ß2-Adrenergic receptor agonist induced hepatic steatosis in mice: modeling nonalcoholic fatty liver disease in hyperadrenergic states.

Nonalcoholic fatty liver disease (NAFLD) is a spectrum of disorders ranging from hepatic steatosis [excessive accumulation of triglycerides (TG)] to nonalcoholic steatohepatitis, which can progress to cirrhosis and hepatocellular carcinoma. The molecular pathogenesis of steatosis and progression to more severe NAFLD remains unclear. Obesity and aging, two principal risk factors for NAFLD, are associated with a hyperadrenergic state. ß-Adrenergic responsiveness in liver increases in animal models of obesity and aging, and in both is linked to increased hepatic expression of ß2-adrenergic receptors (ß2-ARs). We previously showed that in aging rodents intracellular signaling from elevated hepatic levels of ß2-ARs may contribute to liver steatosis. In this study we demonstrate that injection of formoterol, a highly selective ß2-AR agonist, to mice acutely results in hepatic TG accumulation. Further, we have sought to define the intrahepatic mechanisms underlying ß2-AR mediated steatosis by investigating changes in hepatic expression and cellular localization of enzymes, transcription factors, and coactivators involved in processes of lipid accrual and disposition-and also functional aspects thereof-in livers of formoterol-treated animals. Our results suggest that ß2-AR activation by formoterol leads to increased hepatic TG synthesis and de novo lipogenesis, increased but incomplete ß-oxidation of fatty acids with accumulation of potentially toxic long-chain acylcarnitine intermediates, and reduced TG secretion-all previously invoked as contributors to fatty liver disease. Experiments are ongoing to determine whether sustained activation of hepatic ß2-AR signaling by formoterol might be utilized to model fatty liver changes occurring in hyperadrenergic states of obesity and aging, and thereby identify novel molecular targets for the prevention or treatment of NAFLD.NEW & NOTEWORTHY Results of our study suggest that ß2-adrenergic receptor (ß2-AR) activation by agonist formoterol leads to increased hepatic TG synthesis and de novo lipogenesis, incomplete ß-oxidation of fatty acids with accumulation of long-chain acylcarnitine intermediates, and reduced TG secretion. These findings may, for the first time, implicate a role for ß2-AR responsive dysregulation of hepatic lipid metabolism in the pathogenetic processes underlying NAFLD in hyperadrenergic states such as obesity and aging.

Lipin proteins and glycerolipid metabolism: Roles at the ER membrane and beyond.

The regulation of glycerolipid biosynthesis is critical for homeostasis of cellular lipid stores and membranes. Here we review the role of lipin phosphatidic acid phosphatase enzymes in glycerolipid synthesis. Lipin proteins are unique among glycerolipid biosynthetic enzymes in their ability to transit among cellular membranes, rather than remain membrane tethered. We focus on the mechanisms that underlie lipin protein interactions with membranes and the versatile roles of lipins in several organelles, including the endoplasmic reticulum, mitochondria, endolysosomes, lipid droplets, and nucleus. We also review the corresponding physiological roles of lipins, which have been uncovered by the study of genetic lipin deficiencies. We propose that the growing body of knowledge concerning the biochemical and cellular activities of lipin proteins will be valuable for understanding the physiological functions of lipin proteins in health and disease. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá.

Transcript Abundance of Putative Lipid Phosphate Phosphatases During Development of Trypanosoma brucei in the Tsetse Fly.

African trypanosomes (Trypanosoma brucei spp.) cause devastating diseases in sub-Saharan Africa. Trypanosomes differentiate repeatedly during development in tsetse flies before gaining mammalian infectivity in fly salivary glands. Lipid phosphate phosphatases (LPPs) are involved in diverse biological processes, such as cell differentiation and cell migration. Gene sequences encoding two putative T. brucei LPP proteins were used to search the T. brucei genome, revealing two additional putative family members. Putative structural features and transcript abundance during parasite development in tsetse fly were characterized. Three of the four LPP proteins are predicted to have six transmembrane domains, while the fourth shows only one. Semiquantitative gene expression revealed differential regulation of LPPs during parasite development. Transcript abundance for three of the four putative LPP genes was elevated in parasites infecting salivary glands, but not mammalian-infective metacyclic cells in fly saliva, indicating a potential role of this family in parasite establishment in tsetse salivary glands.

