Hepatic Cyclic AMP Research Articles

The impact of glucagon to support postabsorptive glucose flux and glycemia in healthy rats and its attenuation in male Zucker diabetic fatty rats.

Hyperglucagonemia is a hallmark of type 2 diabetes (T2DM), yet the role of elevated plasma glucagon (P-GCG) to promote excessive postabsorptive glucose production and contribute to hyperglycemia in patients with this disease remains debatable. We investigated the acute action of P-GCG to safeguard/support postabsorptive endogenous glucose production (EGP) and euglycemia in healthy Zucker control lean (ZCL) rats. Using male Zucker diabetic fatty (ZDF) rats that exhibit the typical metabolic disorders of human T2DM, such as excessive EGP, hyperglycemia, hyperinsulinemia, and hyperglucagonemia, we examined the ability of hyperglucagonemia to promote greater rates of postabsorptive EGP and hyperglycemia. Euglycemic or hyperglycemic basal insulin (INS-BC) and glucagon (GCG-BC) clamps were performed in the absence or during an acute setting of glucagon deficiency (GCG-DF, ∼10% of basal), either alone or in combination with insulin deficiency (INS-DF, ∼10% of basal). Glucose appearance, disappearance, and cycling rates were measured using [2-3H] and [3-3H]-glucose. In ZCL rats, GCG-DF reduced the levels of hepatic cyclic AMP, EGP, and plasma glucose (PG) by 50%, 32%, and 50%, respectively. EGP fell in the presence GCG-DF and INS-BC, but under GCG-DF and INS-DF, EGP and PG increased two- and threefold, respectively. GCG-DF revealed the hyperglucagonemia present in ZDF rats lacked the ability to regulate hepatic intracellular cyclic AMP levels and glucose flux, since EGP and PG levels fell by only 10%. We conclude that the liver in T2DM suffers from resistance to all three major regulatory factors, glucagon, insulin, and glucose, thus leading to a loss of metabolic flexibility.NEW & NOTEWORTHY In postabsorptive state, basal plasma insulin (P-INS) and plasma glucose (PG) act dominantly to increase hepatic glucose cycling and reduce endogenous glucose production (EGP) and PG in healthy rats, which is only counteracted by the acute action of basal plasma glucagon (P-GCG) to support EGP and euglycemia. Hyperglucagonemia, a hallmark of type 2 diabetes (T2DM) present in Zucker diabetic fatty (ZDF) rats, is not the primary mediator of hyperglycemia and high EGP as commonly thought; instead, the liver is resistant to glucagon as well as insulin and glucose.

Moderate Calorie Restriction Enhances Hepatic Glucagon Sensitivity in Aged Mice

Abstract Chronic calorie restriction (CR) without malnutrition delays the onset of aging, extends lifespan, and improves metabolic function in many species. These CR-induced benefits have largely concentrated on the role of insulin signaling, while ignoring its counter-regulatory hormone, glucagon. Like insulin, hyperglucagonemia and decreased glucagon sensitivity are associated with impaired glucose homeostasis and decreased longevity. Conversely, activation of target molecules downstream of glucagon signaling such as AMPK and FGF21 are known to ameliorate these age-related impairments in metabolic function. To investigate the potential role of glucagon receptor signaling in CR-induced improvements in aging, we have implemented a moderate 15% CR in the mouse. Our studies show that a 15% calorie restriction initiated at 4 months of age enhances hypoglycemia-stimulated glucagon secretion (P <.01) and decreases basal serum glucagon (P <.01), while having no effect on glucagon receptor expression at the liver in 26-month-old mice. Consistent with enhanced hepatic glucagon sensitivity, CR increases glucagon-stimulated hepatic cyclic AMP production (P <.05). Glucagon is a primary regulator of AMPK activation and FGF21 release, both of which have been proposed as key molecules to account for CR-induced benefits to aging. CR increases both hepatic AMPK activation (P <.05) and FGF21 mRNA expression (P <.05). Additionally, CR reduces hepatic lipid accumulation (P <.05), and decreases fasting respiratory quotient (P <.001), indicating an increase in lipid oxidation. Our studies demonstrate that a moderate (15%) CR regimen enhances glucagon sensitivity and decreases hepatic lipid accumulation in aged mice. Thus, we propose glucagon signaling as a mediator of CR-induced improvements in aging.

