THE DIFFERENTIAL BEHAVIOR OF GLUCAGON AGONISTS AND ANTAGONISTS ON NORMAL AND DIABETIC LIVER: EVIDENCE FOR CYCLIC-AMP - INDEPENDENT EVENTS.

Abstract

A nonrecirculatory liver slice perifusion system has been developed and utilized for investigating glucagon-stimulated glycogenolysis in normal and diabetic states. It has been shown here that slices maintained in this system experience a controlled environment with respect to temperature and pH and remain viable throughout a three-hour experimental period based upon their maintenance of intracellular potassium levels. Although glycogen content falls by 40%, slices exhibit significant glycogenolysis in a dose-response manner upon challenge with glucagon, with maximal concentrations eliciting a 2.2-fold stimulation. This system, which permits nonrecirculatory challenge of liver tissue and subsequent analysis of both intracellular events and overall physiological responses, is extremely useful for examining hormonal mechanisms operating for glucagon, particularly at low concentrations. Using this methodology, liver slices challenged with glucagon exhibit a biphasic dose-response for glycogenolysis. While the second phase parallels cAMP (cyclic adenosine 3':5'-monophosphate) accumulation and cAMP-PK (cAMP-dependent protein kinase) activation, the first is mediated independent of cAMP. Trinitrophenylhistidine-1, homoarginine-12-glucagon (THG), which can antagonize glucagon-stimulated adenylate cyclase, exhibits 50% partial agonist activity for cAMP production and cAMP-PK but full agonism for glycogenolysis. Separation between these events is only two-fold indicating a cAMP-mediated process. [Des-amino-fYRKKE]-glucagon, ([Des-amino-His¹,D-Phe⁴,Tyr⁵,Arg¹²,Lys¹⁷·¹⁸,Glu²¹]-glucagon), another adenylate cyclase antagonist, does not stimulate cAMP or cAMP-PK up to 25 μM yet still elicits glycogenolysis. These results demonstrate that glucagon does indeed stimulate both cAMP-independent as well as cAMP-dependent glycogenolysis in normal liver. In diabetic systems, glucagon elicits attenuated adenylate cyclase activity in liver plasma membranes with reduction in basal activity and extent of stimulation. Maximal stimulation of cAMP production is also reduced by half in liver slices, but in both systems (normal vs. diabetic) EC₅₀ values for cAMP production are identical. Neither THG nor [des-amino-fYRKKE]-glucagon stimulate cAMP production or cAMP-PK in diabetic liver slices. While THG lowers blood glucose levels in vivo, [des-amino-fYRKKE]-glucagon acts as an agonist. These results suggest that the mechanisms which operate for glucagon-stimulated glycogenolysis in normal liver are attenuated in the diabetic state. Furthermore, antagonism of cAMP production alone is insufficient to antagonize glucagon's overall physiological action.