Effects of Dexamethasone and Glucagon after Long-Term Exposure on Cyclic AMP Phosphodiesterase 4 in Cultured Rat Hepatocytes

doi:10.1016/S0898-6568(99)00039-X

Cellular Signalling

Volume 11, Issue 9, September 1999, Pages 685-690

https://doi.org/10.1016/S0898-6568(99)00039-X Get rights and content

Abstract

67% of total cAMP phosphodiesterase activity (PDE) in cultured rat hepatocytes could be detected in the cytosol, 15% in plasma membrane, 15% in `dense vesicle,' and 3% in endoplasmatic reticulum fractions. Up to 84% of the PDE activity of the cytosol is represented by the rolipram-sensitive PDE 4. ICI 118233-inhibited PDE 3 was found predominantly in membranes. We were able to show that dexamethasone acts on the PDE 4 in cytosolic and plasma membrane fractions whereas glucagon effected the PDE 4 of the cytosol and the PDE 3 in `dense vesicle' membranes. Primary culture of hepatocytes was used to study long-term effects of dexamethasone and glucagon on PDE 4 activity. Addition of dexamethasone (0.1 μM) at the beginning of cultivation leads to a decrease of total PDE 4 activity whereas after 24 h precultivation no dexamethasone effect could be observed. Glucagon effects on PDE 4 were investigated in 20 h precultured hepatocytes. Maximal stimulation was achieved after 2 h of exposure. PDE 4 subtypes A, B , D and, to a lesser degree, subtype C could be detected by RT-PCR analysis. The results of semiquantitative RT-PCR show that the presence of dexamethasone during the first 24 h of cultivation reduced selectively the transcription of PDE 4D, whereas glucagon was without any effect. Also the translation of PDE 4D was reduced as shown in the Western blot. We would like to discuss the way that dexamethasone influences PDE 4D expression—most likely in combination with other factors such as cytokines—during the time of cell plating, whereas glucagon actions are part of metabolic regulations via phosphorylation reactions.

Introduction

Glucagon and glucocorticoids play an important role in regulation of glycogen metabolism in rat liver. The action of glucagon is mediated by the second messenger cAMP. The intracellular cAMP level is a dynamic equilibrium maintained by synthesis via adenylyl cyclases and degradation via cAMP phosphodiesterases (PDE). PDE could be divided in seven types each with different subtypes 1, 2. Recently two further types were described 3, 4. PDE types show different expression patterns in various cells tissues and organs 1, 2. The subject of this study is the low Km cAMP-specific, rolipram-inhibited PDE 4 which could also be affected by hormones. In Sertoli cells PDE 4 was found to be stimulated by FSH [5] and in plasma membrane fractions of rat liver by insulin [6]. According to Heyworth et al. [7] PDE 4 in plasma membranes does not seem to be stimulated by short-term treatment with glucagon. On the other hand decreased PDE 4 activities after glucagon application in presence of GTP were observed by Robles-Flores et al. [8]. The rolipram-sensitive PDE 4 was characterized as the PDE type up-regulated by cAMP-increasing agents in rat Sertoli cells [5], in human U937 cells [9], or in rat sceletal myoblasts [10]. Long-term treatment with dexamethasone decreased a low Km PDE in homogenates of cultured hepatocytes 11, 12, but the PDE type was not specified.

The aim of our studies was to investigate effects of dexamethasone and glucagon on PDE 4 in primary cultured hepatocytes after long-term exposure. Our results show that dexamethasone decreased PDE 4 activity only after addition at the beginning of cultivation during the time of plating. Glucagon was found to be able to stimulate the cytosolic rolipram-inhibited PDE 4. The subtypes A, B , C, and D of PDE 4 could be detected in hepatocytes. Semiquantitative RT-PCR analysis showed that in presence of dexamethasone the transcription of PDE 4D was found to be decreased selectively whereas glucagon did not influence the transcription of PDE 4 subtypes. The translation of PDE 4D was also reduced. We would like to discuss the way that dexamethasone seems to suppress PDE 4D expression in combination with other factors such as cytokines during cell plating, whereas glucagon acts directly via phosphorylations.

