Per Belfrage

Structure, localization, and regulation of cGMP-inhibited phosphodiesterase (PDE3)

cAMP and cGMP mediate biological responses initiated by diverse extracellular signals. By catalyzing hydrolysis of the 39–59phosphodiester bond of cyclic nucleotides, cyclic nucleotide phosphodiesterases (PDEs) regulate intracellular concentrations and effects of these second messengers. PDEs include a large group of structurally related enzymes (reviewed in Refs. 1–3). These enzymes belong to at least seven related gene families (PDEs 1–7) (Fig. 1), which differ in their primary structures, affinities for cAMP and cGMP, responses to specific effectors, sensitivities to specific inhibitors, and mechanisms of regulation (1–3). Most families are comprised of more than one gene; 14 different PDE genes have been identified. Within different families, tissue-specific mRNAs are generated from the same gene by the use of different transcription initiation sites or by alternative mRNA splicing. Although some aspects of different PDE families will be discussed, this review emphasizes the PDE3 family, including structure-function information and regulation of the adipocyte PDE3, which plays a key role in the antilipolytic action of insulin. Mammalian PDEs share a common structural organization, with a conserved catalytic core (;270 amino acids) usually located in the C-terminal half (Fig. 1) (4). This region is much more similar within an individual PDE family (.80% amino acid identity) than between different PDE families (;25–40% identity) (1–4). The catalytic core is thought to contain common structural elements important for hydrolysis of the cyclic nucleotide phosphodiester bond, as well as family-specific determinants responsible for differences in substrate affinities and inhibitor sensitivities among the different gene families. It contains a PDE-specific sequence motif, HD(X)2H(X4)N, and two consensus Zn -binding domains, the second of which overlaps the PDE motif (3, 5). PDE5 contains tightly bound Zn, which supports catalytic activity (5). The precise role of Zn or other divalent cations in catalytic function of other PDEs has not been defined. Mutagenesis of the first histidine of the PDE sequence motif abolished activity of a recombinant PDE4 expressed in Escherichia coli (6). Histidineand sulfhydryl-modifying reagents inhibited PDE3 activity (7). The widely divergent N-terminal portions of PDEs (Fig. 1) contain determinants that confer regulatory properties specific to the different gene families, e.g. calmodulin-binding domains (PDE1); two non-catalytic cyclic nucleotide-binding domains (PDEs 2, 5, and 6); N-terminal membrane-targeting (PDE4) or hydrophobic membrane-association (PDE3) domains; and calmodulin (PDE1)-, cyclic AMP (PDEs 1, 3, and 4)-, and cGMP (PDE5)-dependent protein kinase phosphorylation sites, etc. (Fig. 1) (1–3). Most cells contain representatives of several PDE families in different amounts, proportions, and subcellular locations (1–3). In some instances a specific PDE regulates a unique cellular function, e.g. photoreceptor PDE6 in cGMP-dependent initiation of visual transduction. In individual cells, different PDEs, with their different responses to regulatory signals, participate in integrating multiple inputs in the complex modulation and termination of cyclic nucleotide signals and responses, e.g. their magnitude and duration, their functional and spatial compartmentation, and their attenuation by short-term feedback or long-term desensitization.

Hormone-sensitive lipase and monoacylglycerol lipase are both required for complete degradation of adipocyte triacylglycerol

The respective roles of monoacylglycerol lipase and hormone-sensitive lipase in the sequential hydrolysis of adipose tissue triacylglycerols have been examined. An adipose tissue preparation, containing both lipases in approximately the same proportion as in the intact tissue, hydrolyzed emulsified tri- or dioleoylglycerol to fatty acids and glycerol, with little accumulation of di- or monooleoylglycerol. Selective removal of the monoacylglycerol lipase by immunoprecipitation markedly reduced the glycerol release. Isolated hormone-sensitive lipase hydrolyzed acylglycerols with a marked accumulation of monoacylglycerol in accordance with the positional specificity of this enzyme (Fredrikson, G. and Belfrage, P. (1983) J. Biol. Chem. 258, 14253-14256). Addition of increasing amounts of isolated monoacylglycerol lipase led to a corresponding increase in glycerol release, due to hydrolysis of the monoacylglycerols formed. The reaction proceeded to completion when the relative proportion of the two lipases was similar to that in the intact tissue. These findings indicate that hormone-sensitive lipase catalyzes the hydrolysis of triacylglycerol in the rate-limiting step of adipose tissues lipolysis, and of the resulting diacylglycerol, whereas the action of monoacylglycerol lipase is required in the final hydrolysis of the 2-monoacylglycerols produced.

