Increased glucagon receptors in chronically hypersomatotrophic and hyperglucagonemic rats

The effect of increased levels of growth hormone on glucagon binding by isolated hepatocytes and on the cellular cyclic AMP response to glucagon was evaluated in rats bearing growth hormone-secreting tumor (Mt-T-W15) and in rats treated with rat growth hormone. An increased binding, due to an increased number of receptors, was observed in both groups of animals. Glucagon binding did not correlate with plasma glucagon levels, suggesting a failure of down regulation, possibly due to an effect of growth hormone and insulin on the number of receptors. Tumor-bearing and growth hormone-treated rats had larger hepatocytes so that, when hormone binding was expressed in terms of square micrometer of membrane surface, it appeared decreased. When the tumor was removed the increase in the number of glucagon receptors per cell persisted, even though the average cell size returned toward normal. It is suggested that this retention of the receptors may have been the result of continuing hyperinsulinism. Basal cAMP levels were elevated in hepatocytes of tumor-bearing and growth hormone-treated animals, possibly due to cell hypertrophy. On the other hand, the maximum cAMP response to glucagon was not altered by the experimental procedures. A negative effect of insulin on cAMP accumulation may explain this apparent paradox. Indeed, hepatocytes isolated from rats following tumor removal, but with continuing hyperinsulinemia, had a lower maximum cAMP response, even though the glucagon binding per cell or per unit of cell surface was increased.     

Increased activity of phosphate-dependent glutaminase in liver mitochondria as a result of glucagon treatment of rats.

1. Injection of rats with glucagon leads to an increased effective activity of glutaminase in subsequently isolated liver mitochondria. 2. This effect of glucagon is manifested as a decreased requirement of glutaminase for phosphate in the presence of HCO3-. The HCO3--concentration-dependence is unchanged. 3. The effect of glucagon is lost on disruption of the mitochondria. 4. In accordance with previous reports, incubation of mitochondria in hypo-osmotic media also increases the effective activity of glutaminase. Glucagon increases glutamine hydrolysis at intermediate osmolarities of the suspending medium, but does not affect glutaminase activity when it is already maximally activated by hypo-osmotic conditions. 5. From this and previous work, it seems that hypo-osmotic incubation conditions, EDTA and glucagon may all activate glutaminase by a common mechanism. It is postulated that this mechanism involves modification of the interaction of glutaminase with the mitochondrial inner membrane.     

Characterization of glucagon receptors in Golgi fractions of fetal rat liver

The present study was designed to determine if Golgi fractions from fetal rat liver contain glucagon receptors and to characterize the properties of such receptors. Purification patterns of liver plasma membranes and Golgi fractions from fetal and adult rats were similar, as verified by morphological and biochemical approaches. Glucagon binding was greater in plasma membranes of adult than fetal rats, while in Golgi fractions glucagon binding was similar in both groups. The modifications in in glucagon binding reflect changes in glucagon receptors. Glucagon association and glucagon receptor inactivation by liver membranes were similar in the two groups of animals, while glucagon degradation was lower in fetal than in adult rats.     

Characterization of glucagon receptors in Golgi fractions of rat liver: evidence for receptors that are uncoupled from adenylyl cyclase

Glucagon receptors have been identified and characterized in intermediate (Gi) and heavy (Gh) Golgi fractions from rat liver. At saturation, plasma membranes bound 3500 fmol of hormone/mg of membrane protein, while Gi and Gh bound 24 and 60 fmol of 125I-glucagon/mg of protein, respectively. Half-maximal saturation of binding to plasma membranes, Gi, and Gh occurred at approximately 4, 10, and 20 nM 125I-glucagon, respectively. Trichloroacetic acid precipitation of intact, but not degraded, glucagon was used to correct binding isotherms for hormone degradation. After such correction, half-maximal saturation of binding to plasma membranes, Gi, and Gh was observed in the presence of approximately 2, 7, and 14 nM hormone, respectively. After 90 min of dissociation in the absence of guanosine 5'-triphosphate (GTP), 86% of 125I-glucagon remained bound to plasma membranes, whereas only 42% remained bound to Golgi membranes. GTP significantly increased the fraction of 125I-glucagon released from plasma membranes but only slightly augmented the dissociation of hormone from Golgi fractions. 125I-Glucagon/receptor complexes solubilized from plasma membranes fractionated by gel filtration as high molecular weight (Kav = 0.16), GTP-sensitive complexes and lower molecular weight (Kav = 0.46), GTP-insensitive complexes. 125I-Glucagon complexes solubilized from Golgi membranes fractionated almost exclusively as the lower molecular weight species. The lower affinity of Golgi than plasma membrane receptors for hormone, the ability of glucagon to stimulate plasma membrane, but not Golgi membrane, adenylyl cyclase, and the near absence of high molecular weight, GTP-sensitive complexes in solubilized Golgi membranes demonstrate that plasma membrane contamination of Golgi fractions cannot account for the 125I-glucagon binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased atypical PKC activity in endurance-trained human skeletal muscle