Adipose expression of CREB3L3 modulates body weight during obesity

We found the hepatic transcription factor Cyclic-AMP Responsive Element Binding Protein 3-like-3 (CREB3L3) to be expressed in adipose tissue, and selectively downregulated in the more metabolically protective subcutaneous adipose tissue in obese mice and humans. We sought to elucidate the specific role of this factor in adipose biology. CREB3L3 fat-specific knockout mice were fed a high-fat diet to induce obesity and metabolic dysfunction. Additionally, we injected a flip-excision adeno-associated virus directly into the subcutaneous inguinal adipose tissue of Adiponectin-Cre mice to create a depot-specific overexpression model for further assessment. Fat-specific ablation of CREB3L3 enhanced weight gain and insulin resistance following high-fat feeding, as fat-specific knockout mice expended less energy and possessed more inflammatory adipose tissue. Conversely, inguinal fat CREB3L3 overexpression deterred diet-induced obesity and ameliorated metabolic dysfunction. Together, this study highlights the relevance of CREB3L3 in obese adipose tissue and demonstrates its role as a powerful body weight modulator.

Effects of oral antidiabetics on cancer incidence and mortality

Introduction: Type 2 diabetes mellitus is a chronic progressive disease that can be controlled by any oral hypoglycemic, with biguanides, thiazolidinediones and sulfonylureas being the main classes of these drugs in the pharmaceutical market. Epidemiological analyzes suggest that diabetics are at higher risk of developing tumors than normoglycemics. A large number of studies have shown the link between oral antidiabetic drugs and developmental rates and mortality in cancer cases. Objective: To present clinical and preclinical results on the relationship between oral hypoglycemic agents and cancer. Methodology: A bibliographical survey was carried out of the main academic journals, with prevalence of articles from the last five years. Results and Discussion: Metformin is a biguanide that phosphorylates hepatic cyclic AMP, a mechanism also proposed as antineoplastic. Epidemiological studies associate the use of metformin with the lower incidence of hepatocellular and pancreatic cancers in diabetic patients. Other studies have shown a 21% lower mortality rate in women with breast cancer and 24% lower in men with prostate cancer who took metformin. Thiazolidinediones are receptor agonists, predominantly present in adipocytes, capable of increasing glucose uptake and improving insulin sensitivity. Randomized trials have shown that pioglitazone was associated with a significant reduction in breast cancer, however, there are concerns regarding its use related to the increase in the incidence of bladder cancer. Sulphonylureas, on the other hand, increase insulin secretion by pancreatic β cells. In an observational study, glibenclamide was associated with an increased risk of 20% to 25% in the overall incidence of cancer. Conclusion: The use of oral hypoglycemic agents may decrease the incidence and mortality of certain types of cancer. However, more studies are needed to elucidate its possible effects on the neoplastic process.

Effects of Chinese Medicinal Formula BNG-1 on Phosphodiesterase 3B Expression, Hepatic Steatosis, and Insulin Resistance in High Fat Diet-induced NAFLD Mice.

Background: Chinese medicinal formula BNG-1, a non-specific inhibitor of phospho-diesterases (PDEs), can be considered as a potential anti-inflammatory agent. The present study was aimed at determining the effects of BNG-1 on the development of non-alcoholic fatty liver disease (NAFLD) in mice.Design and Methods: Male CD1 mice were randomly divided into seven groups, the control Con (4) and Con (8)+saline groups were fed a standard control diet for four or eight weeks; the experimental HFD (4) and HFD (8)+saline groups were fed a high fat diet for four or eight weeks; the HFD (8)+LBNG, HFD (8)+MBNG, and HFD (8)+HBNG groups received a high fat diet along with low, moderate or high doses of BNG-1 (0.026, 0.035, and 0.052g/30g body weight) which was administered for the last four weeks of an eight-week experimental period. After the end of experiment, blood and tissue samples were taken and analyzed.Results: Mice in the HFD (4) group had higher levels of alanine aminotransferase (ALT), plasma and hepatic triglyceride and cholesterol, and homeostasis model assessment-estimated insulin resistance (HOMA-IR) compared with mice in the Con (4) group. Mice receiving the high fat diet along with the BNG-1 supplement had decreased body weight gains and lower visceral fat weights compared with the HFD (8)+saline group. They had also significantly reduced levels of abnormal ALT and HOMA-IR, and improved blood lipid profile. BNG-1-treated mice exhibited reduced hepatic lipid accumulation, lower oxidative stress, and decreased expression of pro-inflammatory cytokines (TNF-α and IL-1β). Furthermore, BNG-1 treatment resulted in down-regulation of hepatic cyclic-AMP dependent PDE3B and up-regulation of PDE3B expression in epididymis adipose tissue.Conclusions: BNG-1 mediated changes in PDE3B expression along with reduction in oxidative stress and inflammation. BNG-1 may ameliorate insulin resistance and hepatic steatosis in the NAFLD mouse model.

