Mechanism for the stimulation of gluconeogenesis by fatty acids in perfused rat liver.

Communicated by B. Chance, May 6, 1966 The pathway of gluconeogenesis is distinguished from that of glycolysis by several enzyme reactions which overcome the energy barriers preventing a direct reversal of glycolysis.I These reactions are* catalyzed by pyruvic carboxylase, PEP-carboxykinase, fructose-1,6-diphosphatase, and glucose-6-phosphatase. Considerable emphasis has been placed on the possibility that control of the rate of gluconeogenesis may be exerted at one or more of these sites since (a) the liver content of these enzymes in vivo increases after some hours or days under conditions of enhanced gluconeogenesis,2 and (b) the activities of pyruvic carboxylase and fructose-1,6-diphosphatase are regulated by cofactor requirements in vitro. Fructose diphosphatase is inhibited by its substrate and by AMP3' 4 while pyruvic carboxylase is activated by acetyl CoA and inhibited by ADP.6 However, proof that these sites control gluconeogenic flux in the intact liver has so far been lacking. Recently, it has been shown that fatty acids cause an immediate increase of glucose production from alanine or lactate both in liver slices6 and in the perfused rat liver.7' 8 This response is unlikely to be caused by changes of enzyme content and provides a suitable means of studying the effect of rapidly acting control mechanisms on the gluconeogenic enzyme sequence. In order to gain further insight into the nature of the mechanisms involved, an emulsion of oleic acid in bovine serum albumin was added to an isolated blood-free perfused rat liver preparation, and the frozen tissue analyzed for metabolic intermediates. Results reported here indicate that in short-term experiments, the rate of gluconeogenesis can be controlled at the glyceraldehyde-3-P dehydrogenase step by the cellular level of DPNH. Materials and Methods. -Male Wistar rats, 180-220 gm in weight, were anesthetized with sodium pentobarbital (50 mg per kg body wt) 18-24 hr after food deprivation. The portal vein and bile duct were cannulated, with the time of hypoxia during the dissection procedure restricted to about 30 sec. The perfusion fluid was Krebs bicarbonate buffer containing 4 per cent bovine serum albumin (fraction V, Sigma Chemical Co.), 5 X 10-5M EDTA, and 10 mM L (+) alanine. This was filtered through a Millipore filter (0.45 ,.) immediately prior to use. Initially, the recirculating volume of medium was 100 ml, and 2-ml aliquots were removed periodically for chemical analyses. Perchloric acid extracts were prepared by adding 2 ml of 8 per cent (v/v) perchloric acid to a 1-ml aliquot of perfusate, centrifuging for 10 min at 27,000 9, and neutralizing the supernatant with a solution containing 3N K2CO3 and 0.5M triethanolamine. The perfusion apparatus was modified from that described by Miller et al.9 The equilibration of oxygen between the gaseous and liquid phaseswas imperfect with this apparatus (the affluent fluid being only 45-50% saturated with oxygen), but the high flow rate through the liver (40-60 ml/min) allowed an adequate extraction of oxygen from the medium, as judged from the effluent oxygen tension, which was in the range of 150-200 mm Hg.