Exercise training may modulate protein content and enzyme activities in skeletal muscle. However, it is not known whether atypical protein kinase C (aPKC) is affected by training. Thus, we investigated aPKC, extracellular-regulated protein kinase 1/2 (ERK 1/2), and P38 mitogen-activated protein kinase (P38 MAPK) activities and expression in skeletal muscle from untrained and endurance-trained subjects at rest and after 20min of cycle exercise (80% of VO(2peak)). Activities of aPKC (P<0.05) and ERK 1/2 (P=0.06), but not phosphorylation of P38 MAPK, were higher in trained than in sedentary subjects at rest. Exercise increased the activities of ERK 1/2 (P<0.01) and aPKC (P<0.05) and the phosphorylation (Thr180/Tyr182) of P38 MAPK (P<0.01) similarly in muscle from trained and sedentary subjects. Protein expression of the kinases was similar in trained and sedentary muscle. The increased aPKC activity in exercise-trained subjects could be important in explaining the enhanced insulin action in these individuals.     

Ventilatory CO2 response in endurance-trained rats

A CO2 rebreathing technique was used to assess possible changes in the ventilatory response to CO2 in rats following a 14-week swim training program. Over the final 9 weeks, the rats swam 1 hr per day with a weight of 2.5% of the body weight attached to the tail. Ventilation was measured by a barometric method in awake, restrained rats in a total body plethysmography at CO2 concentrations of 0, 2, 4, 6, and 8%, with an initial O2 concentration of approximately 100%. Ventilation increased in the trained rats with increasing CO2 from 775ml . min-1 . kg-1 at 0% CO2 to 1,387 ml . min-1 . kg-1 at 8% CO2. This increase was a consequence of a 34% increase in tidal volume and a 32% increase in breathing frequency. In comparison with a group of sedentary control rats, there was a significantly higher ventilation and tidal volume at 0% CO2; however, this difference disappeared with increasing levels of CO2. A significantly lower resting heart rate was observed in the exercised (296 +/- 44 beats . min-1, mean +/- SD) compared to the sedentary control rats 380 +/- 42). It was concluded that, while the normal training response of resting bradycardia was observed following this duration and intensity of training, endurance swimming had no significant effect on the ventilatory response to CO2 in the rat.     

Decreased density of beta1-adrenergic receptors in preneoplastic and neoplastic liver lesions of F344 rats

There is some evidence that rodent hepatocarcinogenesis is accompanied by changes in the adrenergic responsiveness of liver cells to catecholamines. In this study, immunohistochemical expression of beta1-adrenergic receptors (beta1-ARs) has been examined in spontaneous and chemically induced preneoplastic and neoplastic liver lesions of female and male Fischer 344 rats. An antibody specific for beta1-AR subtype was used. The study was carried out on archival formalin-fixed and paraffin-embedded livers from rats used in a previous study of hepatocarcinogenesis. One control group given distilled water by gavage, and two experimental groups, one initiated with a single dose of diethylnitrosamine (DEN) and one initiated with DEN and continuously treated with phenobarbital (PB) were examined. Rats were sacrificed after 2, 4, 8 and 21 months of experimentation. All types of liver putative preneoplastic lesions examined (basophilic, glycogen-retaining, or mixed cell foci) show a lower density of beta1-ARs than the surrounding normal liver parenchyma, either in control and in DEN-treated or DEN+PB-treated rats. No immunostaining is detectable in several altered cell foci. Hepatocellular adenomas and hepatocellular carcinomas also show a very low density of beta1-ARs, extensive areas completely devoid of beta1-ARs being mingled with areas showing a weak immunostaining.     