Central thyrotropin-releasing hormone increases hepatic cyclic AMP through vagal-cholinergic and prostaglandin-dependent pathways in rats

Central neuropeptides play roles in many physiologic regulations through the autonomic nervous system. We have demonstrated that central thyrotropin-releasing hormone (TRH), one of neuropeptides, induces a stimulation of hepatic proliferation through vagal-cholinergic pathways. Since cAMP is known to play an important role in the hepatic proliferation, effect of central TRH on hepatic cAMP was investigated. Rats were intracisternally injected with either a TRH analog, RX-77368 (1–100 ng), or saline. The liver was removed 2–72 h after the TRH analog and hepatic cAMP content was determined by radioimmunoassay. In some experiments, pretreatment with hepatic vagotomy, atropine methyl nitrate, or 6-hydroxydopamine (6-OHDA) was performed. Hepatic cAMP was dose-dependently increased by intracisternal TRH analog (5–100 ng) with a peak response occurring 12 h postinjection. The central TRH-induced increase in hepatic cAMP was abolished by vagotomy, atropine and indomethacin, but not by 6-OHDA. Intravenous injection of the TRH analog (10 ng) did not affect hepatic cAMP. These results demonstrate that TRH acts in the brain to increase hepatic cAMP through vagal-cholinergic and prostaglandin-dependent pathways, suggesting that central TRH modulates hepatic functions through cAMP-mediated signaling pathways.

β-Adrenergic, Hormonal, and Nervous Influences on Cryoprotectant Synthesis by Liver of the Freeze-Tolerant Wood FrogRana sylvatica

The effects of various catecholamines, α- and β-adrenergic agonists and antagonists, hormones, hormone receptor blockers, and other inhibitors, as well as destruction of the nervous system by pithing, were analyzed for their ability to modulate the freezing-induced cryoprotectant output by liver of the wood frogRana sylvatica.Of the numerous treatments tried only intraperitoneal injections of glucagon (a glycogenolytic hormone) and propranolol (a blocker of β-adrenergic receptors), both of which modulate hepatic cyclic AMP levels, altered cryoprotectant output. Glucagon significantly enhanced glucose output into the blood during freezing (by 3.5-fold compared with sham-injected frogs), whereas propranolol significantly reduced the hyperglycemia, liver and blood glucose concentrations being reduced by 55 and 42%, respectively, compared with values for untreated frozen frogs. However, treatment with the α-adrenergic blocker phentolamine did not block freezing-induced cryoprotectant output, indicating that Ca2+-mediated mechanisms are not involved in the hyperglycemic response by liver in these frogs. The mechanism of propranolol action was to suppress the activation of liver glycogen phosphorylase so that the amount of active phosphorylaseain liver of propranolol-treated frogs after freezing exposure was only 5% of the corresponding activity in liver of untreated frozen frogs. The β-adrenergic nature of catecholamine regulation of blood glucose levels was confirmed by the ability of the β-adrenergic agonist isoproterenol to mimic the hyperglycemia induced by epinephrine or norepinephrine injection in unfrozen frogs at 3°C, whereas the α-adrenergic agonist phenylephrine was ineffective. The data indicate that the cryoprotectant output response during freezing ofR. sylvaticais mediated by cAMP-dependent mechanisms within the liver under the control of plasma hyperglycemic hormones.

Beta-adrenergic receptors and angiotensinogen gene expression in mouse hepatoma cells in vitro.

