Effects of chronic glucagon administration on rat lipoprotein composition

Male adult rats of the Wistar strain received daily at 9 a.m. and 5 p.m. 10 micrograms of Zn-protamine glucagon (Novo) for 21 days by subcutaneous injections. Plasma levels of cholesterol, triacylglycerol and phospholipids were decreased by 47, 40 and 21%, respectively. Lipoproteins were separated by sequential ultracentrifugation. Concentrations of cholesterol, phospholipids and proteins were decreased in chylomicrons, VLDL, LDL2 (1.040-1.063 g/ml) and HDL, LDL2 being the most affected by glucagon treatment (-70%). Triacylglycerol levels were decreased only in chylomicrons and VLDL. The relative proportions of cholesterol, triacylglycerol, phospholipids and proteins in lipoproteins were virtually unchanged by glucagon, suggesting a reduced number of some lipoprotein particles in plasma. However, lipoproteins of glucagon-treated rats were depleted in cholesteryl esters, while the proportion of triacylglycerol increased in LDL and HDL. Apo E contents were decreased in plasma, LDL1 (1.006-1.040 g/ml), LDL2 and HDL, whereas apo B100 proportions increased in VLDL and LDL1 in glucagon-treated rats. Glucagon appeared to be a potent hypolipidemic agent affecting mainly the apo-E-rich lipoproteins.     

Effects of chronic glucagon administration on cholesterol and bile acid metabolism

Male adult Wistar rats received daily, at 9 a.m. and 5 p.m., 10 micrograms of Zn-protamine glucagon for 21 days by subcutaneous injections. The blood glucose level was not significantly modified. Cholesterol and triacylglycerol levels were decreased by 40 and 70% in plasma but not in the liver. The rates of cholesterol turnover processes were determined in vivo with an isotope balance method. Internal secretion of cholesterol (13.8 +/- 0.5 mg/day per rat in control rats and 22.4 +/- 0.9 mg/day per rat in glucagon-treated rats) and cholesterol transformation into bile acids were strikingly increased by chronic administration of glucagon. Biliary secretion rates of bile acids measured by a wash-out method were increased by 139%, while the intestinal bile acid pool was not changed. The enterohepatic cycle number was increased from five per day in control rats to nine per day in glucagon-treated rats. An increased turnover rate of the exchangeable cholesterol would explain the hypocholesterolemic effect of glucagon.     

Effects of hormones and pyruvate on the rates of secretion of very-low-density lipoprotein triacylglycerol and cholesterol by rat hepatocytes

The secretion of very-low-density lipoprotein (VLDL) triacylglycerol and cholesterol was determined under various conditions in hepatocytes prepared from rats maintained on a controlled lighting and feeding schedule. The rate of lipogenesis in hepatocytes prepared from rats during the feeding period was 2-3-fold higher than that in cells prepared immediately before the animals had access to food. However, there were no corresponding changes in the rates of secretion of triacylglycerol and cholesterol. Pyruvate alone stimulated triacylglycerol secretion but had no effect on the secretion of cholesterol. Despite its stimulation of lipogenesis, insulin suppressed the secretion of both triacylglycerol and cholesterol. This effect on triacylglycerol secretion was more pronounced when lipogenesis was enhanced in the presence of pyruvate. Thus, insulin may act to alleviate hypertriglyceridaemia, which may arise during periods of increased hepatic lipogenesis. The inhibitory effect of glucagon on cholesterol secretion was much less pronounced than that on the secretion of triacylglycerol. The inhibitory effects of glucagon were reversed by pyruvate on cholesterol secretion differed according to whether glucagon was present or absent. These results suggest that the rate of hepatic VLDL triacylglycerol secretion is not necessarily coupled to the rate of lipogenesis in the liver; nor is there any obligatory coupling between the output of triacylglycerol and cholesterol associated with VLDL.     