Nascent high density lipoproteins from liver perfusates of orotic acid-fed rats

Uniformly fatty livers from orotic acid-fed rats secreted almost no very low density lipoproteins (VLDL) but normal amounts of nascent high density lipoproteins (HDL) accumulated in perfusates. When lecithin:cholesterol acyltransferase (LCAT) was inhibited, nascent HDL were uniformly discoidal and lacked cholesteryl esters. Lipid and apoprotein compositions of nascent HDL from normal and fatty livers were similar whether LCAT was inhibited or not. Apolipoprotein B-100 was not detected in perfusates of uniformly fatty livers, but small amounts of apolipoprotein B-48 were present in HDL2 fractions. Nascent lipoproteins were not seen in Golgi compartments, but lipid-rich particles were clearly evident in endoplasmic reticulum cisternae adjacent to the cis face of the Golgi complex, suggesting that orotic acid blocks VLDL secretion by preventing translocation of nascent particles from the endoplasmic reticulum to the cis Golgi compartment. The accumulation of normal amounts of discoidal HDL in liver perfusates despite virtual absence of triglyceride-rich lipoproteins in Golgi secretory compartments, the space of Disse, and the perfusate is inconsistent with the concept that nascent HDL are exclusively a product of surface remnants cast off during lipolysis of chylomicrons and VLDL.     

Induction of liver cell proliferation in intact rats by amines and glucagon

Administration of isoproterenol followed by glucagon induced DNA synthesis in the liver of intact rats 12-fold over that of non-treated rats. The mixture of heparin, histamine or putrescine, and serotonin followed by glucagon increased DNA synthesis to the same level as in the case of isoproterenol. The stimuli for the induction of liver DNA synthesis appear to be composed of two steps. The first step is stimulated by some catecholamines, the mast-cell components etc., and the second step by glucagon. Each step alone resulted only in a slight increase in DNA synthesis. These results suggest that the substances as mentioned above may be involved in the regulation of liver cell proliferation, and that liver cell proliferation can be induced markedly when both steps of stimuli are provided.     

Arginine stimulated insulin and glucagon release from islets transplanted into the liver of diabetic rats

We investigated the ability of intraportal transplanted islets to release insulin and glucagon after stimulation with arginine. Furthermore, the islet volume and hormone content of the recipient pancreas were analyzed. Three months after syngeneic portal islet transplantation the liver of STZ-diabetic rats was perfused in vitro in the presence of different arginine concentrations. Transplanted islets preserve their functional integrity for at least three months indicated by a stimulus adequate insulin release and contribute substantially to the observed amelioration of the diabetic state. The islet and B-cell volume as well as the insulin and glucagon content of the recipient pancreas are still markedly decreased three months after islet transplantation when compared with healthy controls.     

Leukotriene and purinergic receptors are involved in the hyperpolarizing effect of glucagon in liver cells

The pancreatic hormone glucagon hyperpolarizes the liver cell membrane. In the present study, we investigated the cellular signalling pathway of glucagon-induced hyperpolarization of liver cells by using the conventional microelectrode method. The membrane potential was recorded in superficial liver cells of superfused mouse liver slices. In the presence of the K⁺ channel blockers tetraethylammonium (TEA, 1 mmol/l) and Ba²⁺ (BaCl₂, 5 mmol/l) and the blocker of the Na⁺/K⁺ ATPase, ouabain (1 mmol/l), no glucagon-induced hyperpolarization was observed confirming previous findings. The hyperpolarizing effect of glucagon was abolished by the leukotriene B₄ receptor antagonist CP 195543 (0.1 mmol/l) and the purinergic receptor antagonist PPADS (5 μmol/l). ATPγS (10 μmol/l), a non-hydrolyzable ATP analogue, induced a hyperpolarization of the liver cell membrane similar to glucagon. U 73122 (1 μmol/l), a blocker of phospholipase C, prevented both the glucagon- and ATPγS-induced hyperpolarization. These findings suggest that glucagon affects the hepatic membrane potential partly by inducing the formation and release of leukotrienes and release of ATP acting on purinergic receptors of the liver cell membrane.     

