The role of PP5 and PP2C in cardiac health and disease

Protein phosphatases are important, for example, as functional antagonists of β-adrenergic stimulation of the mammalian heart. While β-adrenergic stimulations increase the phosphorylation state of regulatory proteins and therefore force of contraction in the heart, these phosphorylations are reversed and thus force is reduced by the activity of protein phosphatases. In this context the role of PP5 and PP2C is starting to unravel. They do not belong to the same family of phosphatases with regard to sequence homology, many similarities with regard to location, activation by lipids and putative substrates have been worked out over the years. We also suggest which pathways for regulation of PP5 and/or PP2C described in other tissues and not yet in the heart might be useful to look for in cardiac tissue. Both phosphatases might play a role in signal transduction of sarcolemmal receptors in the heart. Expression of PP5 and PP2C can be increased by extracellular stimuli in the heart. Because PP5 is overexpressed in failing animal and human hearts, and because overexpression of PP5 or PP2C leads to cardiac hypertrophy and KO of PP5 leads to cardiac hypotrophy, one might argue for a role of PP5 and PP2C in heart failure. Because PP5 and PP2C can reduce, at least in vitro, the phosphorylation state of proteins thought to be relevant for cardiac arrhythmias, a role of these phosphatases for cardiac arrhythmias is also probable. Thus, PP5 and PP2C might be druggable targets to treat important cardiac diseases like heart failure, cardiac hypertrophy and cardiac arrhythmias.     

Glucosamine induces lipid accumulation and adipogenic change in C2C12 myoblasts

Hyperglycemia-induced activation of hexosamine biosynthesis pathway (HBP) has been implicated in the development of insulin resistance in skeletal muscles. In the present study, the content of uridine-5'-diphospho-N-acetylglucosamine, the end product of the HBP, was elevated in skeletal muscle of obese diabetic KKA(y) mice, compared with control mice. To elucidate the effect of elevated HBP in the skeletal muscle, we treated C2C12 myoblasts with glucosamine, an intermediate metabolite of the HBP. Glucosamine induced lipid accumulation and significantly increased the mRNA expression levels of peroxisome proliferator-activated receptor gamma, adiponectin, and aP2 in C2C12 myoblasts. Similar mRNA changes were observed in skeletal muscles of Sprague-Dawley rats treated with glucosamine infusion. Our results provide a possible explanation of hyperglycemia-induced insulin resistance in skeletal muscle.     

Nanoparticle-mediated intracellular lipid accumulation during C2C12 cell differentiation

In this report, we sought to elucidate whether multiwall carbon nanotubes are involved in the modulation of the proliferation and differentiation of the skeletal muscle cell line C2C12. Skeletal muscle is a major mass peripheral tissue that accounts for 40% of total body weight and 50% of energy consumption. We focused on the differentiation pathway of myoblasts after exposure to a vapor-grown carbon fiber, HTT2800, which is one of the most highly purified carbon nanotubes. This treatment leads in parallel to the expression of a typical adipose differentiation program. We found that HTT2800 stimulated intracellular lipid accumulation in C2C12 cells. We have also shown by quantified PCR analysis that the expression of adipose-related genes was markedly upregulated during HTT2800 exposure. Taken together, these results suggest that HTT2800 specifically converts the differentiation pathway of C2C12 myoblasts to that of adipoblast-like cells.     

FGF21 deletion exacerbates diabetic cardiomyopathy by aggravating cardiac lipid accumulation

Fibroblast growth factor 21 (FGF21) plays an important role in energy homoeostasis. The unaddressed question of FGF21's effect on the development and progression of diabetic cardiomyopathy (DCM) is investigated here with FGF21 knockout (FGF21KO) diabetic mice. Type 1 diabetes was induced in both FGF21KO and C57BL/6J wild‐type (WT) mice via streptozotocin. At 1, 2 and 4 months after diabetes onset, the plasma FGF21 levels were significantly decreased in WT diabetic mice compared to controls. There was no significant difference between FGF21KO and WT diabetic mice in blood glucose and triglyceride levels. FGF21KO diabetic mice showed earlier and more severe cardiac dysfunction, remodelling and oxidative stress, as well as greater increase in cardiac lipid accumulation than WT diabetic mice. Western blots showed that increased cardiac lipid accumulation was accompanied by further increases in the expression of nuclear factor (erythroid‐derived 2)‐like 2 (Nrf2) and its target protein CD36, along with decreases in the phosphorylation of AMP‐activated protein kinase and the expression of hexokinase II and peroxisome proliferator‐activated receptor gamma co‐activator 1α in the heart of FGF21KO diabetic mice compared to WT diabetic mice. Our results demonstrate that FGF21 deletion‐aggravated cardiac lipid accumulation is likely mediated by cardiac Nrf2‐driven CD36 up‐regulation, which may contribute to the increased cardiac oxidative stress and remodelling, and the eventual development of DCM. These findings suggest that FGF21 may be a therapeutic target for the treatment of DCM.     