Glucagon- and dibutyryl cyclic AMP-produced inhibition of cholesterol ester hydrolase in isolated rat hepatocytes: role of calcium

The regulation of neutral cytosolic cholesterol ester hydrolase was studied in isolated rat liver cells. Addition of glucagon to cell suspensions caused a decrease in the enzyme activity which was significant at 1 nM concentration. The cyclic nucleotide analogue bibutyryl cyclic AMP (10 and 100 microM) also inhibited the esterase activity. In the absence of calcium, glucagon did not produce any effect on the enzyme. To see if calcium was involved in a regulatory mechanism, cholesterol ester hydrolase activity was measured in cytosol from cells preincubated in a medium without calcium and containing EGTA. This treatment produced a marked reduction in cytosolic Ca2+ concentration with a concomitant threefold stimulation of the esterase activity. Readdition of calcium to Ca2(+)-deprived cells diminished the activation due to calcium deficiency. The present results suggest that 1) cholesterol ester hydrolase could be modulated by a cAMP-mediated mechanism elicited by glucagon in which Ca2+ appears to be involved and 2) the enzyme activity may also be regulated by changes in the intracellular calcium concentration.     

Glycemic control in mice with targeted disruption of the glucagon receptor gene.

The action of glucagon in the liver is mediated by G-coupled receptors. To examine the role of glucagon in glucose homeostasis, we have generated mice in which the glucagon receptor was inactivated (GR(-/-) mice). Blood glucose levels were somewhat reduced in GR(-/-) mice relative to wild type, in both the fed and fasted state. Plasma insulin levels were not significantly affected. There was no significant effect on fasting plasma cholesterol or triglyceride levels associated with deletion of the glucagon receptor. Glucose tolerance, as assessed by an oral glucose tolerance test, improved. Plasma glucagon levels were strikingly elevated in both fed and fasted animals. Despite a total absence of glucagon receptors, these animals maintained near-normal glycemia and normal lipidemia, in the presence of circulating glucagon concentrations that were elevated by two orders of magnitude.     

Glucagon receptor antagonism induces increased cholesterol absorption

Glucagon and insulin have opposing action in governing glucose homeostasis. In type 2 diabetes mellitus (T2DM), plasma glucagon is characteristically elevated, contributing to increased gluconeogenesis and hyperglycemia. Therefore, glucagon receptor (GCGR) antagonism has been proposed as a pharmacologic approach to treat T2DM. In support of this concept, a potent small-molecule GCGR antagonist (GRA), MK-0893, demonstrated dose-dependent efficacy to reduce hyperglycemia, with an HbA_1c reduction of 1.5% at the 80 mg dose for 12 weeks in T2DM. However, GRA treatment was associated with dose-dependent elevation of plasma LDL-cholesterol (LDL-c). The current studies investigated the cause for increased LDL-c. We report findings that link MK-0893 with increased glucagon-like peptide 2 and cholesterol absorption. There was not, however, a GRA-related modulation of cholesterol synthesis. These findings were replicated using structurally diverse GRAs. To examine potential pharmacologic mitigation, coadministration of ezetimibe (a potent inhibitor of cholesterol absorption) in mice abrogated the GRA-associated increase of LDL-c. Although the molecular mechanism is unknown, our results provide a novel finding by which glucagon and, hence, GCGR antagonism govern cholesterol metabolism.     

Metabolic consequences of atenolol and propranolol in treatment of essential hypertension.

A six-month study of triglyceride, cholesterol, free fatty acid (FFA), glucose, insulin, growth hormone, and glucagon concentrations was carried out in asymptomatic hypertensive normal-weight men randomly allocated to treatment with atenolol or propranolol. A highly significant increase in the basal plasma triglyceride concentration was observed in propranolol-treated patients after three and six months'' treatment, with a smaller but significant increase in atenolol-treated subjects after six months'' treatment. The changes in triglyceride concentration could not be ascribed to variations in plasma insulin, growth hormone, or glucagon concentrations. Basal FFA concentrations were reduced during the first three months of treatment in both groups but returned to pretreatment levels after six months. Plasma cholesterol concentrations were unchanged by either agent.     

