Charles R. Park

THE ROLE OF CYCLIC AMP IN THE INTERACTION OF GLUCAGON AND INSULIN IN THE CONTROL OF LIVER METABOLISM

It is now well established that insulin exerts direct effects on mammalian liver to inhibit the production of glucose and urea and to promote the uptake of potassium ions. It has been proposed that these effects of insulin may be partly due to a decrease in liver cyclic AMP.l The proposal is based on the following observations: ( 1) insulin produces a small but significant decrease in the level of cyclic AMP in the perfused rat liver; (2) depletion of insulin in vivo by treatment with insulin antiserum or alloxan results in a twofold increase in liver cyclic AMP; (3) exogenous cyclic AMP and hormones such as glucagon and epinephrine which raise the level of cyclic AMP produce effects on the liver which are opposite to those caused by insulin; (4) insulin antagonizes the actions of epinephrine, glucagon, or cyclic AMP in the perfused liver; and ( 5 ) insulin reduces the accumulation of liver cyclic AMP in the presence of glucagon. In this article we will present more recent observations on the interaction of glucagon, epinephrine, and insulin in the control of hepatic metabolism. The investigations have employed the isolated rat liver perfused by a modification of the technique of Mortimore.* The perfusion medium consisted of Krebs-Henseleit bicarbonate buffer containing 3 % bovine albumin and 20% bovine erythrocytes. Livers were from fed rats weighing 100-150 g and the perfusion flow rate was about 7 ml per minute. The perfusions were carried out in two ways. In most cases, livers were perfused for one hour with recirculating medium and hormones were infused into the portal vein at a constant rate. In these experiments, the hormone concentrations were calculated by dividing the quantity of hormone infused during the hour by the final volume of perfusate. Since this does not allow for degradation of hormone, the values are doubtlessly overestimated. In the second type of experiment livers were perfused initially for 20 minutes with recirculating media containing no additions in order to establish steady metabolic rates and levels of cyclic AMP. The perfusion system was then changed to one with nonrecirculating medium by diverting the perfusate leaving the liver into a beaker. After a six-minute control period, infusions of hormone were commenced and livers and effluent media were sampled at designated intervals. In these experi-

The action of insulin on the transport of glucose through the cell membrane

Abstract The properties of the process for the transport of monosaccharides through the cell membrane in the erythrocyte and muscle are reviewed. Transport appears to be distinct from simple physical diffusion and involves an interaction of the sugar with a specific site on the cell membrane. The process is reversible and free sugar is liberated at either surface of the membrane. There is no evidence that sugars are moved against concentration gradients under ordinary circumstances and no metabolic energy is required. In fact, a reduction in the level of phosphate bond energy in muscle leads to more rapid transport of glucose and other sugars. Competition for transport can be shown among the hexoses and pentoses which have been tested, indicating that all these sugars cross the membrane by a common system. Transport antecedes and is distinct from phosphorylation by the hexokinase system and may be regarded as the first step in glucose metabolism. A sugar carrier system in the membrane provides an attractive working concept of the function of the transport process. A characteristic action of insulin is the stimulation of glucose uptake in muscle. This effect must be due to acceleration of whatever step is ratelimiting in the uptake process. Present evidence indicates that membrane transport is predominantly rate-limiting in the absence of insulin and that the hormone accelerates this step. Increased entrance of glucose provides more substrate for the hexokinase system and results in a rise in the metabolism of the sugar. The effect of insulin and other hormonal factors on transport can be particularly well observed by technics employing non-metabolizable analogs of glucose. Such studies show that a very broad range of transport rates is under hormonal control. The acceleratory action of insulin is very rapid and probably occurs through a primary interaction at the cell surface. The elucidation, however, of transport and the insulin effect on the level of specific molecular interactions remains a problem for the future.

Effects of glucagon on cyclic AMP and carbohydrate metabolism in livers from diabetic rats

Abstract Cyclic AMP metabolism in normal and diabetic livers was studied using the isolated perfused rat liver and plasma membrane and supernatat fractions from rat liver. Liver from alloxan- or streptozotocin-diabetic rats had increased tissue levels of cyclic AMP and showed increased release of cyclic AMP during perfusion. Addition of glucagon or cyclic AMP to the medium produced little or no increase in the high rates of glucose production and lactate gluconeogenesis in diabetic livers. Low concentrations of glucagon (2·10 −10 M or less) did not increase tissue accumulation or release of cyclic AMP in livers from diabetic rats but were effective in normal livers. Higher concentrations of the hormone produced normal responses in diabetic livers. Basal or fluoride-stimulated adenylate cyclase activity in plasma membranes isolated from diabetic livers was not altered, but the enzyme was subnormally normally responsive to concentrations of glucagon within the range 10 −10 −10 −8 M. Insulin (10 −11 −10 −6 M) added in vitro was without effect on basal or glucagon-stimulated plasma membrane adenylate cyclase. Neither modulation of the Mg 2+ concentration, nor addition of Ca 2+ , GTP, theophylline or ouabain caused the emergence of an insulin effect. Rat liver plasma membranes contained phosphodiesterase activity with two apparent K m values of about 0.5 and 70 μM. The activity of the low K m enzyme was decreased in plasma membranes from diabetic rats and was increased by insulin treatment of the rats in vivo. Both low and high K m activities were also decreased in supernatant fractions from livers of diabetic rats. Insulin at 10 −9 or 10 −7 M concentration had no effect in vitro on plasma membrane phosphodiesterase activities.