We have previously reported that addition of 8-bromocyclic AMP enhances the stimulatory effect of dexamethasone on the expression of the angiotensinogen gene in mouse hepatoma cells in vitro. Isoproterenol is known to stimulate the synthesis of hepatic intracellular cyclic AMP via beta-adrenergic receptors. To study the possible effect of beta-adrenergic receptors on the expression of the angiotensinogen gene in mouse hepatoma cells, we transiently transfected them with a fusion gene with the 5'-flanking region of the angiotensinogen gene linked to a bacterial chloramphenicol acetyltransferase coding sequence as a reporter, pOCAT (ANG N-1498/+18). The addition of isoproterenol (10(-9) to 10(-5) mol/L) alone had no stimulatory effect on the expression of pOCAT (ANG N-1498/+18). In the presence of dexamethasone (10(-6) mol/L), however, isoproterenol enhanced the stimulatory effect on the dexamethasone on the expression of pOCAT (ANG N-1498/+18). The enhancing effect of isoproterenol was inhibited by the presence of propranolol (beta 1- and beta 2-adrenergic receptor antagonist) and ICI 118,551 (beta 2-adrenergic receptor antagonist) but not by the presence of atenolol (beta 1-adrenergic receptor antagonist). Furthermore, the addition of Rp-cAMP (an inhibitor of protein kinase A I and II) blocked the enhancing effect of isoproterenol. These studies demonstrated that isoproterenol enhances the stimulatory effect of dexamethasone on the expression of the angiotensinogen gene in mouse hepatoma cells via beta 2-adrenergic receptor and cyclic AMP-dependent protein kinase pathways. Our data may be important in understanding the molecular mechanism(s) of the stimulatory effect of catecholamines/glucocorticoid-induced expression of the angiotensinogen gene in the liver.

Effects of islet amyloid polypeptide on hepatic insulin resistance and glucose production in the isolated perfused rat liver.

The impact of (pancreatic) islet amyloid polypeptide on glucose metabolism and insulin sensitivity was examined in isolated rat livers perfused in a non-recirculating system. Continuous infusion of 10(-7) mol/l islet amyloid polypeptide affected neither basal nor glucagon (10(-9) mol/l)-stimulated glucose output by livers from fed rats, but it did increase the hepatic cyclic AMP release within 44 min (7.91 +/- 12.07 vs control: 0.07 +/- 0.03 pmol x 100 g body weight-1). The effect of the peptide on the ability of insulin to inhibit glucagon-induced hepatic glycogenolysis was measured in three experimental groups (n = 6). As expected glucagon (7 x 10(-11) mol/l) increased integral hepatic glucose release within 84 min (763.4 +/- 161.7 vs -25.7 +/- 73.2 mumol x 100 g body weight-1 in the control group, p less than 0.001), while insulin (100 mU/l) decreased the glucagon-stimulated glucose production (395.2 +/- 180.0 mumol x 100 g body weight-1, p less than 0.01). Simultaneous infusion of 10(-7) mol/l islet amyloid polypeptide however, was not able to reverse insulin-dependent inhibition of glucagon-stimulated hepatic glucose output (370.0 +/- 102.5 mumol x 100 g body weight-1, NS) or to enhance lactate-induced gluconeogenesis of livers from 24 h fasted rats (n = 8). The glucose production stimulated by 10(-9) mol/l glucagon was slightly greater in islet amyloid polypeptide-pre-treated livers than in a control group without addition of islet amyloid polypeptide (5 min: 3.60 +/- 3.36 vs 1.67 +/- 1.28 mumol.min-1 x 100 g body weight-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Longer-term regulation of pyruvate dehydrogenase kinase in cultured rat hepatocytes

The activities of pyruvate dehydrogenase (PDH) kinase and of PDH kinase activator protein (KAP) were increased 2-2.4-fold during 25 h of culture of hepatocytes from fed rats with glucagon plus n-octanoate. PDH kinase activity in hepatocytes from starved rats (initially 2.2 x fed control) fell during 25 h of culture in medium 199 (to 1.5 x fed control), but was maintained by glucagon plus octanoate. Dibutyryl or 8-bromo cyclic AMP increased PDH kinase activity 2-2.2-fold in hepatocytes from fed rats, but phenylephrine and isoproterenol (isoprenaline) were without effect. Insulin blocked the action of glucagon to increase PDH kinase activity and decreased the effect of octanoate and octanoate plus glucagon. It is suggested that the effects of starvation to increase activities of PDH kinase and of KAP in liver are mediated by alterations in circulating concentrations of glucagon, fatty acids and insulin and in hepatic cyclic AMP.

