Stimulatory Effect Of Glucagon Research Articles

The stimulatory effects of glucagon on cortisol and GH secretion occur independently from FGF-21.

Glucagon stimulation test (GST) is used to assess the hypothalamo-pituitary-adrenal (HPA) and growth hormone (GH) axes with an incompletely defined mechanism. We aimed to assess if glucagon acted through fibroblast growth factor-21 (FGF-21) to stimulate cortisol and GH secretion. The secondary outcome was to determine the relationship of FGF-21 with variable GH responses to GST in obesity. A total of 26 healthy participants; 11 obese (body mass index (BMI) > 30 kg/m2) and 15 leans (BMI < 25 kg/m2) were included. Basal pituitary and target hormone levels were measured and GST was performed. During GST, glucose, insulin, cortisol, GH, and FGF-21 responses were measured. The mean age of the participants was 26.3±3.6 years. Glucagon resulted in significant increases in FGF-21, glucose, insulin, cortisol, and GH levels. The levels of basal cortisol, GH, FGF-21, and IGF-1 were similar in the two groups. The peak GH and area under the curve (AUC)(GH) responses to GST in the obese group were lower than those of the normal-weight group with a different pattern of response. There were no differences between the groups in terms of peak cortisol, AUC(cortisol), peak insulin, AUC(insulin), peak FGF-21, and AUC(FGF21). Obesity was associated with significantly increased glucose and insulin responses and slightly decreased FGF-21 response to glucagon. Obesity was associated with blunted and delayed GH, but preserved cortisol responses to GST. This is the first study showing that glucagon stimulates the HPA and GH axis independently from FGF-21. The delayed GH response to GST in obesity does not seem to be related to FGF-21.

198-OR: The Stimulatory Effect of Glucagon on Insulin Secretion Is Glucose Dependent in Healthy Human Subjects

Glucagon is generally considered to be a counter-regulatory hormone with a primary role to raise blood glucose concentrations by increasing endogenous glucose production (EGP). However, recent preclinical findings support a paracrine action of glucagon to act directly on pancreatic beta cells to promote nutrient-stimulated insulin secretion. In mice, the divergent actions of glucagon are dependent on glycemic status: during fasting glucagon acts predominantly at the liver to raise blood glucose, whereas in the fed state when glycemia is elevated, the insulinotropic effects predominate to lower glucose. These mechanistic findings have not been extended to human physiology. Therefore, we designed a study to test the hypothesis that in healthy humans glucagon actions are also dependent on ambient glycemia. Subjects received glucagon (100 ng/kg) during fasting euglyemia or experimental hyperglycemia (~150 mg/dl) on separate days. On both days, glycemia was maintained for 60 min prior to a glucagon infusion for an additional 60 min. Blood glucose was monitored at bedside and plasma samples were analyzed for C-peptide and di-deuterated glucose enrichment to calculate HGP. During fasting euglycemia, blood glucose was 85 mg/dl and glucagon elicited an increase to a 60 minute average of 132 mg/dl with a peak at ~156 mg/dl at the end of the infusion. Concordant with increased glycemia was an immediate increase in HGP (1.8x) and a delayed increase in C-peptide AUC (1.5x). For the hyperglycemia studies, blood glucose was maintained at 150 mg/dl before and during glucagon administration. Here, glucagon caused a rapid and robust increase in C-peptide AUC (4x) and a more gradual increase in HGP (2x). This glycemic-dependent behavior of glucagon suggests a physiologic role to promote prandial insulin secretion in healthy subjects. This raises the possibility that this incretin function of glucagon plays a role in β-cell function in diabetic subjects. Disclosure S. M. Gray: None. G. Zhang: None. W. Bennett: None. J. Tong: None. J. Campbell: None. D. A. D’alessio: Advisory Panel; Self; Eli Lilly and Company, Sun Pharmaceutical Industries Ltd., Research Support; Self; Eli Lilly and Company, Merck &amp; Co., Inc. Funding National Institutes of Health (F32DK121420, R01DK101991)

The stimulatory effects of glucagon on cortisol and GH secretion occur independently from FGF-21

The stimulatory effects of glucagon on cortisol and GH secretion occur independently from FGF-21

The stimulatory effects of glucagon on cortisol and GH secretion occur independently from FGF-21