A HPLC-fluorescence detection method for determination of phosphatidic acid phosphohydrolase activity: application in human myocardium.

Phosphatidic acid phosphohydrolase (PAP) catalyzes the dephosphorylation of phosphatidic acid (PA) to diacylglycerol, the second messenger responsible for activation of protein kinase C. Despite the crucial role of PAP lipid signaling, there are no data on PAP signaling function in the human heart. Here we present a nonradioactive assay for the investigation of PAP activity in human myocardium using a fluorescent derivative of PA, 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphate (BODIPY-PA), as substrate in an in vitro PAP-catalyzed reaction. Unreacted BODIPY-PA was resolved from the PAP products by a binary gradient HPLC system and BODIPY-diacylglycerol was detected by fluorimetry. The reaction proceeded at a linear rate for up to 60 min and increased linearly with increasing amounts of cardiac protein in a range of 0.25 to 8.0 microg. This assay proved to be sensitive for accurate quantitation of total PAP activity, PAP-1 activity, and PAP-2 activity in human atrial tissue and right ventricular endomyocardial biopsies. Total PAP activity was approximately fourfold higher in ventricular myocardium than in atrial tissue. There was negligible PAP-1 activity in atrial myocardium compared with ventricular myocardium, indicating regional differences in activities and distribution pattern of PAP-1 and PAP-2 in the human heart.

Colorimetric determination of pure Mg(2+)-dependent phosphatidate phosphatase activity.

The malachite green-molybdate reagent was used for a colorimetric assay of pure Mg2(+)-dependent phosphatidate phosphatase activity. This enzyme plays a major role in fat metabolism. Enzyme activity was linear with time and protein concentration, and with the concentration of water-soluble dioctanoyl phosphatidate. The colorimetric assay was used to examine enzyme inhibition by phenylglyoxal, propranolol, and dimethyl sulfoxide. Pure enzyme and a water-soluble phosphatidate substrate were required for the assay, which should be applicable to a well-defined large-scale screen of Mg2(+)-dependent phosphatidate phosphatise inhibitors (or activators).

Enzymatic analysis of lipid phosphate phosphatases.

Lipid phosphate monoesters including phosphatidic acid, lysophosphatidic acid, sphingosine 1-phosphate and ceramide 1-phosphate are intermediates in phosho- and sphingo-lipid biosynthesis and also play important roles in intra- and extra-cellular signaling. Dephosphorylation of these lipids terminates their signaling actions and, in some cases, generates products with additional biological activities or metabolic fates. The key enzymes responsible for dephosphorylation of these lipid phosphate substrates are collectively termed lipid phosphate phosphatases (LPPs). They are integral membrane enzymes with a core domain of six transmembrane spanning alpha-helices linked by extramembrane loops. LPPs are oriented in the membrane with their N- and C-termini facing the cytoplasm. LPPs exhibit isoform and cell specific localization patterns being variably distributed between endomembrane compartments (primarily the endoplasmic reticulum and Golgi apparatus) and the plasma membrane. The active site of these enzymes is formed from residues within two of the extramembrane loops and faces the lumen of endomembrane compartments or, when localized to the plasma membrane, towards, the extracellular space. Biochemical, pharmacological, cell biological and genetic studies identify roles for LPPs in both intracellular lipid metabolism and the regulation of both intra- and extra-cellular signaling pathways that control cell growth, survival and migration. This article describes procedures for the expression of LPPs in insect and mammalian cells and their analysis by SDS-PAGE and Western blotting. The most straightforward way to determine LPP activity is to measure release of the substrate phosphate group. We described methods for the synthesis and purification of [(32)P]-labeled LPP substrates. We describe the use of both radiolabeled and fluorescent lipid substrates for the detection, quantitation and analysis of the enzymatic activities of the LPPs measured using intact or broken cell preparations as the source of enzyme.

Assaying lipid phosphate phosphatase activities.