Acidosis, cardiac stunning and its prevention by oxygen

When during aerobic perfusion of the 5 Hz paced rat Langendorff heart, under constant aortic pressure of 8.3 kPa, the pH of the medium is changed from 7.5 to 7.0 a short period of positive inotropy is followed by a dramatic loss of contractility. The hearts, rapidly frozen after 10 min pH 7.0 perfusion, show moderate loss of high-energy phosphates and accumulation of lactate and glycerol-3-phosphate, indicative of tissue anaerobiosis. This can be overcome by including fluorocarbon, an O2 vehicle, in the media. The transient positive inotropy is interpreted as H(+)-induced release of plasmalemma-bound Ca2+ into the cytosol. The accompanying morphologic alterations are as described in this issue by Vandeplassche and Borgers (1990) and by Verkleij et al. (1990).     

Protein kinase C inhibits Ca2+ accumulation in cardiac sarcoplasmic reticulum

It is now recognized that phorbol esters are negative inotropic agents in mammalian heart which presumably act via stimulation of Ca2(+)-activated phospholipid-dependent protein kinase (PKC). The goal in the present study was to identify the underlying cellular processes. Digitonin-permeabilized cultured neonatal rat ventricular myocytes were used to study biochemical and functional effects of phorbol esters on cardiac sarcoplasmic reticulum (SR). These cells contracted spontaneously at 3 microM Ca2+. Beating was inhibited by 10 microM ryanodine and was insensitive to 1 microM nifedipine. Thus, beating behavior results from the phasic oscillation of Ca2+ transport by SR in this preparation. Phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), decreased frequency by 30%, suggesting that Ca2+ transport by SR had been reduced. Whereas cAMP stimulated the rate of oxalate-supported 45Ca2+ uptake 2-fold, phorbol esters, TPA, and phorbol 12,13-dibutyrate inhibited this process by about 45%. The effects of phorbols were specific: (a) the alpha-analogues of TPA and phorbol 12,13-dibutyrate were inactive; and (b) the phorbol esters had no effect on Ca2+ transport in cells that had been depleted of PKC. TPA decreased oxalate-stimulated Ca2+ uptake over the entire range of Ca2+ concentrations, from 0.1 to 10 microM, by at least 70% without shifting the half-maximal effective Ca2+ concentration. Taken together these results indicate that the effects of phorbol ester on cardiac contraction are due to decreased Ca2+ transport by the SR and that these responses are mediated by PKC. These studies support the interpretation that the negative inotropic effects of phorbol esters are due, in part, to decreased SR function.     

Increased FAT/CD36 cycling and lipid accumulation in myotubes derived from obese type 2 diabetic patients

Background

Permanent fatty acid translocase (FAT/)CD36 relocation has previously been shown to be related to abnormal lipid accumulation in the skeletal muscle of type 2 diabetic patients, however mechanisms responsible for the regulation of FAT/CD36 expression and localization are not well characterized in human skeletal muscle.

Methodology/Principal Findings

Primary muscle cells derived from obese type 2 diabetic patients (OBT2D) and from healthy subjects (Control) were used to examine the regulation of FAT/CD36. We showed that compared to Control myotubes, FAT/CD36 was continuously cycling between intracellular compartments and the cell surface in OBT2D myotubes, independently of lipid raft association, leading to increased cell surface FAT/CD36 localization and lipid accumulation. Moreover, we showed that FAT/CD36 cycling and lipid accumulation were specific to myotubes and were not observed in reserve cells. However, in Control myotubes, the induction of FAT/CD36 membrane translocation by the activation of (AMP)-activated protein kinase (AMPK) pathway did not increase lipid accumulation. This result can be explained by the fact that pharmacological activation of AMPK leads to increased mitochondrial beta-oxidation in Control cells.

Conclusion/Significance

Lipid accumulation in myotubes derived from obese type 2 diabetic patients arises from abnormal FAT/CD36 cycling while lipid accumulation in Control cells results from an equilibrium between lipid uptake and oxidation. As such, inhibiting FAT/CD36 cycling in the skeletal muscle of obese type 2 diabetic patients should be sufficient to diminish lipid accumulation.     