Activities of enzymes of lipid metabolism in Morris hepatoma 7800 C1 cells

(1) The rate of palmitate oxidation in the 7800 C1 Morris hepatoma cells was about 60% of the activity observed in hepatocytes. The stimulatory effect of glucagon in hepatocytes was not observed in the hepatoma cells. The rate of fatty acid synthesis from [2-14C]acetate in the hepatoma cells was 1/20 of the activity in hepatocytes. The conversion of [2-14C]acetate to cholesterol was not different in the two kinds of cell. (2) Acetyl-CoA carboxylase and fatty acid synthetase were significantly decreased in the hepatoma cells. The hepatoma cells had, however, raised activities of malate dehydrogenase (decarboxylating), and glucose-6-phosphate and 6-phosphogluconate dehydrogenases. (3) The activities of the enzymes were not affected by different concentrations of glucose or palmitate in the culture medium. Insulin, dexamethasone, triiothyronine and glucagon had no effect on the enzyme activities. This is in contrast to the adaptation of the peroxisomal beta-oxidation system, which is induced by fatty acids and modified by hormones.     

Binding of glucagon to lipid bilayers

At physiological pH and temperature, glucagon binds to liposomes composed of egg phosphatidylcholine and cholesterol (2:1 mol/mol) in a highly specific manner. The chemical reactivities of the functional groups were determined over the concentration range of 1.0 X 10(-6)-3.0 X 10(-8) M by the method of competitive labelling with 1-fluoro-2,4-dinitrobenzene as the labelling reagent. At concentrations above 3 X 10(-7) M, the amino terminal histidine and the two tyrosine residues showed a marked decrease in reactivity in the presence of liposomes, but the reactivity of the Lys-12 N epsilon-amino group was unaltered. At lower concentrations the Lys-12 reactivity also decreased markedly, owing to a change in the environment of this group. These results indicated that two different forms of glucagon existed over the concentration range studied. Both in the absence and presence of liposomes the Lys-12 N epsilon-amino groups showed a transition in reactivity at 1.8 X 10(-7) M. In the presence of liposomes the other functional groups also showed a transition in reactivity at 2 X 10(-7) M but the change was much smaller. The pattern of reactivities were consistent with the X-ray crystallographic structure of the type 2 glucagon trimer being the predominant species at 10(-6) M, with free monomeric glucagon occurring at 3 X 10(-8) M. A trimerization constant of 4 X 10(13) M-2 at pH 7.5 and 37 degrees C was determined.(ABSTRACT TRUNCATED AT 250 WORDS)     

Secretion of glucagon and insulin in hypophysectomized rats: effect of tolbutamide.

Normal and hypophysectomized (hypox) rats, fed ad libitum, received intraperitoneal injections of tolbutamide (75 mg/kg/day) or of saline for 6 weeks. 24 h after the last injection, blood samples were taken for glucose, insulin and glucagon determinations. In normal rats, tolbutamide treatment did not alter serum glucose, insulin and glucagon, although it suppressed the secretion of insulin and glucagon by the pancreatic islets. In hypox rats, tolbutamide decreased serum glucose and insulin, elevated serum glucagon and stimulated the secretion of glucagon, but not that of insulin by the pancreatic islets. In addition, tolbutamide treatment increased the glucagon response to arginine in normal, but not in hypox rats. The serum glucose response to arginine was decreased by tolbutamide treatment and by hypophysectomy and, thus, appeared independent of the glucagon rise or preexisting glucagon level. We conclude that tolbutamide treatment decreased the secretion of glucagon and insulin in normal rats and stimulated that of glucagon in hypox rats, perhaps because of the low levels of insulin in the serum and in the pancreas of the latter. Our results are compatible with the hypothesis that the pancreatic action of tolbutamide is influenced by the pituitary.     