63 PARADOXICAL EFFECTS OF GLUCAGON ON DE NOVO PURINE SYNTHESIS IN RAT LIVER

In the prereplicative period of regenerating rat liver after a 70% hepatectomy, “the rate of de novo purine synthesis” (“purine synthesis”) increases by 2.4-fold in parallel with plasma glucagon (2.5-fold), hepatic cyclic AMP (cAMP) (2-fold) in the second peak, 5-phosphoribosyl 1-pyrophosphate (PRPP) (3.0-fold) and specific activity of amidophosphoribosyltransferase (ATase) (1.8-fold) (1). Based on these correlations, it was hypothesized that glucagon promotes liver regeneration by increasing “purine synthesis”. To test this hypothesis, the effect of infusion or bolus administration of glucagon or dibutyryl cAMP (Bt2cAMP) was studied in rat liver in regard to “purine synthesis”, ATase activity, concentrations of cAMP, PRPP and purine ribonucleotides. “Purine synthesis” increased (3.5-fold) by infusion for 12 hours (2), but was transiently inhibited by bolus administration (0.5-fold), in spite of increased cAMP (5- vs. 50-fold) and PRPP (1.5- vs. 2.4-fold) concentrations in a dose-dependent and time-dependent manner in both conditions (3). All the other factors were unchanged. These paradoxical effects of glucagon or Bt2cAMP on “purine synthesis” suggest that the mechanism of regulation of “purine synthesis” includes differential phosphorylation of proteins including ATase depending on the difference of magnitude and duration of cAMP increase. (1) Am.J.Physiol. 252, G373, 1986, (2) Am.J.Physiol. 253, E684, 1987, (3) Metabolism, 35, 758, 1986

Glycogenolytic and haemodynamic responses to heat-aggregated immunoglobulin G and prostaglandin E2 in the perfused rat liver.

Infusion of heat-aggregated immunoglobulin G (HAG) into perfused livers from fed rats caused transient increases in hepatic glycogenolysis and portal-vein pressure, accompanied by a transient increase in hepatic glycogen phosphorylase alpha content. The hepatic responses to HAG were inhibited by indomethacin (2 microM). In contrast, HAG was without effect on phosphorylase alpha content and glucose output in isolated hepatocytes. HAG infusion caused a transient decrease in hepatic cyclic AMP. Lowering the extracellular Ca2+ concentration to 6 or 50 microM attenuated markedly the glycogenolytic and haemodynamic responses to HAG; efflux of Ca2+ from the liver was not observed in response to HAG. Co-infusion of the specific platelet-activating-factor antagonist U-66985 (1-O-octadecyl-2-O-acetyl-sn-glycero-3-phosphoric acid 6'-trimethylammoniumhexyl ester) did not attenuate the glycogenolytic response to HAG. Infusion of prostaglandin E2 caused increases in glucose output, portal-vein pressure and the reduction state of the cytosolic NAD(H) redox couple similar to those seen with HAG. The present study suggests that the glycogenolytic activation after HAG infusion may be an indirect consequence of the haemodynamic response of the hepatic vasculature to stimulation of the reticuloendothelial cells of the liver.

Acute effects of starvation and treatment of rats with anti-insulin serum, glucagon and catecholamines on the state of phosphorylation of hepatic 3-hydroxy-3-methylglutaryl-CoA reductase in vivo.