Nephrotoxicity and its prevention by vitamin E in ferric nitrilotriacetate-promoted lipid peroxidation

Iron and aluminum complexes of nitrilotriacetic acid cause severe nephrotoxicity in Wistar rats. In addition, a high incidence of renal cell carcinoma is seen in ferric nitrilotriacetate-treated animals. The present study was performed to see if lipid peroxidation is involved in ferric nitrilotriacetate toxicity. Ferric nitrilotriacetate had more bleomycin-detectable 'free' iron than any ferric salt, while iron complexed with desferrioxamine or ferric chondroitin sulfate had none. The toxicity of ferric nitrilotriacetate in vivo was more pronounced in vitamin E-deficient rats. A thiobarbituric acid-reactive substance was present in the kidneys of vitamin E-deficient rats in amounts markedly elevated compared to vitamin E-sufficient, or vitamin E-supplemented rats. Non-complexed nitrilotriacetate or aluminum nitrilotriacetate did not produce any thiobarbituric acid-reactive substance in vitamin E-sufficient rats died by the 58th day of administration. We suggest that the iron-stimulated production of free radicals leading to lipid peroxidation is the major cause of ferric nitrilotriacetate-mediated renal toxicity. Vitamin E, a known scavenger of free radicals, is effective in protecting against this iron-induced toxicity.     

Differential regulation of single CFTR channels by PP2C, PP2A, and other phosphatases

Cystic fibrosistransmembrane conductance regulator (CFTR)Cl channel activitydeclines rapidly when excised from transfected Chinese hamster ovary(CHO) or human airway cells because of membrane-associated phosphataseactivity. In the present study, we found that CFTR channels usuallyremained active in patches excised from baby hamster kidney (BHK) cellsoverexpressing CFTR. Those patches with stable channel activity wereused to investigate the regulation of CFTR by exogenous proteinphosphatases (PP). Adding PP2A, PP2C, or alkaline phosphatase toexcised patches reduced CFTR channel activity by >90% but did notabolish it completely. PP2B caused weak deactivation, whereas PP1 hadno detectable effect on open probability(P_o).Interestingly, the time course of deactivation by PP2C was identical tothat of the spontaneous rundown observed in some patches afterexcision. PP2C and PP2A had distinct effects on channel gating;P_o declinedduring exposure to exogenous PP2C (and during spontaneous rundown, whenit was observed) without any change in mean burst duration. Bycontrast, deactivation by exogenous PP2A was associated with a dramaticshortening of burst duration similar to that reported previously inpatches from cardiac cells during deactivation of CFTR by endogenousphosphatases. Rundown of CFTR-mediated current across intact T84epithelial cell monolayers was insensitive to toxic levels of the PP2Ainhibitor calyculin A. These results demonstrate that exogenous PP2C is a potent regulator of CFTR activity, that its effects on single-channel gating are distinct from those of PP2A but similar to those of endogenous phosphatases in CHO, BHK, and T84 epithelial cells, and thatmultiple protein phosphatases may be required for complete deactivationof CFTR channels.

     

不同运动方式对肥胖大鼠肝脏脂质沉积及FGF21分泌的影响*

目的: 研究持续性运动训练(CT)与高强度间歇运动训练(HIIT)对正常和肥胖大鼠血清和肝脏FGF21蛋白含量及肝脏脂肪代谢的影响。方法: 雄性SD大鼠随机分为两组:普通饲料及45%高脂饲料喂养,8周后以普通饲料喂养,大鼠体重增加20%为肥胖造模成功标准。将正常大鼠随机分为正常安静组(LC)、正常高强度间歇运动训练组(LHI)、正常持续性运动训练组(LCT),肥胖大鼠随机分为肥胖安静组(OC)、肥胖高强度间歇运动训练组(OHI)及肥胖持续性运动训练组(OCT),每组10只,运动干预组大鼠进行8周不同方式负重游泳运动训练干预,末次运动干预间隔24 h后取血液检测血清炎症因子、FGF21水平,取肝脏组织检测脂质含量、脂代谢酶含量及FGF21表达水平。结果: 与LC组比较,OC组大鼠体重、血清炎症因子、肝脏甘油三酯(TG)含量显著增高(P＜0.05),LHI组肝脏TG含量显著降低,LCT组肝脏FGF21表达水平显著增高(P＜0.05)。与OC组比较,OHI组大鼠肝脏TG含量显著降低(P＜0.05),线粒体CPT-1β、β-HAD酶含量显著升高(P＜0.05),OCT组大鼠肝脏LPL、FAT/CD36酶含量显著增高,血清、肝脏FGF21水平均显著上升(P＜0.05)。结论: 两种运动方式均能降低正常、肥胖大鼠体重及肥胖大鼠肝脏脂质沉积现象,其中HIIT上调线粒体脂肪氧化水平,显著降低正常、肥胖大鼠肝脏TG含量,而CT通过提高正常、肥胖大鼠肝脏FGF21蛋白表达及血清FGF21水平,促进肝脏摄取脂肪酸,对缓解肥胖大鼠肝脏脂质沉积效果有限。     