Interaction of adrenal steroids and glucagon on gluconeogenesis in perfused rat liver

Abstract A stimulatory effect of glucagon on gluconeogenesis in normal rats has been demonstrated by studies in vivo and in vitro ( Kalant, 1956 ; Salter et al ., 1957 ; Izzo and Glasser, 1961 ; Exton et al . 1966 ; Garcia et al . 1966 ; Sokal, 1966 ). However, livers from fasted adrenalectomized rats perfused with lactate or pyruvate fail to show this response ( Exton et al ., 1966 ; Eisenstein, 1967 ). This led us to investigate the possibility suggested by Friedmann and Wertheimer (1966) that glucocorticoids play a permissive role in the regulation of gluconeogenesis by glucagon. This paper presents data supporting this view. Injection of dexamethasone into adrenalectomized rats 30 minutes prior to sacrifice restored the response to glucagon in vitro almost to normal. In perfused livers from adrenalectomized rats, addition of dexamethasone to the perfusion medium caused a rapid rise in gluconeogenesis in the presence of glucagon but had no effect, except at high levels, in the absence of glucagon.

Hormonal control of hepatic gluconeogenesis.

Publisher Summary This chapter describes the hormonal control of hepatic gluconeogenesis, which is the process whereby lactate, pyruvate, glycerol, and certain amino acids are converted into glucose and glycogen. The liver is the major site of gluconeogenesis although the kidney becomes important during prolonged starvation. The most important function of gluconeogenesis is the maintenance of blood glucose levels during times when food intake is restricted and/or glycogen stores are depleted. Hormonal control of gluconeogenesis can be divided for convenience into three categories. The first involves the regulation of substrate supply. All gluconeogenic substrates reach the liver in subsaturating concentration. Thus, the regulation of substrate release into the blood from the extrahepatic tissues will directly affect hepatic glucose formation. The regulation of the level of cyclic AMP (cAMP) could also explain the inhibitory effect of insulin on gluconeogenesis. There is also evidence that insulin can suppress gluconeogenesis by a mechanism that is independent of changes in cAMP.

Stimulation by glucagon of the incorporation of U-14 C-labeled substrates into glucose by isolated hepatocytes from fed rats☆

The effect of glucagon on the incorporation of U-14C-labeled lactate, pyruvate or alanine into glucose has been studied using isolated hepatocytes from livers of fed rats. Rates of incorporation into glucose were about the same as observed in perfused liver preparations provided precautions were taken to avoid depletion of certain metabolities by the preparative procedures. With each substrate, stimulation of the incorporation into glucose by a maximally effective concentration of glucagon (10 nM) was associated with about a 75% reduction in the substrate concentration required for a half-maximal rate and with about a 30% increase in maximum rate. Consequently, the hormone caused a substantial (2--4-fold) stimulation when any one of the above substrates was present at a near physiological concentration, but brought about only a relatively small stimulation (1.4-fold) when very high substrate concentrations were used. Provision of cytoplasmic reducing equivalents (by ethanol addition), or of precursor for acetyl-coenzyme A formation (by acetate addition)-stimulated incorporation of labeled alanine into glucose and their effects were additive with that of glucagon. This suggested that provision of either of these intermediates was not a means by which the hormone increased the incorporation of labeled substrate into glucose. NH4+ stimulated the incorporation of 20 mM [U-14C] lactate into glucose 2-fold, probably by promoting glutamate synthesis and thus enhancing the transamination of oxaloacetate to aspartate. Evidence was obtained to support the view that glucagon also increases glutamate production (presumably from endogenous protein). However, the stimulation of incorporation into glucose from 20 mM [U-14C] lactate by NH4+ plus glucagon was synergistic. This suggested that glucagon also stimulated the incorporation of labeled substrate into glucose by additional means. Stimulation of the incorporation of [U-14C] alanine into glucose by beta-hydroxybutyrate plus glucagon was also synergistic. This suggested that another action of glucagon may be to provide more intramitochondrial reducing potential.

Advantages of deuterium modification of nitroxide spin labels for biological epr studies

Abstract The spin label, perdeuterio-N-(1-oxy 1-2,2,6,6-tetramenthyl-4-piperidinyl)maleimide (DMSL) was synthesized and its EPR and saturation transfer EPR spectra were compared to those of the hydrogen analogue, HMSL- The labels were studied as freely tumbling entities and also bound to bovine serum albumin (BSA). Significant gains in spectral resolution and detectability were observed for DMSL relative to HMSL.