The fraction of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase in the dephosphorylated (active) form in rat liver in vivo was measured after various experimental treatments of animals. Intraperitoneal injection of glucose (to raise serum insulin concentrations) into rats 4 h into the light phase (L-4) resulted in a transient (30 min) increase in the expressed (E)/total (T) activity ratio of HMG-CoA reductase without any change in total activity (obtained after complete dephosphorylation of the enzyme). Conversely, intravenous injection of guinea-pig anti-insulin serum into rats 4 h into the dark phase (D-4) significantly depressed the E/T ratio within 20 min. Intravenous injection of glucagon into normal rats at this time point did not affect the degree of phosphorylation of the enzyme, in spite of a 10-fold increase in hepatic cyclic AMP concentration induced by the hormone treatment. A 3-fold increase in the concentration of the cyclic nucleotide induced by adrenaline infusion was similarly ineffective in inducing any change in expressed or total activities of hepatic HMG-CoA reductase. However, when insulin secretion was inhibited, either by the induction of streptozotocin-diabetes or by simultaneous infusion of somatostatin, glucagon treatment was able to depress the expressed activity of HMG-CoA reductase (i.e. it increased the phosphorylation of the enzyme). Therefore insulin appears to have a dominant role in the regulation of the phosphorylation state of hepatic HMG-CoA reductase. In apparent corroboration of this suggestion, short-term 4 h food deprivation of animals before D-4 resulted in a marked decrease in the E/T activity ratio of reductase, which was not affected further by an additional 8 h starvation. By contrast, the total activity of the enzyme was not significantly affected by 4 h starvation, but was markedly diminished after 12 or 24 h starvation. Longer-term starvation also produced a chronic increase in the degree of phosphorylation of the enzyme. These results are discussed in relation to the role of reversible phosphorylation in the control of hepatic HMG-CoA reductase activity in vivo.

Stimulation of glycogenolysis by adenine nucleotides in the perfused rat liver.

Infusion of adenine nucleotides and adenosine into perfused rat livers resulted in stimulation of hepatic glycogenolysis, transient increases in the effluent perfusate [3-hydroxybutyrate]/[acetoacetate] ratio, and increased portal vein pressure. In livers perfused with buffer containing 50 microM-Ca2+, transient efflux of Ca2+ was seen on stimulation of the liver with adenine nucleotides or adenosine. ADP was the most potent of the nucleotides, stimulating glucose output at concentrations as low as 0.15 microM, with half-maximal stimulation at approx. 1 microM, and ATP was slightly less potent, half-maximal stimulation requiring 4 microM-ATP. AMP and adenosine were much less effective, doses giving half-maximal stimulation being 40 and 20 microM respectively. Non-hydrolysed ATP analogues were much less effective than ATP in promoting changes in hepatic metabolism. ITP, GTP and GDP caused similar changes in hepatic metabolism to ATP, but were 10-20 times less potent than ATP. In livers perfused at low (7 microM) Ca2+, infusion of phenylephrine before ATP desensitized hepatic responses to ATP. Repeated infusions of ATP in such low-Ca2+-perfused livers caused homologous desensitization of ATP responses, and also desensitized subsequent Ca2+-dependent responses to phenylephrine. A short infusion of Ca2+ (1.25 mM) after phenylephrine infusion restored subsequent responses to ATP, indicating that, during perfusion with buffer containing 7 microM-Ca2+, ATP and phenylephrine deplete the same pool of intracellular Ca2+, which can be rapidly replenished in the presence of extracellular Ca2+. Measurement of cyclic AMP in freeze-clamped liver tissue demonstrated that adenosine (150 microM) significantly increased hepatic cyclic AMP, whereas ATP (15 microM) was without effect. It is concluded that ATP and ADP stimulate hepatic glycogenolysis via P2-purinergic receptors, through a Ca2+-dependent mechanism similar to that in alpha-adrenergic stimulation of hepatic tissue. However, adenosine stimulates glycogenolysis via P1-purinoreceptors and/or uptake into the cell, at least partially through a mechanism involving increase in cyclic AMP. Further, the hepatic response to adenine nucleotides may be significant in regulating hepatic glucose output in physiological and pathophysiological states.

The effects of stress and injury on the activity of phosphoenolpyruvate carboxykinase in the liver of the rat.

