoproteins (TGRL) on hepatic insulin sensitivity in HepG2 cells. Incubation of HepG2 cells with purified TGRL particles induced hepatocellular triglyceride accumulation paralleled by diminished insulin-stimulated glycogen content and glycogen synthase activity. Accordingly, insulin-induced inhibition of glycogen synthase phosphorylation as well as insulin-induced GSK-3 and AKT phosphorylation were reduced by TGRL. The effects of TGRL were dependent on the presence of apolipoproteins and more pronounced for denser TGRL. Moreover, TGRL effects required the presence of heparan sulfate-proteoglycans on the cell membrane and lipase activity but were independent of the cellular uptake of TGRL particles by receptors of the LDL receptor family. We suggest postprandial lipemia to be an important factor in the pathogenesis of NAFLD.
Subject(s)
Insulin Resistance/physiology , Lipoproteins/chemistry , Lipoproteins/metabolism , Liver/metabolism , Postprandial Period/physiology , Receptors, LDL/metabolism , Triglycerides/metabolism , Adult , Fatty Liver/metabolism , Fatty Liver/physiopathology , Glycogen/metabolism , Glycogen Synthase/metabolism , Glycogen Synthase Kinase 3/metabolism , Hep G2 Cells , Humans , Hypertriglyceridemia/metabolism , Male , Non-alcoholic Fatty Liver Disease , Proto-Oncogene Proteins c-akt/metabolismABSTRACT
In the present study, we investigated the mechanisms by which resistin (100 nM, 1 h) affects glycogen synthesis in L6 skeletal muscle cells. The activity of glycogen synthase, the major enzyme in glycogen synthesis, is determined by both its covalent phosphorylation and allostery through intracellular glucose-6-phosphate. Covalent phosphorylation of glycogen synthase was not altered by resistin and, accordingly, phosphorylation of GSK-3alpha/beta and Akt remained unchanged. The rate of glucose-6-phosphate formation, however, was decreased by resistin both in the absence and presence of insulin; in the absence of insulin, resistin decreased glucose-6-phosphate formation by reducing hexokinase type I activity without affecting glucose uptake; by contrast, in the presence of insulin, resistin decreased glucose-6-phosphate formation by reducing the Vmax of glucose uptake without changing hexokinase type I activity. In conclusion, short-term resistin incubation impairs glycogen synthesis by reducing the rate of glucose-6-phosphate formation involving, however, differential mechanisms in basal and insulin-stimulated states.
Subject(s)
Glycogen/biosynthesis , Hypoglycemic Agents/pharmacology , Insulin/pharmacology , Muscle, Skeletal/drug effects , Resistin/pharmacology , Animals , Cells, Cultured , Glucose/metabolism , Glucose-6-Phosphate/metabolism , Glycogen Synthase/metabolism , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Hexokinase/metabolism , Immunoblotting , Muscle, Skeletal/cytology , Muscle, Skeletal/metabolism , Phosphorylation , Proto-Oncogene Proteins c-abl/metabolism , Proto-Oncogene Proteins c-akt/metabolism , RatsABSTRACT
We developed a gel filtration assay for the determination of glycogen synthase activity in cultured cells or tissue homogenates. Compared to the commonly used filter paper assay, the gel filtration assay resulted in a more than 5-fold reduction of background levels leading to an--at least--twofold increase in precision. These benefits allow the gel filtration method to detect differences of +/-5% in enzyme activity out of 300 microg total cell protein. In addition to high precision and sensitivity, the method's additional salient advantages include lesser expenditure of time and labour and reduced exposure time of the personnel to radioactivity.