Effect of dietary carbohydrates on insulin and glucagon receptors in a new model of noninsulin-dependent diabetes-SHR/N-corpulent rat

A new congenic strain of rat, the SHR/N-corpulent, provides a good model for noninsulin-dependent diabetes and was used in the present study. Corpulent rats as compared to their lean littermates are obese, hyperlipidemic, and severely hyperinsulinemic, and show an age-dependent loss of glucose tolerance. Mild fasting hyperglycemia is seen only in corpulent rats fed sucrose. Since dietary sucrose is more lipogenic than starch and since insulin and glucagon are involved in lipid and carbohydrate metabolism, we studied the effect of the type of dietary carbohydrate on insulin and glucagon levels and their receptors in lean and corpulent SHR/N rats. A significant phenotypic effect was observed (corpulent greater than lean) on plasma levels of triglyceride, cholesterol, and insulin. Dietary sucrose increased these parameters in corpulent rats but not in lean rats. Insulin and glucagon binding to liver plasma membranes was lower in corpulent rats than in lean; decreases were due to fewer receptors without a significant change in affinity. Thus, in corpulent rats, in addition to hyperinsulinemia, fewer glucagon receptors and their failure to be regulated by plasma glucagon levels appear to contribute to the hyperlipidemia. Furthermore, the hyperglycemia observed in sucrose-fed corpulent rats may be due to extreme resistance to insulin despite lower plasma glucagon and fewer glucagon receptors.     

Further studies concerning glucagon-induced hypocholesterolemia.

Some of the possible mechanisms responsible for the hypocholesterolemic effect of glucagon were investigated. Glucagon was found to inhibit the intestinal absorption of cholesterol. In addition, it was found to either hasten the rate of egress of lipoprotein cholesterol from the blood into the liver or to retard the rate of re-entry of cholesterol from the liver into the blood. The data do not distinguish between these two possibilities, which indeed may occur simultaneously.     

Glucagon induced inactivation of phosphofructokinase and its counteraction by insulin in isolated hepatocytes from the domestic fowl (Gallus domesticus)

The effects of glucagon and insulin on phosphofructokinase activity in isolated chicken hepatocytes were studied. Phosphofructokinase activity was decreased in extracts of hepatocytes exposed to glucagon both at subsaturating (0.2 mM) and saturating (5 mM) concentrations of fructose 6-phosphate. Both effects were still present after Sephadex G-25 gel filtration and subsequent ammonium sulfate precipitation. Half-maximal effects of glucagon were found between 10(-11) and 10(-10) M glucagon. Insulin alone had no effect but decreased the action of glucagon.     

Effects of glucagon and insulin on fatty acid synthesis and glycogen degradation in the perfused liver of normal and genetically obese (ob/ob) mice.

1. Rapid effects of hormones on glycogen metabolism and fatty acid synthesis in the perfused liver of the mouse were studied. 2. In perfusions lasting 2h, of livers from normal mice, glucagon in successive doses, each producing concentrations of 10(-10) or 10(-9)M, inhibited fatty acid and cholesterol synthesis. In perfusions lasting 40--50 min, in which medium was not recycled, inhibition of fatty acid synthesis was only observed with glucagon at concentrations greater than 10(-9)M. This concentration was about two orders of magnitude higher than that required for the stimulation of glycogen breakdown. Glucagon did not inhibit the activity of acetyl-CoA carboxylase, assayed 10 or 20 min after addition of glucagon (10(-9) or 10(-10)M). It is proposed that the action of glucagon on hepatic fatty acid biosynthesis could be secondary in time to depletion of glycogen. Insulin prevented the effect of glucagon (10(-10)M) on glycogenolysis, but not that of vasopressin. 3. Livers of genetically obese (ob/ob) mice did not show significant inhibition of lipid biosynthesis in response to glucagon, although there was normal acceleration of glycogen breakdown. This resistance to glucagon action was not reversed by food deprivation. Livers of obese mice exhibited resistance to the counteraction by insulin of glucagon-stimulated glycogenolysis, which was reversible by partial food deprivation.     

Role of endocrine pancreas in temperature acclimation

Role of endocrine pancreas in temperature acclimation in rats was investigated. Plasma glucagon level increased and insulin level decreased in cold-acclimated rats (CA). The reverse was observed in heat-acclimated rats (HA). In the pancreas there were no changes in glucagon and insulin in CA, but a decrease in glucagon and an increase in insulin were found in HA. Plasma insulin/glucagon molar ratio (I/G) declined in CA and rose in HA. Pancreatic I/G rose in HA. Acute cold exposure elevated plasma glucagon, but did not affect plasma insulin. Pancreatic glucagon, insulin and I/G were not influenced by acute cold exposure, while plasma I/G decreased. Plasma I/G was inversely correlated with both blood free fatty acids and glucose levels. These results suggest that endocrine pancreas is closely associated with metabolic acclimation to cold and heat through its regulation of the metabolic direction to catabolic phase in cold acclimation and to anabolic phase in heat acclimation.     