Glucagon stimulation test (GST) is used to assess the hypothalamo-pituitary-adrenal (HPA) and growth hormone (GH) axes with an incompletely defined mechanism. We aimed to assess if glucagon acted through fibroblast growth factor-21 (FGF-21) to stimulate cortisol and GH secretion. The secondary outcome was to determine the relationship of FGF-21 with variable GH responses to GST in obesity. A total of 26 healthy participants; 11 obese (body mass index (BMI) > 30 kg/m2) and 15 leans (BMI < 25 kg/m2) were included. Basal pituitary and target hormone levels were measured and GST was performed. During GST, glucose, insulin, cortisol, GH, and FGF-21 responses were measured. The mean age of the participants was 26.3±3.6 years. Glucagon resulted in significant increases in FGF-21, glucose, insulin, cortisol, and GH levels. The levels of basal cortisol, GH, FGF-21, and IGF-1 were similar in the two groups. The peak GH and area under the curve (AUC)(GH) responses to GST in the obese group were lower than those of the normal-weight group with a different pattern of response. There were no differences between the groups in terms of peak cortisol, AUC(cortisol), peak insulin, AUC(insulin), peak FGF-21, and AUC(FGF21). Obesity was associated with significantly increased glucose and insulin responses and slightly decreased FGF-21 response to glucagon. Obesity was associated with blunted and delayed GH, but preserved cortisol responses to GST. This is the first study showing that glucagon stimulates the HPA and GH axis independently from FGF-21. The delayed GH response to GST in obesity does not seem to be related to FGF-21.

The stimulatory effects of glucagon on cortisol and GH secretion occur independently from FGF-21

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Glucagon Activates mTOR1/2 Signaling via an EPAC/Rap1 Signaling Axis in Hepatocyte Cultures

Activation of the protein kinase mTOR (mechanistic target of rapamycin) in complex 1/2 (mTORC1/2) in the liver is repressed during fasting and rapidly stimulated in response to feeding. The effect of a meal on hepatic mTORC1/2 is largely mediated by an increase in plasma levels of nutrients (i.e. amino acids) and growth factors (i.e. insulin). By contrast, fasting is associated with elevated plasma levels of glucagon, which is conventionally viewed as playing a counter-regulatory role to insulin. However, glucagon and insulin secretion are both rapidly increased by consumption of a protein meal. In fact, amino acid-induced glucagon secretion is many times greater than that seen with fasting. Herein we investigated the impact of insulin and glucagon on mTORC1/2 activation in rat H4IIE and human HepG2 cell cultures. As expected, insulin enhanced phosphorylation of the mTORC1 substrates p70S6K1 and 4E-BP1. Surprisingly, the effect of glucagon on mTORC1 signaling was biphasic, wherein there was an acute increase in phosphorylation of p70S6K1 and 4E-BP1 over the first hour of exposure, followed by latent suppression. Notably, the stimulatory effect of glucagon on mTORC1 was not additive with insulin, suggesting a convergent signaling axis. Glucagon-induced mTORC1 activation was associated with enhanced cAMP levels and required activation of the glucagon receptor, PI3K/Akt, and exchange protein activated by cAMP (EPAC). An EPAC-specific cAMP analog as well as PKA inhibition enhanced activation of mTORC1/2. EPAC acts as the guanine nucleotide exchange factor for the small GTPase Rap1, and in H4IIE cultures, transient expression of Rap1 was sufficient to stimulate activation of mTORC1/2. Finally, we found that glucagon addition to culture medium was sufficient to promote Rap1-GTP loading in H4IIE cells. Overall, the findings support that glucagon elicits biphasic regulation of hepatic mTORC1/2 by an EPAC-dependent increase in Rap1-GTP which is followed by a repression of mTORC1.

