ABSTRACT
Glucokinase (GK) plays a critical role in controlling blood glucose; GK activators have been shown to stimulate insulin secretion acutely both in vitro and in vivo. Sustained stimulation of insulin secretion could potentially lead to ß-cell exhaustion; this study examines the effect of chronic GK activation on ß-cells. Gene expression and insulin secretion were measured in rodent islets treated in vitro with GKA71 for 72 h. Key ß-cell gene expression was measured in rat, mouse and global GK heterozygous knockout mouse islets (gk(del/wt)). Insulin secretion, after chronic exposure to GKA71, was measured in perifused rat islets. GKA71 acutely increased insulin secretion in rat islets in a glucose-dependent manner. Chronic culture of mouse islets with GKA71 in 5 mmol/l glucose significantly increased the expression of insulin, IAPP, GLUT2, PDX1 and PC1 and decreased the expression of C/EBPß compared with 5 mmol/l glucose alone. Similar increases were shown for insulin, GLUT2, IAPP and PC1 in chronically treated rat islets. Insulin mRNA was also increased in GKA71-treated gk(del/wt) islets. No changes in GK mRNA were observed. Glucose-stimulated insulin secretion was improved in perifused rat islets following chronic treatment with GKA71. This was associated with a greater insulin content and GK protein level. Chronic treatment of rodent islets with GKA71 showed an upregulation of key ß-cell genes including insulin and an increase in insulin content and GK protein compared with glucose alone.
Subject(s)
Enzyme Activation/drug effects , Enzyme Activators/pharmacology , Glucokinase/metabolism , Insulin-Secreting Cells/physiology , Sulfones/pharmacology , Thiadiazoles/pharmacology , Up-Regulation/genetics , Animals , Enzyme Assays , Glucokinase/chemistry , Glucose/metabolism , Insulin/genetics , Insulin/metabolism , Insulin Secretion , Insulin-Secreting Cells/drug effects , Insulin-Secreting Cells/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Rats , Rats, Wistar , Tissue Culture Techniques , Transcription, Genetic/drug effectsSubject(s)
Adenosine Triphosphate/metabolism , Adrenergic beta-Agonists/pharmacology , Cysteine Endopeptidases/metabolism , Multienzyme Complexes/metabolism , Muscle, Skeletal/drug effects , Animals , Denervation , Hydrolysis , Muscle, Skeletal/enzymology , Muscle, Skeletal/innervation , Muscle, Skeletal/metabolism , Proteasome Endopeptidase Complex , Rats , Ubiquitins/metabolismSubject(s)
Adrenergic beta-2 Receptor Agonists , Adrenergic beta-Agonists/pharmacology , Actins/biosynthesis , Animals , Cell Line , Clenbuterol/pharmacology , Kinetics , Muscle, Skeletal , Prenalterol/pharmacology , Propanolamines/pharmacology , Rats , Time Factors , Tropomyosin/biosynthesis , Troponin/biosynthesis , Troponin TABSTRACT
In vivo, hormone-sensitive lipase (HSL) is known to be phosphorylated on two sites termed the regulatory and basal sites. However, the intracellular role of the basal site or the identity of the protein kinase phosphorylating this site has not been established. We show that 5-amino-4-imidazolecarboxamide ribonucleoside (AICAR) markedly activates cellular AMP-activated protein kinase (AMPK) in a time- and dose-dependent manner. As expected for an agent that activates AMPK intracellularly, AICAR had no effect on the basal activity of HSL. However, preincubation of adipocytes with AICAR led to a reduced response of these cells to the lipolytic agent isoprenaline. AICAR was also shown to profoundly inhibit lipogenesis through increased phosphorylation of acetyl-CoA carboxylase (ACC). Thus it appears that in addition to regulating lipogenesis, AMPK also plays an important antilipolytic role by regulating HSL in rat adipocytes.