Leukotriene and purinergic receptors are involved in the hyperpolarizing effect of glucagon in liver cells

The pancreatic hormone glucagon hyperpolarizes the liver cell membrane. In the present study, we investigated the cellular signalling pathway of glucagon-induced hyperpolarization of liver cells by using the conventional microelectrode method. The membrane potential was recorded in superficial liver cells of superfused mouse liver slices. In the presence of the K+ channel blockers tetraethylammonium (TEA, 1 mmol/l) and Ba2+ (BaCl2, 5 mmol/l) and the blocker of the Na+/K+ ATPase, ouabain (1 mmol/l), no glucagon-induced hyperpolarization was observed confirming previous findings. The hyperpolarizing effect of glucagon was abolished by the leukotriene B4 receptor antagonist CP 195543 (0.1 mmol/l) and the purinergic receptor antagonist PPADS (5 micromol/l). ATPgammaS (10 micromol/l), a non-hydrolyzable ATP analogue, induced a hyperpolarization of the liver cell membrane similar to glucagon. U 73122 (1 micromol/l), a blocker of phospholipase C, prevented both the glucagon- and ATPgammaS-induced hyperpolarization. These findings suggest that glucagon affects the hepatic membrane potential partly by inducing the formation and release of leukotrienes and release of ATP acting on purinergic receptors of the liver cell membrane.     

Decreased liver cytosol glucocorticoid receptors in protein-deficient rats

A low protein diet (5% as compared to a control 21% protein diet, $\text{ad libitum}$) caused a significant decrease in the concentration of liver cytoplasmic glucocorticoid receptors; the equilibrium dissociation constant (Kd) did not change. The maximum decrease occurred in two weeks and was reversible upon substitution of the low protein by a control diet. This influence of protein deficiency could not be attributed to elevated plasma corticosterone levels since a comparable increase in plasma corticosterone of calorie-deficient rats (21% protein diet in restricted quantity) did not decrease glucocorticoid receptors and the difference in receptor levels of control and protein deficient animals persisted following adrenalectomy. These results suggest that glucocorticoids might not exert their usual biologic effects in the presence of protein malnutrition.     

Aldrin epoxidase activity in liver microsomes from normal or streptozotocin-diabetic rats: Comparison with activity in isolated hepatocytes from normal rats incubated with glucagon

Aldrin epoxidase activity in liver microsomes from streptozotocin-diabetic rats is only 40% of that from normal rats. Epoxidation of aldrin has also been assayed in freshly isolated hepatocytes from normal rats. Addition of 10^–7 M glucagon to the incubation medium leads to a decreased aldrin epoxidase activity. Owing to the previously reported phosphorylation of a purified cytochrome P-450 isozyme, it is postulated that the cytochrome P-450 dependent aldrin epoxidase may be regulated by a glucagon induced phosphorylation process.     

Increased uptake of alpha-hydroxy aldehyde-modified low density lipoprotein by macrophage scavenger receptors

Reactive aldehydes can be formed during the oxidation of lipids, glucose, and amino acids and during the nonenzymatic glycation of proteins. Low density lipoprotein (LDL) modified with malondialdehyde are taken up by scavenger receptors on macrophages. In the current studies we determined whether alpha-hydroxy aldehydes also modify LDL to a form recognized by macrophage scavenger receptors. LDL modified by incubation with glycolaldehyde, glyceraldehyde, erythrose, arabinose, or glucose (alpha-hydroxy aldehydes that possess two, three, four, five, and six carbon atoms, respectively) exhibited decreased free amino groups and increased mobility on agarose gel electrophoresis. The lower the molecular weight of the aldehyde used for LDL modification, the more rapid and extensive was the derivatization of free amino groups. Approximately 50-75% of free lysine groups in LDL were modified after incubation with glyceraldehyde, glycolaldehyde, or erythrose for 24-48 h. Less extensive reductions in free amino groups were observed when LDL was incubated with arabinose or glucose, even at high concentration for up to 5 days. LDL modified with glycolaldehyde and glyceraldehyde labeled with (125)I was degraded more extensively by human monocyte-derived macrophages than was (125)I-labeled native LDL. Conversely, LDL modified with (125)I-labeled erythrose, arabinose, or glucose was degraded less rapidly than (125)I-labeled native LDL. Competition for the degradation of LDL modified with (125)I-labeled glyceraldehyde was nearly complete with acetyl-, glycolaldehyde-, and glyceraldehyde-modified LDL, fucoidin, and advanced glycation end product-modified bovine serum albumin, and absent with unlabeled native LDL.These results suggest that short-chain alpha-hydroxy aldehydes react with amino groups on LDL to yield moieties that are important determinants of recognition by macrophage scavenger receptors.