The effects of stress (diethyl ether anaesthesia for 4-8 min, or intravenous injection of 0.05 ml of a dimethyl sulphoxide/water mixture) and of a scald injury given under ether anaesthesia on hepatic PEPCK (phosphoenolpyruvate carboxykinase, EC 4.1.1.32) were studied in the post-absorptive rat. Injury raised PEPCK activity by about 70% in 2 h and by over 100% in 4 h, over three times as fast as in animals that had only been handled (controls). The two stresses, both of types commonly imposed in animal experiments, had almost as much effect as injury for the first 2 h, although much less thereafter. The roles of sympathetic stimulation and corticosterone in mediating these rises were studied by using alpha beta-blockers and trilostane respectively as inhibitors. (Trilostane only decreased corticosterone concentrations to a little above control values.) The ether-induced increase was somewhat decreased by alpha beta-blockade, but was only eliminated by combined alpha beta-blockade and trilostane. After injury, however, PEPCK synthesis was unaffected by either alpha beta-blockade or trilostane, although it was decreased by their combined action; and it seems that either corticosterone or sympathetic stimulation was sufficient to stimulate PEPCK synthesis maximally. Stimulation by corticosterone was much greater than reported previously by others, for reasons that are discussed. Sympathetic stimulation may have been mediated by glucagon and cyclic AMP, since injury raised portal glucagon concentrations, and stress and injury raised those of hepatic cyclic AMP. PEPCK synthesis was, however, stimulated despite increases in portal insulin concentration, and was not related to the [insulin]/[glucagon] ratio. Thus stress and injury over-rode normal control mechanisms.

Hepatic cylic AMP generation and ornithine decarboxylase induction by glucagon and beta adrenergic agonists

The relationship of hepatic ornithine decarboxylase (ODC) activity to cyclic AMP levels and nutritional status was studied in the preweanling rat. Previous studies demonstrated that 2 hr without food causes a loss of hepatic ODC induction after glucagon or catecholamine injection. Isoproterenol or glucagon administration produced increased hepatic cyclic AMP and tyrosine aminotransferase activity which were not prevented by nutritional deprivation. Blockade of hepatic β 2 receptors by the selective antagonist ICI 118, 551 prevented increased cAMP levels and ODC activity after isoproterenol administration. Blockade of β 1 receptors by atenolol did not prevent increased cAMP levels or ODC induction by isoproterenol although it did block activation of cardiac ODC. The phosphodiesterase inhibitor R020-1724 increased hepatic cAMP levels as well as ODC and TAT activities, although the increase in ODC activity was attenuated by nutritional deprivation. R020-1724 also potentiated the induction of hepatic ODC after glucagon or isoproterenol administration. Administration of 8-bromo cAMP elevated hepatic ODC activity regardless of nutritional status but also elevated serum levels of growth hormone and corticosterone. Hepatic ODC induction by glucagon or β 2 agonists can be dissociated from changes in cAMP levels during nutritional deprivation.

Decreases in hepatic fructose-2,6-bisphosphate level and fructose-6-phosphate,2-kinase activity in diabetic mice: A close relationship to the development of ketosis

Hyperglycemic mice with streptozotocin diabetes were divided into two groups according to the presence or absence of ketosis. No difference in blood glucose level between two groups was observed in this experiment. However, hepatic fructose-2,6-P 2 level and fructose-6-P,2-kinase activity were decreased only in ketotic diabetic mice. Similar decreases in those indices were observed in 48-h starved normal mice. In ketotic diabetes, insulinization for 24 h was required to normalize fructose-2,6-P 2 level and fructose-6-P,2-kinase activity, while glucose administration normalized altered fructose-2,6-P 2 metabolism in starvation only in 30 min. Hepatic cyclic AMP was increased neither in ketotic nor in non-ketotic diabetic mice. These results indicate that the decrease in hepatic fructose-2,6-P 2 level in diabetes is apparently related to the occurrence of ketosis, but not to hyperglycemia. The mechanisms of the decrease in fructose-6-P,2-kinase activity in ketotic diabetes and starvation are discussed.

Age-related changes in the control of hepatic cyclic AMP levels by alpha 1- and beta 2-adrenergic receptors in male rats.

Hepatocytes from juvenile male rats (80-110 g) showed a 12-fold elevation of cAMP in response to epinephrine, which was mediated by beta 2-adrenergic receptors. In these cells, either alpha 1- or beta 2-adrenergic stimulation alone activated phosphorylase and glucose release although the alpha 1-phosphorylase response was 10-fold more sensitive to epinephrine and resulted in more rapid (by 10-20 s) activation of the enzyme. This suggests that the beta 2-adrenergic response is functionally unimportant for glycogenolysis, even in juvenile rats. beta 2-Adrenergic stimulation did, however, produce an increase in the rate of gluconeogenesis from [U-14C] lactate in these cells. Aging in the male rat was associated with attenuation of the beta 2-adrenergic cAMP response coupled with the emergence of an alpha 1-receptor-mediated accumulation of cAMP. The order of potency displayed by the alpha 1-adrenergic/cAMP system to adrenergic agonists and antagonists was identical with that of the alpha 1-adrenergic/Ca2+ system. These data suggest that, in maturity, hepatic alpha 1-receptors become linked to 2 separate transduction mechanisms, namely Ca2+ mobilization and cAMP generation. Calcium depletion of hepatocytes from adult, but not juvenile, male rats increased the alpha 1-component of the cAMP response to epinephrine, but under these conditions, alpha 1-activation of phosphorylase occurred more slowly than in calcium-replete cells. Blockade of alpha 2-adrenergic receptors did not significantly modify catecholamine effects on hepatocyte cAMP or phosphorylase a levels in male rats at any age studied, suggesting a lack of functional significance for these receptors in the regulation of glycogenolysis.