1925-P: Beneficial Effects of a Novel Long-Acting Glucagon Analog, HM15136, on Obesity and Obesity-Related Metabolic Disorders in Animal Models

Obesity is a major risk factor for a number of metabolic disorders such as hyperlipidemia, glucose intolerance, and T2DM. Recent researches indicated a stimulatory effect of glucagon (GCG) on energy expenditure and appetite inhibition, suggesting the utilization of GCG as a pharmacological therapy for obesity and related metabolic disorders. In line with this, we previously observed that chronic treatment of HM15136, a novel long-acting glucagon analog, conferred efficient body weight loss (BWL) in DIO mice. Here, we further investigated the potential benefits of HM15136 in obesity-related metabolic disorders. In DIO mice, time-course change in fat mass, liver triglyceride (TG), and fasting blood glucose (FBG) was determined. Chronic treatment of HM15136 was associated with progressive BWL (-37.5% vs. vehicle (Veh) at day17) and fat mass reduction with little effect on lean mass, confirming its potent anti-obesity effect. Interestingly, despite marginal BWL at initial drug treatment period, significant reduction in blood TG (-56.5% vs. Veh at day2) and subsequent liver TG (-41.5% vs. Veh at day7) was achieved, and maintained thereafter. Moreover, the TG lowering both in blood and liver was followed by sustainable FBG lowering, suggesting that rapid depletion of lipid, the main sources for gluconeogenesis, might contribute to FBG control by HM15136. To evaluate the potential effect on post-prandial glucose control, oral glucose tolerance test (oGTT) was performed, and reduced BG level during oGTT was observed as BWL occurred. Time-dependent reduction in HOMA-IR evidenced the insulin sensitivity improvement. To our knowledge, this is the first study demonstrating the potential benefits of chronic GCG engagement in the management of BW, blood lipid, and even BG. Based on these results, HM15136 may be a novel option for the management of obesity and related metabolic disorders. Human study is ongoing for the clinical relevance of these findings. Disclosure J. Lee: Employee; Self; Hanmi Pharmaceutical. S. Lee: Employee; Self; Hanmi Pharmaceutical. J. Choi: Employee; Self; Hanmi Pharmaceutical. E. Park: Employee; Self; Hanmi Pharmaceutical. J. Lee: Employee; Self; Hanmi Pharmaceutical. S. Lee: Employee; Spouse/Partner; Hanmi Pharmaceutical. S. Bae: Employee; Self; Hanmi Pharmaceutical. D. Kim: Board Member; Self; Hanmi Pharmaceutical. I. Choi: Board Member; Self; Hanmi Pharmaceutical.

991-P: Potential of a Novel Long-Acting Glucagon Analog, HM15136, for the Treatment of Obesity

Obesity is considered as a main risk factor for metabolic syndrome. Although there are several anti-obesity drugs, their main mode of action (MoA) is appetite suppression. Thus, body weight loss (BWL) efficacy is limited with benefits fading when the drugs stopped. Glucagon (GCG) has been used to treat hypoglycemia. Recent studies indicated a stimulatory effect of GCG on energy expenditure, suggesting its utilization as an anti-obese drug. Since BWL is a key for insulin resistance (IR) reversal and blood glucose (BG) normalization, we hypothesized that sustained GCG treatment may reduce hyperglycemic risk via IR improvement following efficient BWL. To investigate this hypothesis, we developed a novel long-acting GCG analog, HM15136, and evaluated the effect of HM15136 on BW and BG in vivo. In obese animals, HM15136 demonstrated dose-dependent BWL (-21 ∼ -53% vs. Veh. for DIO mice, -10 ∼ -64% vs. Veh. for DIO rats). Of note, despite an initial increase, BG level rapidly normalized. As to the responsible MoA, a reduced blood lipid and HOMA-IR were observed. In vitro studies demonstrated the increased FGF-21 and adiponectin expression by HM15136 in 3T3-L1 adipocytes. These results suggest that HM15136 could improve insulin sensitivity via multiple mechanisms, leading to normal BG maintenance under chronic GCG receptor stimulation. To further investigate the therapeutic potential of HM15136, BWL efficacy was compared to liraglutide: Greater BWL was observed with HM15136 (-34% and -21% vs. Veh. for HM15136 and liraglutide, respectively). Indirect calorimetry demonstrated enhanced energy expenditure by HM15136. Most interestingly, efficient BWL achieved by HM15136 was maintained during 4 weeks drug-holiday, compared to liraglutide. In conclusion, our results demonstrate the potential for prolonged GCG receptor stimulation with agent like HM15136 as a novel therapeutic option for obesity treatment. Human studies are needed to confirm these findings and to evaluate the potential safety of HM15136. Disclosure J. Kim: None. J. Kim: None. S. Lee: None. J. Lee: None. S. Lee: Employee; Self; Hanmi Pharm. Co., Ltd. I. Choi: Employee; Self; Hanmi Pharm. Co., Ltd.