Lipid phosphate molecules such as phosphatidate, lysophosphatidate, and diacylglycerol pyro phosphate play roles as signaling molecules in prokaryotic and eukaryotic cells. The cellular processes by which lipid phosphate molecules signal may be attenuated through the action of lipid phosphate phosphatase enzymes. The levels of lipid phosphate phosphatase activities may be used as a marker of signaling events in the cell. In this chapter we describe enzymatic assays that are routinely used to measure the activities of phosphatidate phosphatase, lysophosphatidate phosphatase, and diacylglycerol pyrophosphate phosphatase. These activities are measured by following the release of water-soluble radioactive inorganic phosphate from chloroform-soluble radioactive lipid phosphate substrate following a simple chloroform/methanol/ water phase partition.

Alterations of sarcolemmal phospholipase D and phosphatidate phosphohydrolase in congestive heart failure.

Phospholipase D 2 (PLD2) is the major PLD isozyme associated with the cardiac sarcolemmal (SL) membrane. Hydrolysis of SL phosphatidylcholine (PC) by PLD2 produces phosphatidic acid (PA), which is then converted to 1,2 diacylglycerol (DAG) by the action of phosphatidate phosphohydrolase type 2 (PAP2). In view of the role of both PA and DAG in the regulation of Ca(2+) movements and the association of abnormal Ca(2+) homeostasis with congestive heart failure (CHF), we examined the status of both PLD2 and PAP2 in SL membranes in the infarcted heart upon occluding the left coronary artery in rats for 1, 2, 4, 8 and 16 weeks. A time-dependent increase in both SL PLD2 and PAP2 activities was observed in the non-infarcted left ventricular tissue following myocardial infarction (MI); however, the increase in PAP2 activity was greater than that in PLD2 activity. Furthermore, the contents of both PA and PC were reduced, whereas that of DAG was increased in the failing heart SL membrane. Treatment of the CHF animals with imidapril, an angiotensin-converting enzyme (ACE) inhibitor, attenuated the observed changes in heart function, SL PLD2 and PAP2 activities, as well as SL PA, PC and DAG contents. The results suggest that heart failure is associated with increased activities of both PLD2 and PAP2 in the SL membrane and the beneficial effect of imidapril on heart function may be due to its ability to prevent these changes in the phospholipid signaling molecules in the cardiac SL membrane.

Detection of functional PTEN lipid phosphatase protein and enzyme activity in squamous cell carcinomas of the head and neck, despite loss of heterozygosity at this locus.

The human tumour suppressor gene PTEN located at 10q23 is mutated in a variety of tumour types particularly metastatic cases and in the germline of some individuals with Cowdens cancer predisposition syndrome. We have assessed the status of PTEN and associated pathways in cell lines derived from 19 squamous cell carcinomas of the head and neck. Loss of heterozygosity is evident at, or close to the PTEN gene in 5 cases, however there were no mutations in the remaining alleles. Furthermore by Western analysis PTEN protein levels are normal in all of these SCC-HN tumours and cell lines. To assess the possibility that PTEN may be inactivated by another mechanism, we characterized lipid phosphatase levels and from a specific PIP3 biochemical assay it is clear that PTEN is functionally active in all 19 human SCCs. Our data strongly suggest the possibility that a tumour suppressor gene associated with development of SCC-HN, other than PTEN, is located in this chromosomal region. This gene does not appear to be MXI-1, which has been implicated in some other human tumour types. PTEN is an important negative regulator of PI3Kinase, of which subunit alpha is frequently amplified in SCC-HN. To examine the possibility that PI3K is upregulated by amplification in this tumour set we assessed the phosphorylation status of Akt, a downstream target of PI3K. In all cases there is no detectable increase in Akt phosphorylation. Therefore there is no detectable defect in the PI3K pathway in SCC-HN suggesting that the reason for 3q26.3 over-representation may be due to genes other than PI3K110alpha.

Triacylglycerol biosynthetic enzymes in lean and obese Zucker rats.

