A Novel Assay for Evaluating Glycogenolysis in Rat Adipocytes and the Inability of Insulin To Antagonize Glycogenolysis in This Cell Type

Abstract

We report here on a novel procedure for measuring glycogenolysis in rat adipocytes. In this procedure, cells are incubated for 30 min at 37 degrees C with insulin or vanadate, and with [U-14C]glucose to label the glycogen pool with radioactive glucose. The cells are washed and preincubated for an additional 1 h, before being assayed. The extent of glycogenolysis is determined by the decrease in radioactivity in precipitated glycogen, which was quite substantial under experimental conditions facilitating glycogenolysis. From the assay, we determined the following. (a) Glycogenolysis is activated in rat adipocytes in response to lipolytic hormones (i.e. catecholamines and adrenocorticotropic hormone). (b) Other agents and conditions elevating intracellular adenosine 3',5'-monophosphate levels (i.e. cholera toxin, dibutyryladenosine 3',5'-monophosphate, and isobutylmethylxanthine) also activate glycogenolysis. (c) Glycogenolysis (as opposed to lipolysis) is activated at concentrations of adrenocorticotropic hormone or isoproterenol 7-11-fold lower and at adenosine 3',5'-monophosphate concentrations 7-fold lower. (d) Calyculin A, a specific inhibitor of protein phosphatase 1, activates glycogenolysis as well. Calyculin A also activates lipolysis at an equimolar potency. (e) Insulin does not antagonize glycogenolysis in rat adipocytes. In conclusion, the assay allowed us to compare glycogenolysis to lipolysis within the same cell, and to find that the sensitivity to hormones and adenosine 3',5'-monophosphate was about 1 order of magnitude higher for glycogenolysis than for lipolysis. A more striking finding was the inability of insulin to antagonize glycogenolysis in the rat adipose cell, an effect which occurs readily in liver and muscle cells via protein phosphatase 1-activating machinery. This rules out a role for adipose protein phosphatase 1 activation in the mechanism by which insulin antagonizes lipolysis and supports the contention that the insulin effect in lowering adenosine 3',5'-monophosphate levels is the central mechanism by which insulin antagonizes lipolysis.

The activation of p38 MAPK by the β‐adrenergic agonist isoproterenol in rat epididymal fat cells

“…3 ) shows that half maximal activation of p38 MAPK occurred at 13±3 nM isoproterenol ( n =3 separate cell preparations). This EC 50 for the activation of p38 MAPK was similar to that for the stimulatory effects of isoproterenol on cyclic AMP‐dependent protein kinase [17], lipolysis [17]and glycogen breakdown [18].…”

Independent Signal-Transduction Pathways for Vanadate and for Insulin in the Activation of Glycogen Synthase and Glycogenesis in Rat Adipocytes1

“…It induces a conformational change that favors dephosphorylation and activation of glycogen synthase by PP1G (43). Recently, however, we found that lipolytic hormones activate glycogenolysis in rat adipocytes (53). It was believed that this enzymatic activity is absent in nongluconeogenic tissues such as fat.…”

Mechanism of the stimulatory action of okadaic acid on lipolysis in rat fat cells

“…In 1997, Sekar et al (9) were the first to report that okadaic acid activated lipolysis in rat fat cells in the absence of lipolytic hormones. Generally, the mechanisms of action of lipolytic hormones such as catecholamines and ACTH are believed to be mediated by the cAMP cascade: lipolytic hormones activate adenylate cyclase, thereby increasing cAMP formation (10,11).…”