1011-P: Therapeutic Potential of Novel Combination of a Long-Acting Glucagon Analog (HM15136) and Antidiabetic Drugs for the Treatment of Obesity

The obesity epidemic represents a major challenge for public health. However, existing anti-obesity drugs have limited efficacy because they only suppress appetite, emphasizing the development of next generation therapeutic option. Recently, we showed that prolonged glucagon (GCG) receptor activation with HM15136, a long-acting GCG analog, could provide potent body weight loss (BWL) in obese animal models. Considering the stimulatory effect of GCG on glucose production, transient blood glucose (BG) elevation might be important when utilizing HM15136 as a novel anti-obesity drug. Antidiabetic drugs such as metformin, and DPP-4 inhibitor have been widely used for T2DM patients. Considering their safety profile and treatment adherence in addition to BG lowering nature, we hypothesized that combination of antidiabetic drugs might minimize the transient BG elevation by HM15136 and confer additional BWL. To investigate this hypothesis, the present study evaluated the combination effect of HM15136 and antidiabetic medication on BG and BW in vivo. In normal mice, HM15136-induced BG increase was effectively reversed by concomitant treatment of metformin, sitagliptin, or empagliflozin. To further confirm, DIO mice were chronically administered with HM15136 and/or antidiabetic drugs, and normal BG control was continuously maintained in COMBO groups. These results indicate that when combined, antidiabetic drugs could effectively counteract the transient BG elevation by HM15136. As to the BWL efficacy, combination of HM15136 either with metformin, sitagliptin, or empagliflozin provided additional BWL (-18% to -31% by metformin COMBO; -35% to -47% by sitagliptin COMBO; -35% to -40% by empagliflozin COMBO), highlighting the therapeutic benefit of this novel combination. In conclusion, our results demonstrate the prolonged GCG receptor stimulation with antidiabetic medication as a novel therapeutic option for obesity treatment. Disclosure Y. Park: None. J. Lee: None. J. Kim: None. D. Kim: None. Y. Kim: None. I. Choi: Employee; Self; Hanmi Pharm. Co., Ltd.

Effects of glucagon on axoplasmic transport in mouse superior cervical ganglion cells.

The effects of glucagon on the axoplasmic transport of cultured mouse superior cervical ganglion cells were analyzed with the video-enhanced DIC microscope system. Glucagon increased the rate of fast axoplasmic transport by 30% in both anterograde and retrograde directions. The average velocity was increased from 1.36 +/- 0.48 microns/s to 1.74 +/- 0.43 microns/s (anterograde, n = 60) and from 1.37 +/- 0.48 microns/s to 1.62 +/- 0.39 microns/s (retrograde, n = 60). The stimulatory effect of glucagon on the axoplasmic transport was reversed in a glucose-free medium, whereas blocking the citrate cycle by pretreating neuronal cells with malonate did not alter the effect of glucagon. Together with our previous findings, our data suggest that neurotransmitters and hormones play a major role in the regulation of fast axoplasmic transport.

Chronic and transitional regulation of gluconeogenesis and glyconeogenesis by insulin and glucagon in neonatal calf hepatocytes.

Milk-fed calves were used as a source of hepatocytes to establish monlayers to test the effects of chronic (41 h) incubation with no hormone, 100 nM insulin, or 100 nM glucagon on gluconeogenesis (glucose retained as glycogen plus glucose released into the medium) from 2.5 mM [2-(14)C]propionate and 2.0 mM [U-14C]lactate (1.0 mM lactate plus 1.0 mM pyruvate) during a subsequent 3-h (acute) incubation. Media for acute incubations contained no hormone, 0 or 10 nM insulin, or 0, 1, 10, or 100 mM glucagon. Chronic glucagon increased gluconeogenesis from propionate and glyconeogenesis from propionate and lactate compared with chronic exposure to medium with no hormone. A chronic glucagon x acute hormone interaction was manifested as an augmented response to acute glucagon on gluconeogenesis from propionate; a similar potentiation was not evident for gluconeogenesis from lactate. The concentration of glucagon required to acutely stimulate gluconeogenesis was increased by prior incubation with glucagon. Acute glucagon decreased the flux of glucose retained as glycogen regardless of chronic hormone treatment. Chronic incubation with insulin diminished the stimulatory effects of glucagon on gluconeogenesis from lactate. Chronic incubation with insulin did not alter the sensitivity or responsiveness at acute glucagon of gluconeogenesis from propionate. The data demonstrate persisting changes that favor increased basal gluconeogenesis from propionate with chronically elevated glucagon coupled to an increased capacity to respond acutely to glucagon and opposing chronic actions of insulin on lactate metabolism. These data suggest that insulin and glucagon target separate pathways that are unique to metabolism of propionate and lactate.