EPR and saturation transfer EPR studies on glyceraldehyde 3‐phosphate dehydrogenase

Electron paramagnetic resonance (EPR) and saturation transfer–EPR (ST–EPR) techniques were employed to investigate the hydrodynamic properties of glyceraldehyde 3‐phosphate dehydrogenase (GAPDH). Both apo‐ and holoenzyme were spin‐labeled at the active site cysteine‐149 residue with N‐ (1‐oxyl‐2,2,6,6‐tetramethyl‐4‐piperidinyl) ‐ maleimide. The apo‐ and holoenzymes were observed to have the same hydrodynamic structure and the spectroscopic results were consistent with these complexes behaving as spheres with hydrated radii of 41 A. The environment of the paramagnetic electron was significantly more polar in the spin‐labeled holoenzyme than in the spin‐labeled apoenzyme, suggesting that either ionic residues are positioned closer to the active site in the holoenzyme or that ionic segments of coenzyme nicotinamide adenine dinucleotide (NAD+) itself may interact with the paramagnetic electron of the maleimide spin label. The dependence of the phase quadrature second harmonic absorption ST‐EPR signal upon mic...

Effects of adrenalectomy and glucocorticoid replacement on gluconeogenesis in perfused livers from diabetic rats

Abstract 1. 1. The dependence of the increased rate of hepatic gluconeogenesis in diabetes upon glucocorticoid secretion was studied using the perfused rat liver preparation. Adrenalectomy reduced, and glucocorticoid treatment restored, glucose output, gluconeogenesis and glycogen synthesis from lactate, and ureogenesis in livers from alloxan-diabetic rats. Cortisol treatment for 1 h in vivo or in vitro significantly increased glucose output and gluconeogenesis from high or physiological levels of lactate in livers from adrenalectomized diabetic rats. Cortisol treatment for 2 h in vivo increased ureogenesis and glycogen synthesis in such livers, but cortisol infusion for 2 h in vitro produced no effects on these processes. 2. 2. The in vitro effects of cortisol on glucose output and gluconeogenesis were of similar magnitude to the in vivo effects and were abolished by addition of cycloheximide and actinomycin D. This suggests that the direct effects of glucocorticoids on hepatic glucose production and gluconeogenesis are quantitatively important and probably involved increased mRNA and protein synthesis. 3. 3. Measurements of the levels of gluconeogenic intermediates in perfused livers from adrenalectomized diabetic rats indicated restraint of reactions located between pyruvate and phosphopyruvate and possibly between glucose-6- P and glucose. Glucocorticoid treatment in vivo or in vitro produced changes consistent with facilitation of reactions located at these sites. There was no evidence of glucocorticoid action on fructose diphosphatase or phosphofructokinase and changes in adenine nucleotides and citrate were the opposite to those expected if steroid facilitated the net conversion of fructose-1,6- P 2 to fructose-6- P . 4. 4. Adrenalectomy reduced the large increase in phosphoenolpyruvate carboxykinase activity in livers from diabetic rats, but did not alter the small increase in pyruvate carboxylase. Cortisol treatment for 1 h and longer significantly elevated phosphoenolpyruvate carboxykinase activity in livers from adrenalectomized diabetic rats, but did not change pyruvate carboxylase activity. Adrenalectomy and steroid replacement did not modulate the increased levels of acetyl-CoA and cyclic AMP in livers from diabetic rats. 5. 5. It is concluded that the increase in hepatic gluconeogenesis in diabetes is dependent upon secretion of glucocorticoids and that induction of phosphoenolpyruvate carboxykinase is a major mechanism by which the steroid act. The hormones do not alter cyclics AMP levels nor affect pyruvate carboxylase activity by increasing the level of the enzyme or acetyl-CoA.

Mechanism of action of penicillamine in the treatment of avian muscular dystrophy.

Penicillamine, a cysteine analog with a reduced sulfhydryl group, has been used in this laboratory for the treatment of hereditary avian dystrophy. The drug delays the onset of symptoms and alleviates the debilitating aspects of the disease. To study the mechanism of drug action, the effects of penicillamine on white and red muscles of dystrophic chickens were examined with regard to the specific activities of the soluble enzymes glyceraldehyde-3-phosphate dehydrogenase, acetylphosphatase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glutathione reductase, glutathione preoxidase, superoxide dismutase, and catalase. The sulfhydryl contents of the soluble proteins and the concentration of myoglobin were also determined. In white dystrophic muscle (pectoral), there were large alterations in the various enzymatic activities compared to normal levels. In the DISCUSSION, these changes are related to the pathogenesis of the disease and to the adaptive response for protection of the severely affected fast fibers. Red dystrophic muscles (thigh) were minimally involved, in accordance with the known sparing action of the slow fiber type. The results suggested that the disease process in dystrophic muscle may be due to oxidation of the essential sulfhydryl groups of proteins. Penicillamine may produce therapeutic effects by altering the intracellular redox status, thereby promoting better regulation of enzymatic activity, membrane stability, and improved muscle function.