Role of endothelial AADC in cardiac synthesis of serotonin and nitrates accumulation

Serotonin (5-HT) regulates different cardiac functions by acting directly on cardiomyocytes, fibroblasts and endothelial cells. Today, it is widely accepted that activated platelets represent a major source of 5-HT. In contrast, a supposed production of 5-HT in the heart is still controversial. To address this issue, we investigated the expression and localization of 5-HT synthesizing enzyme tryptophan hydroxylase (TPH) and L-aromatic amino acid decarboxylase (AADC) in the heart. We also evaluated their involvement in cardiac production of 5-HT. TPH1 was weakly expressed in mouse and rat heart and appeared restricted to mast cells. Degranulation of mast cells by compound 48/80 did not modify 5-HT cardiac content in mice. Western blots and immunolabelling experiments showed an abundant expression of AADC in the mouse and rat heart and its co-localization with endothelial cells. Incubation of cardiac homogenate with the AADC substrate (5-hydroxy-L-tryptophan) 5-HTP or intraperitoneal injection of 5-HTP in mice significantly increased cardiac 5-HT. These effects were prevented by the AADC inhibitor benserazide. Finally, 5-HTP administration in mice increased phosphorylation of aortic nitric oxide synthase 3 at Ser (1177) as well as accumulation of nitrates in cardiac tissue. This suggests that the increase in 5-HT production by AADC leads to activation of endothelial and cardiac nitric oxide pathway. These data show that endothelial AADC plays an important role in cardiac synthesis of 5-HT and possibly in 5-HT-dependent regulation of nitric oxide generation.     

Adipose tissue sensitization to insulin induced by troglitazone and MEDICA 16 in obese Zucker rats in vivo

The putative role played by insulin sensitizers in modulating adipose tissue lipolysis in the fasting state was evaluated in obese conscious Zucker rats treated with troglitazone or beta,beta'-tetramethylhexadecanedioic acid (MEDICA 16) and compared with nontreated lean and obese animals. The rates of appearance (R(a)) of glycerol and free fatty acid (FFA), primary intra-adipose reesterification, and secondary reuptake of plasma FFA in adipose fat were measured using constant infusion of stable isotope-labeled [(2)H(5)]glycerol, [2,2-(2)H(2)]palmitate, and radioactive [(3)H]palmitate. The overall lipolytic flux (R(a) glycerol) was increased 1.7- and 1.4-fold in obese animals treated with troglitazone or MEDICA 16, respectively, resulting in increased FFA export (R(a) FFA) in the troglitazone-treated rats. Primary intra-adipose reesterification of lipolysis-derived fatty acids was enhanced twofold by insulin sensitizers, whereas reesterification of plasma fatty acids was unaffected by either treatment. Despite the unchanged R(a) FFA in MEDICA 16 or the increased R(a) FFA induced by troglitazone, very low density lipoprotein production rates were robustly curtailed. Total adipose tissue reesterification, used as an estimate of glucose conversion to glyceride-glycerol, was increased 1.9-fold by treatment with the insulin sensitizers. Our results indicate that, in the fasting state, insulin sensitizers induce, in vivo, a significant activation rather than suppression of adipose tissue lipolysis together with stimulation of glucose conversion to glyceride-glycerol.     

Insulin receptor activation through its accumulation in lipid rafts by mild electrical stress

Insulin resistance is due to the reduced cellular response to insulin in peripheral tissues. The interaction of insulin with its receptor is the first step in insulin action and thus the identified target of insulin resistance. It has been well established that defects or mutations in the insulin receptor (IR) cause insulin resistance. Therefore, an IR activator might be a novel therapeutic approach for insulin resistance. Our previous report showed that mild electrical stress (MES) enhanced the insulin‐induced signaling pathway. However, the molecular mechanism of the effect of MES remains unclear. We assessed the effect of MES, which is characterized by low‐intensity direct current, on insulin signaling in vitro and in vivo. Here, we showed that MES activated the insulin signaling in an insulin‐independent manner and improved insulin resistance in peripheral tissues of high fat‐fed mice. Moreover, we found that MES increased the localization of IR in lipid rafts and enhanced the level of phosphorylated Akt in insulin‐resistant hepatic cells. Ablation of lipid rafts disrupted the effect of MES on Akt activation. Our findings indicate that MES has potential as an activator of IR in an insulin‐independent manner, and might be beneficial for insulin resistance in type 2 diabetes. J. Cell. Physiol. 228: 439–446, 2013. © 2012 Wiley Periodicals, Inc.     