Section snippets

Chemicals

³[H]-cAMP (24 Ci/mmol) was supplied by Amersham-Buchler (Braunschweig, Germany), M 199 medium with 25 mM HEPES by Serva (Heidelberg, Germany), and ICI 118233 by D.E. Riley and J. Bebbington, ICI Pharmaceuticals (Macclesfield, U.K.). Monoclonal PDE 4D specific antipeptide antibody was supplied by S. Wolda, ICOS Corporation (Bothell, Washington, USA), and rolipram was kindly given by Dr. Wachtel, Schering AG (Berlin, Germany). Glucagon was obtained from Novo Nordisk Pharma GmbH (Mainz, Germany),

Results

To get information about the subcellular distribution of PDE activity cytosol, plasma membrane, `dense vesicle' membrane and endoplasmatic reticulum membrane fractions were prepared by stepwise centrifugations of the cell homogenate [14]. The main part of PDE avtivity (67%) measured at 1 μM cAMP was found in the cytosol of hepatocytes after 24 h culturing (Table 1). PDE activity of the plasma membrane and `dense vesicle' membrane fractions were equal and reached up to 15% respectively. Only a

Discussion

In a previous study we have used primary culture of rat hepatocytes in order to investigate hormonal short-term and long-term effects on PDE 3 [13]. Pursuing our investigations on hormonal regulation of PDE, we have now studied dexamethasone and glucagon effects after long-term exposure on the PDE 4 in cultured rat hepatocytes. Type specific inhibitors were used to differentiate exactly between PDE 4 or PDE 3 in various subcellular fractions. As shown in Table 1, PDE 4 could be found

Acknowledgements

This study was supported by grant from Deutsche Forschungsgemeinschaft, Bonn, Germany.

References (25)

V.C Manganiello et al.
Arch. Biochem. Biophys.
(1995)
D.A Fisher et al.
Biochem. Biophys. Res. Commun.
(1998)
D.A Fisher et al.
J. Biol. Chem.
(1998)
J.V Swinnen et al.
J. Biol. Chem.
(1991)
T Kovala et al.
J. Biol. Chem.
(1994)
T Hermsdorf et al.
Cell. Signal.
(1998)
S Fleischer et al.
Methods Enzymol.
(1974)
M.D Percival et al.
Biochem. Biophys. Res. Commun.
(1997)
M.M Bradford
Analyt. Biochem.
(1976)
E Huston et al.
J. Biol. Chem.
(1996)

E.G Loten et al.

J. Biol. Chem.

(1978)

E.T Morgan et al.

Biochim. Biophys. Acta

(1994)

Cited by (16)