The regulation of insulin-like growth factor-binding protein 1 messenger ribonucleic acid in cultured rat hepatocytes: the roles of glucagon and growth hormone.

In previous studies it was shown that bovine GH (bGH) suppressed and glucagon stimulated the level of 24- and 30- to 34-kilodalton insulin-like growth factor-binding proteins (IGFBPs) in the media of cultured rat hepatocytes. In the present study we have evaluated the regulation of IGFBP-1 gene expression in primary rat hepatocyte cultures. Glucagon produced a dose-dependent stimulation of hepatocyte IGFBP-1 messenger RNA (mRNA), attaining levels 2- to 6-fold greater than control at a glucagon concentration of 100 ng/ml. GH inhibited the accumulation of IGFBP-1 mRNA in a dose-dependent manner producing, 40-70% inhibition at 50 ng/ml. The effect of glucagon was comparable to and additive with dexamethasone (1 microM). The addition of 3-isobutyl-1-methylxanthine (100 microM) and (Bu)2cAMP (100 microM) augmented IGFBP-1 mRNA levels 5- to 6-fold. 4 beta-Phorbol 12 beta-myristate 13 alpha-acetate (300 nM) was found to inhibit IGFBP-1 mRNA levels by 40-50%. The inhibitory effect of bGH on IGFBP-1 mRNA levels was abolished after preincubation with 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (10 microM) for 24 h, whereas glucagon's stimulatory effect was unaffected. The addition of staurosporine (500 nM) and H-7 (1 mM) abolished the inhibitory effect of GH but also significantly inhibited the stimulatory effect of glucagon, a result consistent with these agents acting on both protein kinase C (PKC) and PKA. In the presence of 10 micrograms/ml cycloheximide, IGFBP-1 gene expression was superinduced by bGH, whereas the effect of glucagon was uninfluenced. Thus the inhibitory action of GH involves, in part, the activation of PKC. Glucagon's stimulatory effect seems to involve the activation of PKA. The inhibitory effect of bGH on IGFBP-1 gene expression may require the continuing synthesis of one or more labile protein(s).

G proteins in rat liver proliferation during cholestasis.

Liver proliferation appears to be dually regulated, in part by cyclic AMP levels. Here we studied the alterations in the stimulatory action of cholera toxin and other agents on the adenylyl cyclase system, as well as the status of Gs and Gi protein subunits during the liver proliferation that follows bile duct ligation in rats. The stimulatory effects of glucagon and vasoactive intestinal peptide (which act through membrane receptors) or guanosine 5'-[beta gamma-imido]triphosphate (which interacts with G proteins) and forskolin (which directly activates the adenylyl cyclase catalytic subunit) on liver adenylyl cyclase activity were blunted in cholestasis. The results indicated an impairment in the stimulatory interaction between the alpha s subunit of Gs protein and the adenylyl cyclase catalytic subunit. Indeed, we observed an important decrease in the stimulation of adenylyl cyclase activity by cholera toxin in cholestasis that was accompanied by a reduced extent of [32P]ADP ribosylation of alpha s protein catalyzed by cholera toxin, as revealed by the poor labeling of the 42,000 Da band in liver membranes from cholestatic rats. However, there was no change in the amount of alpha s or beta proteins as measured with immunoblotting techniques. Experiments on [32P]ADP ribosylation of alpha i subunits of Gi proteins indicated an impairment in liver membranes from cholestatic rats, whereas Western blotting for the detection of alpha i subunits showed decreased alpha i3 and increased alpha i2 levels in this condition. Further efforts are needed to better understand the molecular mechanisms underlying the relationship between the observed divergent expression of Gs and Gi proteins and liver cell proliferation in the cholestatic liver.

Angiotensin II stimulates the synthesis of angiotensinogen in hepatocytes by inhibiting adenylylcyclase activity and stabilizing angiotensinogen mRNA.

