Abstract
The effects of adrenalectomy on glucagon activation of liver glycogen phosphorylase and glycogenolysis were studied in isolated hepatocytes. Adrenalectomy resulted in reduced responsiveness of glycogenolysis and phosphorylase to glucagon activation. Stimulation of cAMP accumulation and cAMP-dependent protein kinase activity by glucagon was unaltered in cells from adrenalectomized rats. Adrenalectomy did not alter the proportion of type I and type II protein kinase isozymes in liver, whereas this was changed by fasting. Activation of phosphorylase kinase by glucagon was reduced in hepatocytes from adrenalectomized rats, although the half-maximal effective concentration of glucagon was unchanged. No difference in phosphorylase phosphatase activity between liver cells from control and adrenalectomized rats was detected. Glucagon-activated phosphorylase declined rapidly in hepatocytes from adrenalectomized rats, whereas the time course of cAMP increase in response to glucagon was normal. Addition of glucose (15 mM) rapidly inactivated glucagon-stimulated phosphorylase in both adrenalectomized and control rat hepatocytes. The inactivation by glucose was reversed by increasing glucagon concentration in cells from control rats, but was accelerated in cells from adrenalectomized rats. It is concluded that impaired activation of phosphorylase kinase contributes to the reduced glucagon stimulation of hepatic glycogenolysis in adrenalectomized rats. The possible role of changes in phosphorylase phosphatase is discussed.
Highlights
Accumulation-In agreement with findings in the perfused rat liver, adrenalectomy led to a decrease in the glucagon responsiveness of glycogenolysis in isolated hepatocytes (Fig. 1)
Earlier studies with the perfused rat liver showed that adrenalectomy led to reduced responsiveness of glycogenolysis to glucagon stimulation
In agreement with Rousseau et al [5], we have ruled out activation of CAMP-dependent protein kinase as a possible site of permissive action
Summary
Hepatocyte Isolation and Incubation-Male obtained from Harlan Industries, Indianapolis. Cell aliquots were taken for phosphorylase a activity and CAMP determinations. CAMP-dependent protein kinase was measured as described by Cherrington et al [14] except that 1 ml of cell suspension was directly homogenized in 2 ml of homogenizing medium instead of first being centrifuged to remove incubation medium. The samples were thawed in 2 volumes of a buffer composed of 250 mrvr sucrose, 100 mM NaF, 10 mM potassium phosphate, and 1 mM ,f-mercaptoethanol at pH 7.2 and containing sufficient heat-stable inhibitor [16] to inhibit CAMP-dependent protein kinase activity in the extract. Phosphorylase phosphatase activity is expressed as nmol of phosphate released/min/g of cells (wet weight).
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