Activation of SIK1 by phanginin A inhibits hepatic gluconeogenesis by increasing PDE4 activity and suppressing the cAMP signaling pathway
2020, Molecular Metabolism
Citation Excerpt :
Although CRTC2 is a widely studied substrate of SIK1 [39], it was recently reported that PDE4D was identified as a new direct substrate for SIK1 in β cells, through which SIK1 terminated cAMP signaling by promoting the degradation of cAMP [22]. Earlier studies suggested that PDE4 was the main PDE isoenzyme responsible for cAMP hydrolysis in the liver, accounting for approximately 80% of total PDE [40], and our data showed that both PDE4D and PDE4B isoforms were highly expressed in the liver. Phanginin A profoundly enhanced PDE4 activity in primary mouse hepatocytes, but there is a limitation in our study that the exact contribution of PDE4D and PDE4B to the increased PDE4 activity was unknown.
Salt-induced kinase 1 (SIK1) acts as a key modulator in many physiological processes. However, the effects of SIK1 on gluconeogenesis and the underlying mechanisms have not been fully elucidated. In this study, we found that a natural compound phanginin A could activate SIK1 and further inhibit gluconeogenesis. The mechanisms by which phanginin A activates SIK1 and inhibits gluconeogenesis were explored in primary mouse hepatocytes, and the effects of phanginin A on glucose homeostasis were investigated in ob/ob mice.
The effects of phanginin A on gluconeogenesis and SIK1 phosphorylation were examined in primary mouse hepatocytes. Pan-SIK inhibitor and siRNA-mediated knockdown were used to elucidate the involvement of SIK1 activation in phanginin A-reduced gluconeogenesis. LKB1 knockdown was used to explore how phanginin A activated SIK1. SIK1 overexpression was used to evaluate its effect on gluconeogenesis, PDE4 activity, and the cAMP pathway. The acute and chronic effects of phanginin A on metabolic abnormalities were observed in ob/ob mice.
Phanginin A significantly increased SIK1 phosphorylation through LKB1 and further suppressed gluconeogenesis by increasing PDE4 activity and inhibiting the cAMP/PKA/CREB pathway in primary mouse hepatocytes, and this effect was blocked by pan-SIK inhibitor HG-9-91-01 or siRNA-mediated knockdown of SIK1. Overexpression of SIK1 in hepatocytes increased PDE4 activity, reduced cAMP accumulation, and thereby inhibited gluconeogenesis. Acute treatment with phanginin A reduced gluconeogenesis in vivo, accompanied by increased SIK1 phosphorylation and PDE4 activity in the liver. Long-term treatment of phanginin A profoundly reduced blood glucose levels and improved glucose tolerance and dyslipidemia in ob/ob mice.
We discovered an unrecognized effect of phanginin A in suppressing hepatic gluconeogenesis and revealed a novel mechanism that activation of SIK1 by phanginin A could inhibit gluconeogenesis by increasing PDE4 activity and suppressing the cAMP/PKA/CREB pathway in the liver. We also highlighted the potential value of phanginin A as a lead compound for treating type 2 diabetes.
Acetylcholine exerts additive and permissive but not synergistic effects with insulin on glycogen synthesis in hepatocytes
2007, FEBS Letters
Citation Excerpt :
Acetylcholine potentiated the increase in glucokinase mRNA caused by insulin (Fig. 5) but had no effect in the absence of insulin (results not shown). The effects of acetylcholine are unlikely to be due to changes in cAMP, because acetylcholine unlike dexamethasone [28] did not affect the activity of PDE3 and PDE4 (Fig. 6). The contribution of the Portal Signal to regulation of hepatic glucose balance has been determined experimentally by comparing rates of hepatic glucose production or uptake when a glucose load is administered either through the portal vein or the hepatic artery/peripheral circulation at the same clamped level of insulin.
Parasympathetic (cholinergic) innervation is implicated in the stimulation of hepatic glucose uptake by portal vein hyperglycaemia. We determined the direct effects of acetylcholine on hepatocytes. Acute exposure to acetylcholine mimicked insulin action on inactivation of phosphorylase, stimulation of glycogen synthesis and suppression of phosphoenolpyruvate carboxykinase mRNA levels but with lower efficacy and without synergy. Pre-exposure to acetylcholine had a permissive effect on insulin action similar to glucocorticoids and associated with increased glucokinase activity. It is concluded that acetylcholine has a permissive effect on insulin action but cannot fully account for the rapid stimulation of glucose uptake by the portal signal.
Insulin and heregulin-β1 upregulate guanylyl cyclase C expression in rat hepatocytes: Reversal by phosphodiesterase-3 inhibition
2001, Cellular Signalling
Guanylyl cyclase C (GC-C) is the receptor for the hormones guanylin and uroguanylin. Although primarily expressed in the rat intestine, GC-C is also expressed in the liver during neonatal or regenerative growth or during the acute phase response. Little is known about the hepatic regulation of GC-C expression. The influence of various hepatic growth or acute phase regulators on GC-C expression was evaluated by immunoblot analysis of protein from primary rat hepatocytes grown in a serum-free medium. Insulin and heregulin-β1 strongly stimulated GC-C expression by 24 h of cell culture. Several different hormones and agents suppressed this action, including transforming growth factor β (TGF-β), as well as inhibitors of phosphatidylinositol 3-kinase (PI-3-kinase) and phosphodiesterase 3 (PDE-3, an insulin- and PI-3-kinase-dependent enzyme). The compartmental downregulation of cAMP levels by PDE-3 may be a critical step in the hormonal action that culminates in GC-C synthesis.