Role of histone deacetylase 2 and its posttranslational modifications in cardiac hypertrophy

Cardiac hypertrophy is a form of global remodeling, although the initial step seems to be an adaptation to increased hemodynamic demands. The characteristics of cardiac hypertrophy include the functional reactivation of the arrested fetal gene program, where histone deacetylases (HDACs) are closely linked in the development of the process. To date, mammalian HDACs are divided into four classes: I, II, III, and IV. By structural similarities, class II HDACs are then subdivided into IIa and IIb. Among class I and II HDACs, HDAC2, 4, 5, and 9 have been reported to be involved in hypertrophic responses; HDAC4, 5, and 9 are negative regulators, whereas HDAC2 is a pro-hypertrophic mediator. The molecular function and regulation of class IIa HDACs depend largely on the phosphorylation-mediated cytosolic redistribution, whereas those of HDAC2 take place primarily in the nucleus. In response to stresses, posttranslational modification (PTM) processes, dynamic modifications after the translation of proteins, are involved in the regulation of the activities of those hypertrophy-related HDACs. In this article, we briefly review 1) the activation of HDAC2 in the development of cardiac hypertrophy and 2) the PTM of HDAC2 and its implications in the regulation of HDAC2 activity. [BMB Reports 2015; 48(3): 131-138]     

PPARgamma2 regulates lipogenesis and lipid accumulation in steatotic hepatocytes

Peroxisome proliferator-activated receptor-gamma (PPARgamma) is considered to be one of the master regulators of adipocyte differentiation. PPARgamma2 is abundantly expressed in mature adipocytes and is elevated in the livers of animals that develop fatty livers. The aim of this study was to determine the ability of PPARgamma2 to induce lipid accumulation in hepatocytes and to delineate molecular mechanisms driving this process. The hepatic cell line AML-12 was used to generate a cell line stably expressing PPARgamma2. Oil Red O staining revealed that PPARgamma2 induces lipid accumulation in hepatocytes. This phenotype is accompanied by a selective upregulation of several adipogenic and lipogenic genes including adipose differentiation-related protein (ADRP), adipocyte fatty acid-binding protein 4, sterol regulatory element-binding protein-1 (SREBP-1), fatty acid synthase (FAS), and acetyl-CoA carboxylase, genes whose expression levels are known to increase in steatotic livers of ob/ob mice. Furthermore, the PPARgamma2-regulated induction of both SREBP-1 and FAS parallels an increase in de novo triacylglycerol synthesis in hepatocytes. Triacylglycerol synthesis and lipid accumulation are further enhanced by culturing hepatocytes with troglitazone in the absence of exogenous lipids. These results correspond with an increase in the lipid droplet protein, ADRP, and the data demonstrate that ADRP functions to coat lipid droplets in hepatocytes as observed by confocal microscopy. Taken together, these observations propose a role for PPARgamma2 as an inducer of steatosis in hepatocytes and suggest that this phenomenon occurs through an induction of pathways regulating de novo lipid synthesis.     

Protein phosphatase 2C (PP2C) function in higher plants

In the past few years, molecular cloning studies have revealed the primary structure of plant protein serine/threonine phosphatases. Two structurally distinct families, the PP1/PP2A family and the PP2C family, are present in plants as well as in animals. This review will focus on the plant PP2C family of protein phosphatases. Biochemical and molecular genetic studies in Arabidopsis have identified PP2C enzymes as key players in plant signal transduction processes. For instance, the ABI1/ABI2 PP2Cs are central components in abscisic acid (ABA) signal transduction. Arabidopsis mutants containing a single amino acid exchange in ABI1 or ABI2 show a reduced response to ABA. Another member of the PP2C family, kinase-associated protein phosphatase (KAPP), appears to be an important element in some receptor-like kinase (RLK) signalling pathways. Finally, an alfalfa PP2C acts as a negative regulator of a plant mitogen-activated protein kinase (MAPK) pathway. Thus, the plant PP2Cs function as regulators of various signal transduction pathways.