Angiotensin II stimulates the hepatic synthesis and secretion of angiotensinogen, the substrate of renin. In the present study performed on freshly isolated rat hepatocytes we demonstrate that this effect of angiotensin II is mainly related to a transient inhibition of adenylylcyclase. Agents known to decrease intracellular cAMP (angiotensin II, vasopressin, guanfacine) or the cAMP-antagonist Rp-adenosine-3',5'-cyclic phosphothioate stimulated, whereas cAMP-stimulating agents (isoproterenol, forskolin, glucagon) or the cAMP-agonist Sp-adenosine-3',5'-cyclic phosphothioate inhibited angiotensinogen synthesis. In contrast, all agents known to affect intracellular concentrations of calcium, as confirmed in Fura-2-loaded hepatocytes (Bay K 8644, calcimycin, calmidazolium, ionomycin, or methoxamine) failed to influence the synthesis of angiotensinogen. The inhibitory effect of angiotensin II as well as the stimulatory effect of glucagon on cAMP were inversely related to angiotensinogen mRNA and angiotensinogen secretion over a wide concentration range of both peptides. Both the angiotensin II-dependent inhibition of cAMP and the angiotensin II-induced increase in angiotensinogen mRNA were abolished by a pertussis toxin pretreatment. In hepatocyte membranes, pertussis toxin ADP-ribosylated a single protein (approximately 41 kDa) probably representing the alpha-subunit of the Gi-protein, coupling inhibitory receptors to adenylylcyclase. We further show that the increase of angiotensinogen mRNA and secretion mainly represents the result of mRNA stabilization, since in a nuclear run-on assay, angiotensin II pretreatment of hepatocytes does not significantly alter the rate of [32P]UTP incorporation into angiotensinogen mRNA, whereas angiotensin II prolonged the half-life of angiotensinogen mRNA in transcription-arrested as well as in [3H]uridine pulse-labeled hepatocytes about 2.5-fold from 80 to 190 min. It is concluded that angiotensin II induces an increase in angiotensinogen synthesis in hepatocytes by stabilizing of angiotensinogen mRNA and that this effect is mediated through inhibition of adenylylcyclase.

Regulation of hepatic lactate balance during exercise

The rate of exchange of lactate across the liver gives important insights into intracellular processes during muscular work. At the onset of exercise hepatic glycogenolysis increases rapidly, resulting in high rates of glycolytic flux and a transient rise in lactate output. With increasing exercise duration, gluconeogenesis is accelerated and the liver gradually shifts from a lactate-producing to a lactate-consuming state. Exercise-induced changes in hormone levels are critical in the regulation of hepatic glycogenolysis and gluconeogenesis and, therefore, net hepatic lactate balance. The fall in insulin stimulates hepatic glycogenolysis, glycolytic flux, and, as a result, hepatic lactate output. On the other hand, the stimulatory effects of glucagon on gluconeogenesis elicit an increase in hepatic lactate uptake. The rise in epinephrine may regulate gluconeogenesis during prolonged exercise by stimulating peripheral lactate mobilization, thereby providing gluconeogenic substrate to the liver. Chronic hepatic-denervation leads to an increase in gluconeogenesis and net hepatic lactate uptake at rest without altering total glucose production. However, the response to exercise is unaffected by the absence of hepatic nerves. Hence, the direction and magnitude of the hepatic lactate balance during exercise yields important information regarding flux through the gluconeogenic and glycolytic pathways, such that high rates of gluconeogenesis correspond to accelerated rates of hepatic lactate uptake and high rates of hepatic glycolytic flux lead to increased rates of hepatic lactate output.

Hormonal regulation of glutathione efflux.