Dexamethasone increases intracellular cyclic AMP concentration in murine T lymphocyte cell lines
2001, Steroids
The effects of the synthetic glucocorticoid dexamethasone on the cAMP content of murine T lymphocyte cell lines has been investigated. Incubation of the 3B4.15 T cell hybrids with dexamethasone results in an average 5-fold increase in intracellular cyclic AMP levels after 6 h of treatment. This phenomenon is abolished in the presence of RU486 and of cycloheximide, indicating that it requires binding of the drug to the intracellular glucocorticoids receptor and de novo protein synthesis. Dexamethasone-induced elevation of intracellular cyclic AMP correlates with both an increase in adenylate cyclase activity and a decrease in phosphodiesterase activity in T cell hybrids. This modulation of cyclic AMP metabolism is independent of serum-derived factors, suggesting that it is not secondary to transmembrane receptor stimulation by an extracellular ligand. We propose that glucocorticoids interfere with the homeostatic control of intracellular cAMP concentration, leading to a sustained increase in the content of this important second messenger in murine T lymphocyte cell lines. This study suggests that elevation of cAMP levels may represent one way by which glucocorticoids modulate the immune response.
PDE4 cAMP-specific phosphodiesterases
2001, Progress in Nucleic Acid Research and Molecular Biology
This chapter discusses the cyclic nucleotide phosphodiesterases (PDE4) cyclic Adenosine monophosphate (cAMP)-specific phosphodiesterases. Inhibitors selective for PDE4 enzymes have proved to be extremely informative in identifying the key role that this enzyme activity plays in regulating a variety of important processes related to the inflammatory processes, cell survival, memory, and learning. Molecular biological studies have proved to be crucial in delineating the diversity of this enzyme family. The fact that this diversity has been sustained during evolution, and that particular PDE4 isoenzymes are selectively expressed in different cells, suggests that the various PDE4 isoforms have distinct roles in regulating particular cellular processes. PDE4B, 4C, and 4D isoenzymes are regulated by growth stimulatory responses mediated as a consequence of extracellular-signal-regulated kinases (ERK) activation. In contrast to this, the commonly expressed PDE4A4/5 long form can be regulated through PI-3 kinase-dependent processes and provides a unique target for caspase-3 action, implying a role in cell survival. Indeed, the fact that caspase-3 activation serves to disrupt the intracellular targeting of this enzyme may serve as a paradigm for a means of altering/ablating the role of a PDE4 enzyme, by removing it from a functionally relevant compartment. In this way, new generations of isoenzyme selective inhibitors might be attained.
Hepatothermic therapy of obesity: Rationale and an inventory of resources
2001, Medical Hypotheses
Hepatothermic therapy (HT) of obesity is rooted in the observation that the liver has substantial capacities for both fatty acid oxidation and for thermogenesis. When hepatic fatty acid oxidation is optimized, the newly available free energy may be able to drive hepatic thermogenesis, such that respiratory quotient declines while basal metabolic rate increases, a circumstance evidently favorable for fat loss. Effective implementation of HT may require activation of carnitine palmitoyl transferase-1 (rate-limiting for fatty acid beta-oxidation), an increase in mitochondrial oxaloacetate production (required for optimal Krebs cycle activity), and up-regulation of hepatic thermogenic pathways. The possible utility of various natural agents and drugs for achieving these objectives is discussed. Potential components of HT regimens include EPA-rich fish oil, sesamin, hydroxycitrate, pantethine, L-carnitine, pyruvate, aspartate, chromium, coenzyme Q10, green tea polyphenols, conjugated linoleic acids, DHEA derivatives, cilostazol, diazoxide, and fibrate drugs. Aerobic exercise training and very-low-fat, low-glycemic-index, high-protein or vegan food choices may help to establish the hormonal environment conducive to effective HT. High-dose biotin and/or metformin may help to prevent an excessive increase in hepatic glucose output. Since many of the agents contemplated as components of HT regimens are nutritional or food-derived compounds likely to be health protective, HT is envisioned as an on-going lifestyle rather than as a temporary Ôquick fixÕ. Initial clinical efforts to evaluate the potential of HT are now in progress.

View all citing articles on Scopus

View full text

Effects of Dexamethasone and Glucagon after Long-Term Exposure on Cyclic AMP Phosphodiesterase 4 in Cultured Rat Hepatocytes

Abstract

Introduction

Section snippets

Chemicals

Results

Discussion

Acknowledgements

Arch. Biochem. Biophys.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Cell. Signal.

Methods Enzymol.

Biochem. Biophys. Res. Commun.

Analyt. Biochem.

J. Biol. Chem.

J. Biol. Chem.

Biochim. Biophys. Acta