In the present investigation, we have compared the potential of triacylglycerol formation from sn-glycerol-3-phosphate (GP) and 2-monoacylglycerol (MG) in liver, adipose tissue and intestine from lean and obese Zucker rats. Microsomal fractions were used to measure the sn-glycerol-3-phosphate acyltransferase (GPAT), diacylglycerol acyltransferase (DGAT) and monoacylglycerol acyltransferase (MGAT) activities and homogenates were used to measure NEM-sensitive and NEM-insensitive phosphatidate phosphohydrolase (PPH) activities. In adipose tissue and liver, the GP pathway served as the major route of glycerolipid formation, with adipose tissue being 5-20-fold more active. The activities of the GP pathway enzymes increased further in response to obesity, with some degree of organ specificity. In adipose tissue of obese rats, the activities of all the pathway enzymes increased; whereas, in liver and intestine, this response was limited to PPH and GPAT, respectively. In contrast with the GP pathway enzymes, obesity in Zucker rats was not associated with alterations in the acylation of 2-monoacylglycerol. Comparison of the activities of MGAT in different intestinal segments indicated that the MG pathway was most active in the jejunum and least active in the ileum and that this pattern did not change in response to obesity. These measurements of the individual enzyme reactions provide evidence that the entire process of esterification via sn-glycerol-3-phosphate is accelerated in the various organs from obese rats and that this perturbation in lipid metabolism may contribute significantly to the increased deposition of body fat noted in this animal model.

Characterization of phosphatidic acid phosphohydrolase in neutrophil subcellular fractions.

This investigation was designed to confirm the presence of PA phosphohydrolase in human neutrophils and to determine the distribution and characteristics of the enzyme in soluble and particulate subcellular fractions of disrupted neutrophils. Enzyme activity was detected in unseparated extracts of sonicated neutrophils. The majority of the recovered activity was recovered in a particulate fraction rich in neutrophil plasma-membrane markers; moderate levels (20%) of the total activity were recovered in the cytosol. While Mg2+ markedly potentiated the cytosolic but not the particulate activity, Ca2+ moderately inhibited both the cytosolic and particulate enzymes. The plasma-membrane-associated activity was absolutely dependent on detergent (0.5% Triton X-100) and displayed an apparent Km of 62 microM for phosphatidic acid. Enzyme activity was markedly inhibited by NaF, not influenced by excess glycerophosphate and slightly attenuated by propranolol, an inhibitor of PA phosphohydrolase in other systems. Preincubation of plasma membranes with N-ethylmaleimide at concentrations up to 25 mM had little effect on enzyme activity. However, activity in cytosolic and microsomal fractions of neutrophils were completely abolished by preincubation with N-ethylmaleimide at concentrations of less than 5 mM. We conclude that neutrophils possess a potent PA phosphohydrolase localized in their plasma membranes. Metabolism of cellular second-messengers by this enzyme may exert a profound effect on the functions of stimulated neutrophils.

Physical evidence for the presence of two forms of phosphatidate phosphohydrolase in rat liver.

Phosphatidate phosphohydrolase (PAP)-catalysed dephosphorylation of phosphatidic acid to diacylglycerol is an important step in glycerolipid metabolism and cell-signalling. Gel filtration chromatography on Superose 6 and anion-exchange chromatography on Mono Q of rat liver subcellular fractions has provided physical evidence for the presence of two distinct forms of PAP activity. One form was sensitive to inhibition by N-ethylmaleimide (NEM), had an apparent M(r) of 540,000 and was eluted from the anion-exchange column by 0.35 M NaCl, while the other was insensitive to inhibition by NEM, had an apparent M(r) of 240,000 and was eluted from the anion-exchange column by 0.15 M NaCl. Studies on the subcellular distribution of these two enzymes, using 5'-nucleotidase as a plasma membrane marker, demonstrated that the NEM-sensitive form was predominantly cytosolic but translocated to the microsomal membranes in response to oleate. The NEM-insensitive form was predominantly located in the plasma membrane but a small proportion (approx. 10%) of total cell activity was present on the endoplasmic reticulum. The implications of these results for the likely roles of the two different forms of PAP in fatty acid esterification and cell-signalling are discussed.

Regulation of beta-adrenergic receptor-mediated processes in fetal rat lung: selective desensitization caused by chronic terbutaline exposure.