The efflux of GSH has been shown previously to be a saturable process in both isolated rat hepatocytes and perfused liver, suggesting a carrier-mediated transport mechanism. The possibility in hormonal regulation of this process has been raised by recent reports. Our present work examined the role of hormones known to affect intracellular signal transduction mechanisms on GSH efflux in cultured rat hepatocytes and perfused rat livers. We found that cAMP-dependent factors, such as cholera toxin (CT), dibutyryl cAMP, forskolin, and glucagon all stimulated GSH efflux in cultured rat hepatocytes. The efflux kinetics were compared in cultured cells incubated with or without CT; the stimulation of GSH efflux was related to a near doubling of the Vmax while exhibiting no significant alteration of the Km. The increase in intracellular cAMP level associated with the threshold for this stimulatory effect was 25% above control. The stimulatory effect of CT could not be blocked by cyclohexamide pretreatment or reversed by colchicine treatment. The stimulatory effect of glucagon was abolished in the presence of ouabain but not in the presence of barium. On the other hand, hormones which act through Ca2+ and protein kinase C, such as phenylephrine and vasopressin, had no effect on GSH efflux in the cultured cells. In the perfused liver model, glucagon (10 nM) and dibutyryl cAMP (8 microM) stimulated sinusoidal GSH efflux to 130 and 144% of control values, respectively, and increased bile flow while not affecting biliary GSH efflux. Finally, the physiological significance of glucagon-mediated stimulation of sinusoidal GSH efflux was assessed by both plasma GSH and glucose levels in response to in vivo glucagon infusion. The threshold dose of glucagon for significant increase in plasma GSH (5.21 pmol/min) was lower than for glucose (15.61 pmol/min). At the highest glucagon infusion rate (261 pmol/min), plasma GSH level doubled while glucose level increased 80%. In conclusion, increased cAMP stimulates GSH efflux in cultured rat hepatocytes and perfused livers. The stimulatory effect of cAMP is exerted at the sinusoidal pole and appears to be mediated by hyperpolarization of hepatocytes by stimulation of Na(+)-K(+)-ATPase. In vivo studies confirmed the importance of cAMP-mediated stimulation of sinusoidal GSH efflux as it resulted in significant elevation of the plasma GSH level.

Glucagon-Like Peptide-1(7-37) does not Stimulate either Hepatic Glycogenolysis or Ketogenesis.

Recent Studies have demonstrated that glucagon-like peptide-1 (GLP)(7-37) has more potent insulinotropic activity than glucagon. We therefore examined the effect of GLP-1(7-37) on liver metabolism using rat liver perfusion system. Ten nM GLP-1(7-37) did not affect glucose, ketone body and cAMP outputs from the perfused liver. Whereas, the same dose of glucagon stimulated these outputs significantly. When 10 nM GLP-1(7-37) perfused 5 min before the administration of 10 nM glucagon, the above stimulatory effects of glucagon were not affected. These results indicate that truncated GLP-1 has no effect on hepatic glycogenolysis and ketogenesis dissociating from its potent insulinotropic activity.

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 β-oxidation system, which is induced by fatty acids and modified by hormones.

Dissociation of the alpha-adrenergic inhibitory effects on glucagon- and secretin-stimulated bile volume and on cyclic adenosine monophosphate production in the isolated perfused rat liver.

In the isolated perfused rat liver, glucagon increased glucose, cAMP, and bile production. Secretin increased cAMP in the effluent more and bile volume less than glucagon, but did not increase glucose output. When glucagon and secretin were infused together, the effect on cAMP was additive, but glucose output and bile volume were the same as with glucagon alone. The stimulation of bile production by glucagon was suppressed by phenylephrine but not by oxymetazoline, whereas the glucagon-induced cAMP increase in the effluent was suppressed by oxymetazoline in a dose-dependent manner, but not by phenylephrine. Glucagon-stimulated glucose output was not suppressed even when cAMP in the effluent was significantly suppressed by oxymetazoline. The secretin-induced stimulation of bile production was suppressed by phenylephrine, but not by oxymetazoline. Secretin-stimulated cAMP was suppressed more by oxymetazoline than by phenylephrine. The bile volume was not suppressed by phenylephrine alone either in the presence or in the absence of sodium taurocholate. These results indicate that the cholestatic effect of adrenergic agents is mediated by alpha 1-adrenergic receptors, whereas their cAMP suppressive effect is mediated by alpha 2-adrenergic receptors. The stimulatory effect of glucagon on cAMP production was more resistant to alpha-adrenergic agonists than to that of secretin. The dissociation by oxymetazoline of the effects of glucagon on cAMP production from its effect on glucose production is unexplained, whereas the significance of the secretin-induced increase in cAMP production is uncertain.

Time dependent stimulatory effect of glucagon on the capacity of urea synthesis in intact rats

Time dependent stimulatory effect of glucagon on the capacity of urea synthesis in intact rats