Alterations of lipid metabolism in healthy volunteers during long-term ethanol intake

Abstract. Nine young, healthy male volunteers were given ethanol (75 g/day) for 5 weeks. The ethanol was divided into five daily doses and taken so that blood ethanol levels never exceeded 0.04% (w/v).

Immunological evidence for the presence of hormone-sensitive lipase in rat tissues other than adipose tissue

A polyclonal rabbit antibody was used to detect hormone-sensitive lipase in rat organs other than white adipose tissue. Inhibition of tissue diacylglycerol lipase activity by the anti-hormone-sensitive lipase, and by NaF, Hg2+ and diisopropyl fluorophosphate, known inhibitors of the hormone-sensitive lipase, demonstrated its presence in the adrenals, ovaries, testes, heart and skeletal muscle, but not in the liver and kidneys. After enrichment by immunoprecipitation an immunoreactive protein, corresponding to the adipose tissue hormone-sensitive lipase 84 kDa subunit, and some additional, higher Mrapp proteins, were detected by Western blotting in the same tissues. The adipose tissue contained greater than 80% of the total hormone-sensitive lipase, with 5-10- and 50-100-fold lower specific activity in the steroid-producing and the muscle tissues, respectively.

Insulin-induced phosphorylation and activation of phosphodiesterase 3B in rat adipocytes: possible role for protein kinase B but not mitogen-activated protein kinase or p70 S6 kinase.

Insulin stimulation of adipocytes results in serine phosphorylation/activation of phosphodiesterase 3B (PDE 3B) and activation of a kinase that phosphorylates PDE 3B in vitro, key events in the antilipolytic action of this hormone. We have investigated the role for p70 S6 kinase, mitogen-activated protein kinases (MAP kinases), and protein kinase B (PKB) in the insulin signaling pathway leading to phosphorylation/activation of PDE 3B in adipocytes. Insulin stimulation of adipocytes resulted in increased activity of p70 S6 kinase, which was completely blocked by pretreatment with rapamycin. However, rapamycin had no effect on the insulin-induced phosphorylation/activation of PDE 3B or the activation of the kinase that phosphorylates PDE 3B. Stimulation of adipocytes with insulin or phorbol myristate acetate induced activation of MAP kinases. Pretreatment of adipocytes with the MAP kinase kinase inhibitor PD 98059 was without effect on the insulin-induced activation of PDE 3B. Furthermore, phorbol myristate acetate stimulation did not result in phosphorylation/activation of PDE 3B or activation of the kinase that phosphorylates PDE 3B. Using Mono Q and Superdex chromatography, the kinase that phosphorylates PDE 3B was found to co-elute with PKB, but not with p70 S6 kinase or MAP kinases. Furthermore, both PKB and the kinase that phosphorylates PDE 3B were found to translocate to membranes in response to peroxovanadate stimulation of adipocytes in a wortmannin-sensitive way. Whereas these results suggest that p70 S6 kinase and MAP kinases are not involved in the insulin-induced phosphorylation/activation of PDE 3B in rat adipocytes, they are consistent with PKB being the kinase that phosphorylates PDE 3B.

Essential role of phosphatidylinositol 3-kinase in insulin-induced activation and phosphorylation of the cGMP-inhibited cAMP phosphodiesterase in rat adipocytes. Studies using the selective inhibitor wortmannin.

Incubation of rat adipocytes with wortmannin, a potent and selective phosphatidylinositol 3‐kinase (PI 3‐kinase) inhibitor, completely blocked the antilipolytic action of insulin (IC50≈ 100 nM), the insulin‐induced activation and phosphorylation of cGMP‐inhibited cAMP phosphodiesterase (cGI‐PDE) as well as the activation of the insulin‐stimulated cGI‐PDE kinase (IC50≈ 10–30 nM). No direct effects of the inhibitor on the insulin‐stimulated cGI‐PDE kinase, the cGI‐PDE and the hormone‐sensitive lipase were observed. These data suggest that activation of PI 3‐kinase upstream of the insulin‐stimulated cGI‐PDE kinase in the antilipolytic insulin signalchain has an essential role for insulin‐induced cGI‐PDE activation/ phosphorylation and anti‐lipolysis.