Interaction of thyroid hormones and cyclic AMP in the stimulation of hepatic gluconeogenesis

The possible interaction of l-3,3′,-5-triiodthyronine (T 3) and cycli AMP on hepatic gluconeogenesis was investigated in perfused livers isolated from hypothyroid rats starved for 24 h. T 3 (1·10 −6) and cyclic AMP (2·10 −4 M) increased hepatic gluconeogenesis from alanine within 30–60 min perfusion time (+85%/ + 90%), both were additive in their action (+191%). Concomitantly, α-amino[ 14C]isobutyric acid as well as net alanine uptake and urea production were elevated by T 3 and by cyclic AMP. T 3 increased the oligomycin-sensitive O 2 consumption and the tissue ‘overall’ ATP/ADP ratio, whereas cyclic AMP showed only a minor effect on cellular energy metabolism. As was observed recently for cyclic AMP, the stimulating action of T 3 on hepatic gluconeogenesis was independent of exogenous Ca 2+ concentration. T 3 by itself affected neither the total nor the protein-bound hepatic cyclic AMP contents, pyruvate kinese ( v:0.15 mM) activation nor the tissue levels of gluconeogenic intermediates. In contrast, cyclic AMP itself — although less effective than in euthyroid livers — decreased pyruvate kinase activity in hypothyroid livers with a concomitant increase in hepatic phospho enolpyruvate concentration. This resulted in a ‘crossover’ between pyruvate and phospho enolpyruvate. Cyclic AMP action was not affected by the further addition of T 3. Glucagon (1·10 −8 M) was less effective in hypo-than in euthyroid livers in increasing endogenous cyclic AMP content, deactivating pyruvate kinase and stimualting glucose production; this is normalized by the further addition of 1-methyl-3-isobutylxanthine (50 μM). It is concluded that T 3 stimulats hepatic gluconeogenesis by a cyclic-AMP-independent mechanism. In addition, the stimulatory action of cyclic AMP and glucagon with respect to hepatic gluconeogenesis is reduced in hypothyroidism. This may be explained by an increase in hepatic phosphodiesterase activity.

Hormonal control of glycogenolysis and the mechanism of action of adrenaline in amphibian liver in vitro

In in vitro cultures of liver from Ambystoma mexicanum glycogenolysis was stimulated by adrenaline, glucagon, and vasopressin in a dose-dependent manner. Maximum activity was seen at 10 −6 M hormone while 10 −9 M was without effect. Dibutyryl cyclic AMP (10 −3 M) stimulated glycogenolysis maximally although 10 −5 M had no effect. The glucose release brought about by adrenaline was blocked by the β-adrenergic antagonist propranolol but not by prazosin or yohimbine which are α 1- and α 2-adrenergic antagonists. Cyclic AMP concentrations in liver were elevated within 1 min of administration of adrenaline and remained elevated for at least 60 min. Phosphorylase a activity was elevated 10 min after addition of adrenaline and remained elevated for at least 6 hr. The rise in hepatic cyclic AMP concentration and phosphorlyase a activity was largely blocked by propranolol. These findings are consistent with adrenaline acting via a β-adrenergic receptor in A. mexicanum. Glycogenolysis in A. mexicanum liver was stimulated by isoprenaline and phenylephrine and in each case the stimulation was reduced in the presence of propranolol but unaffected by phentolamine. High concentrations of methoxamine, a specific α 1-agonist, had no effect upon glycogenolysis. These findings suggest that α-adrenergic receptors play no role in regulation of glycogenolysis in A. mexicanum.