Fetal lung beta-receptors become effectively coupled to lung fluid reabsorption and enzymes involved in surfactant synthesis on the day before birth, a period when circulating catecholamine levels are high. Accordingly, we examined the effects of repeated maternal terbutaline exposure on beta-receptor binding capabilities and beta-receptor-mediated processes in the fetal rat lung. Administration of terbutaline to pregnant rats on gestational day 17-20 produced significant reductions in beta-receptor binding to membrane preparations. Similarly, beta-receptor-mediated stimulation of adenylate cyclase activity and ornithine decarboxylase activity showed marked desensitization in the terbutaline-exposed fetuses. However, the linkage of beta-receptors to lung fluid reabsorption and phosphatidic acid phosphatase, an enzyme involved in surfactant synthesis, did not desensitize with chronic terbutaline pretreatment; both of these processes displayed the normal onset of responsiveness on gestational day 21 in the treated animals, as well as a normal magnitude of response. Hence, beta-receptor-mediated events in the developing lung may be differentially regulated during exposure to agonists, allowing the selective expression or depression of function when circulating catecholamine levels are high.

Prenatal exposure to propranolol via continuous maternal infusion: effects on physiological and biochemical processes mediated by beta adrenergic receptors in fetal and neonatal rat lung.

During lung development, beta adrenergic receptors undergo transient coupling to enzymes and physiological processes which govern respiratory function and trophic responses to neural stimulation. To determine the role of endogenous catecholamines in mediating these processes, we examined the gestational and postnatal effects of chronic propranolol infusion (10 mg/kg/day) throughout fetal development. The effectiveness of receptor blockade in dam and fetus were confirmed through measurements of heart rate and enzymatic stimulatory responses to acute challenge with beta agonists (terbutaline to isoproterenol). Propranolol antagonized the ability of terbutaline to stimulate fetal lung fluid resorption and phosphatidic acid phosphatase, a key enzyme in surfactant synthesis. After birth, basal lung compliance and the compliance response to beta adrenergic stimulation were compromised in the neonates that had been exposed to propranolol before birth, despite the fact that direct receptor antagonism had disappeared by that time. After weaning, animals exposed to prenatal propranolol showed interference with basal activity of ornithine decarboxylase (an enzyme involved in transduction of neuronal and hormonal trophic stimuli) and its response to acute beta adrenergic challenge. These results suggest that endogenous fetal catecholamines participate in perinatal respiratory adaptation to air-breathing and help to program future cellular responsiveness to neuronal input.

Spermine antagonises the effects of dexamethasone, glucagon and cyclic AMP in increasing the activity of phosphatidate phosphohydrolase in isolated rat hepatocytes.

Rat hepatocytes were incubated in monolayer culture, under serum free conditions, for 8 h. Glucagon (10 nM), 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (100 microM) and dexamethasone (100 nM) increased the activity of phosphatidate phosphohydrolase by approx. 2-, 3.6- and 3.3-fold, respectively. Spermine alone had no significant effect. Spermine (2.5 mM) almost completely inhibited the glucagon induced increase in phosphohydrolase activity. It only partially inhibited the dexamethasone and cyclic AMP mediated inductions. Spermidine had no significant effect in this respect. The results are discussed in relation to the known effects of polyamines on glycerolipid synthesis, in particular, and on intermediary metabolism.

Isolation and characterization of multiple forms of Mg2+-dependent phosphatidate phosphohydrolase from rat adipose cytosol.

In the present studies, we have made several unique observations. First, we have shown that cytosolic phosphatidate phosphohydrolase from adipose tissue subjected to butyl-agarose chromatography was resolved into four different components. These components, designated as passthrough (PT), D150, D250 and E, were present in the proportions of 51:7:24:16, respectively, in the rat adipose cytosol. Comparison of the properties of these components revealed some similar properties, and also several differences. These components showed the same pH optimum, required Mg2+ for activity and were inhibited by N-ethylmaleimide, indicating a requirement of active sulfhydryl groups for activity. These components differed from one another with respect to hydrophobicity, sedimentation behavior, Stokes diameter, Km values, thermolability and susceptibility to proteinase treatment. Second, we have shown that each component of this system was associated with lipids which were found to be essential for the catalytic activity. Perturbation of this association by organic solvent or by adding excess amounts of exogenous lipids resulted in the loss of enzyme activity. Finally, we analyzed lipid composition of individual components. These studies suggest that the multi-component system of Mg2+-dependent phosphatidate phosphohydrolase may be a part of the cytomembrane network.