Effects of acute and chronic endotoxin treatment on glucagon and insulin receptors on rat liver plasma membranes

The effects of acute and chronic endotoxin treatment on the plasma levels of insulin and glucagon and their binding to rat liver plasma membranes were examined. Both acute and chronic endotoxin administration increased plasma glucagon levels and decreased the glucagon to insulin molar ratio. Acute, but not chronic, endotoxin decreased blood glucose and insulin levels. Glucagon binding was increased in membranes prepared from the acutely treated rats. However, in membranes obtained from rats treated chronically with endotoxin, only insulin binding was increased. The increases in the binding of both insulin and glucagon were the result of increases in receptor sites.     

Binding and action of glucagon in isolated adipocytes from cortisol-treated rats

Evidence for pre-receptor, receptor and post-receptor glucagon defects was investigated in adipocytes from cortisol-treated rats. A decrease in glucagon binding due to a decreased number of receptors was observed. No changes in receptor affinity were detected. Both, the lipolytic response of glucagon and the ability of glucagon to increase basal and theophylline-stimulated cAMP accumulation remained unaltered. Moreover, a hyperglucagonemia accompanied by an increase in glucagon degradation in the serum of cortisol-treated rats was observed. Such alterations could represent a new mechanism by which glucocorticoids exert their biological actions.     

Prostaglandins suppress VLDL secretion in primary rat hepatocyte cultures: relationships to hepatic calcium metabolism.

In primary cultures of rat hepatocytes, prostaglandin E2 and prostaglandin D2 (PGE2 and PGD2) inhibited the secretion of very low density lipoprotein (VLDL)-associated apoB, triacylglycerol, and cholesterol. These effects were concentration-dependent and remained apparent for at least 3 days of culture without an effect on the apoB/triacylglycerol ratio of the secreted VLDL. Prostaglandins had no effect on the overall synthesis of triacylglycerol but triacylglycerol accumulated within the cells, without intracellular accumulation of apoB. PGE2, when added to the medium together with glucagon, increased the inhibition of VLDL secretion, compared to that observed with glucagon alone. However, PGE2 did not increase the stimulatory effect of glucagon on ketogenesis. Unlike glucagon, the prostaglandins did not inhibit fatty acid synthesis nor did they stimulate ketogenesis or production of cAMP. Thus, of all the parameters of hepatic lipid metabolism studied, PGE2 and PGD2 selectively affected VLDL. Selective inhibition of VLDL secretion was also observed with the calcium antagonist verapamil. The divalent cation ionophore A23187 also inhibited VLDL release but, in contrast, also inhibited fatty acid and cholesterol synthesis. The results suggest that VLDL secretion is modulated at some optimal cell calcium concentration that may be mediated selectively by agents such as prostaglandins.     

Role of glucagon in metabolic acclimation to cold and heat

Plasma glucagon concentration increased in 2 weeks cold-acclimated rats, but it returned to normal value in 4 weeks cold-acclimated ones. Plasma free fatty acid (FFA) and β-hydroxybutyrate concentrations also showed the similar pattern of changes. Plasma glucagon and FFA concentrations decreased in both 2 weeks and 4 weeks heat-acclimated animals. Plasma β-hydroxybutyrate concentration was not changed by heat acclimation. Plasma glucose concentration decreased in heat-acclimated animals, while it was not affected by cold acclimation. There was a significant positive correlation between plasma glucagon and FFA levels as a whole in 2 weeks warm-, cold- and heat-acclimated rats, and in 4 weeks warm- and heat-acclimated ones. These findings would appear to indicate that in both cold and heat acclimation glucagon is closely involved as one member of a hormonal team through regulating lipid and carbohydrate metabolism.