Type III cGMP-inhibited cyclic nucleotide phosphodiesterases (PDE3 gene family)

Seven different but related cyclic nucleotide phosphodiesterase (PDE) gene families have been identified. Type III cGMP-inhibited (cGI) PDEs, the PDE3 gene family, are found in many tissues. cGI PDEs exhibit a high affinity for both cAMP and cGMP, and are selectively and relatively specifically inhibited by certain agents which augment myocardial contractility, promote smooth muscle relaxation and inhibit platelet aggregation. Adipocyte, platelet, and hepatocyte cGI PDE activities are regulated by cAMP-dependent phosphorylation. Insulin-induced phosphorylation/activation of adipocyte and hepatocyte cGI PDEs is thought to be important in acute regulation of triglyceride and glycogen metabolism by insulin. Two distinct cGI PDE subfamilies, products of distinct but related genes, have been identified. They exhibit the domain structure common to PDEs with a carboxyterminal region, conserved catalytic domain and divergent regulatory domain. In their catalytic domains cGI PDEs contain a 44 amino acid insertion not found in other PDE families. The expression of cGIP1 and cGIP2 mRNAs differs in different rat tissues, suggesting distinct functions for the two cGI PDE subfamilies, i.e., cGIP1 in adipose tissue, liver, testis and cGIP2 in myocardium, platelets and smooth muscle.

Regulation of adipose tissue lipolysis: effects of noradrenaline and insulin on phosphorylation of hormone-sensitive lipase and on lipolysis in intact rat adipocytes

In [l] we have demonstrated that hormone-sensitive lipase is phosphorylated in intact rat adipocytes, but the effects of hormones on the extent of this phosphorylation was not studied. The [32P]phosphorylated enzyme protein migrated in a small [“PIphosphopeptide band (1.5% of total) when proteins from adipocytes, incubated with 32Pi, were separated with SDS-PAGE. During the isolation of the [32P] phosphorylated lipase we obtained no indication of heterogeneity of this 84 000 dalton [32P]phosphopeptide band, which suggested that it mainly consisted of [32P]hormone-sensitive lipase [I]. Based on these observations we have derived a method for quantitation of [32P]hormone-sensitive lipase in adipocyte protein extracts. We had developed a technique for continuous monitoring of FFA release from adipocytes by pH-stat titration as a measure of hormone-sensitive lipase activity [2]. In combination, these techniques have enabled us to correlate the time-course of effects of noradrenaline and insulin on phosphorylation and activity of the enzyme, in intact adipocytes. The results suggest that these hormones can regulate lipolysis in the intact cell by altering the extent of phosphorylation of hormonesensitive lipase.

Regulation of protein kinase B in rat adipocytes by insulin, vanadate, and peroxovanadate. Membrane translocation in response to peroxovanadate

Protein kinase B (PKB) (also referred to as RAC/Akt kinase) has been shown to be controlled by various growth factors, including insulin, using cell lines and transfected cells. However, information is so far scarce regarding its regulation in primary insulin-responsive cells. We have therefore used isolated rat adipocytes to examine the mechanisms, including membrane translocation, whereby insulin and the insulin-mimicking agents vanadate and peroxovanadate control PKB. Stimulation of adipocytes with insulin, vanadate, or peroxovanadate caused decreased PKB mobility on sodium dodecyl sulfate-polyacrylamide gels, indicative of increased phosphorylation, which correlated with an increase in kinase activity detected with the peptide KKRNRTLTK. This peptide was found to detect activated PKB selectively in crude cytosol and partially purified cytosol fractions from insulin-stimulated adipocytes. The decrease in electrophoretic mobility and activation of PKB induced by insulin was reversed both in vitro by treatment of the enzyme with alkaline phosphatase and in the intact adipocyte upon removal of insulin or addition of the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin. Significant translocation of PKB to membranes could not be demonstrated after insulin stimulation, but peroxovanadate, which appeared to activate PI 3-kinase to a higher extent than insulin, induced substantial translocation. The translocation was prevented by wortmannin, suggesting that PI 3-kinase and/or the 3-phosphorylated phosphoinositides generated by PI 3-kinase are indeed involved in the membrane targeting of PKB.

Hormone-sensitive lipase of rat adipose tissue: Identification and some properties of the enzyme protein

The cyclic AMP-dependent stimulation of hormonesensitive lipase activity in adipose tissue preparations, originally described by Bizack [ 1 ] and confirmed by others [2,3] has been shown to be mediated via a protein kinase-catalyzed phosphorylation [4,5]. However, direct evidence for the molecular events taking place upon stimulation is lacking since the enzyme protein has not been isolated. We have recently purified monoacylglycerol lipase from rat adipose tissue by solubilization and fractionation in non-ionic detergent [6]. Using the same technique we have been able to separate solubilized hormonesensitive lipase from other lipolytic enzymes of rat adipose tissue, to identify the enzyme protein and to demonstrate some of its properties, notably a protein kinase-catalyzed phosphorylation.