Lipocalin prostaglandin D synthase and PPARγ2 coordinate to regulate carbohydrate and lipid metabolism in vivo

Mice lacking Peroxisome Proliferator-Activated Receptor γ2 (PPARγ2) have unexpectedly normal glucose tolerance and mild insulin resistance. Mice lacking PPARγ2 were found to have elevated levels of Lipocalin prostaglandin D synthase (L-PGDS) expression in BAT and subcutaneous white adipose tissue (WAT). To determine if induction of L-PGDS was compensating for a lack of PPARγ2, we crossed L-PGDS KO mice to PPARγ2 KO mice to generate Double Knock Out mice (DKO). Using DKO mice we demonstrated a requirement of L-PGDS for maintenance of subcutaneous WAT (scWAT) function. In scWAT, DKO mice had reduced expression of thermogenic genes, the de novo lipogenic program and the lipases ATGL and HSL. Despite the reduction in markers of lipolysis in scWAT, DKO mice had a normal metabolic rate and elevated serum FFA levels compared to L-PGDS KO alone. Analysis of intra-abdominal white adipose tissue (epididymal WAT) showed elevated expression of mRNA and protein markers of lipolysis in DKO mice, suggesting that DKO mice may become more reliant on intra-abdominal WAT to supply lipid for oxidation. This switch in depot utilisation from subcutaneous to epididymal white adipose tissue was associated with a worsening of whole organism metabolic function, with DKO mice being glucose intolerant, and having elevated serum triglyceride levels compared to any other genotype. Overall, L-PGDS and PPARγ2 coordinate to regulate carbohydrate and lipid metabolism.     

How does protein phosphorylation regulate photosynthesis?

Phosphorylation of light-harvesting antenna proteins redirects absorbed light energy between reaction centres of photosynthetic membranes. A generally accepted explanation for this is that electrostatic forces drive the more negatively charged, phosphorylated antenna proteins between membrane domains that differ in surface charge. However, structural studies on soluble phosphoproteins indicate that phosphorylated amino acid side chains have specific effects on molecular recognition, by ligand blocking or by intramolecular interactions which alter protein structure. These studies suggest alternative mechanisms for phosphorylation in control of pairwise protein-protein interactions in biological membranes. Thus, in photosynthesis, the surface charge model is only one possible interpretation.     

Isohumulones modulate blood lipid status through the activation of PPARα

Isohumulones derived from hops are the major bitter compounds in beer. It was recently reported that isohumulones activated peroxisome proliferator-activated receptors (PPARs) α and γ in vitro and modulated glucose and lipid metabolism in vivo. In this study, we examined the effects of isomerized hop extract (IHE) primarily containing isohumulones in C57BL/6N male mice and found that such treatment increased their liver weight and reduced their plasma triglyceride and free fatty acid levels. Microarray analysis and quantitative real time PCR (QPCR) showed that IHE dose-dependently upregulated the expression of a battery of hepatic genes that are involved in microsomal ω-oxidation and peroxisomal and mitochondrial β-oxidation. These effects were common in both genders and very similar to those found with the PPARα agonist, fenofibrate (FF). Moreover, these effects were not found in PPARα-deficient mice. Thus, our results strongly suggest that IHE intake upregulates the expression of key genes that are involved in hepatic fatty acid oxidation, and that it ameliorates the blood lipid profile by activating PPARα.     

Inhibition of hepatocyte lipogenesis by nitric oxide donor: could nitric oxide regulate lipid synthesis?

Tissue lipogenesis is variably controlled by substrate supply and hormones. The possibility that nitric oxide (NO) might regulate lipogenesis derives from the action of NO on coenzyme A (CoA) to produce metabolically inactive S-nitrosoCoA. The effect of the nitric oxide donor S-nitrosoglutathione (GSNO) on long chain fatty acid and cholesterol synthesis was measured in isolated cultured rat hepatocytes. [1-14C] Butyrate was used as substrate to measure 14C incorporation into lipids as butyrate is twice as effective as acetate in hepatic lipogenesis and is ketogenic via the Lynen cycle. NO very significantly (P < 0.01) impaired long chain fatty acid and cholesterol synthesis an observation dependent upon time of exposure (3 h pre-incubation or 6 h continuous exposure) and concentration of GSNO (500 microM to 2.0 mM). Decrease in hepatic lipogenesis was paralleled by decrease in ketogenesis. ATP levels remained unchanged following short-term exposure to GSNO. Exposure of hepatocytes to GSNO together with 2.0 mM glutathione significantly diminished the inhibition of lipogenesis induced by GSNO alone. Impairment of lipogenesis by GSNO appears not to be limited by energy supply and now adduced, but not proven, to be operative via the degree of inactivation of cytosolic CoA. NO control of lipogenesis could be clinically important where NO production is increased as in demyelinating diseases, chronic arthritis or colitis and in wasting diseases such as AIDS.     

Hepatic PPARγ and LXRα independently regulate lipid accumulation in the livers of genetically obese mice

The nuclear hormone receptors liver X receptor α (LXRα) and peroxisome proliferator-activated receptor γ (PPARγ) play key roles in the development of fatty liver. To determine the link between hepatic PPARγ and LXRα signaling and the development of fatty liver, a LXRα-specific ligand, T0901317, was administered to normal OB/OB and genetically obese (ob/ob) mice lacking hepatic PPARγ (Pparγ^ΔH). In ob/ob-Pparγ^ΔH and OB/OB-Pparγ^ΔH mice, as well as ob/ob-Pparγ^WT and OB/OB-Pparγ^WT mice, the liver weights and hepatic triglyceride levels were markedly increased in response to T0901317 treatment. These results suggest that hepatic PPARγ and LXRα signals independently contribute to the development of fatty liver.     

PPARα and PPARγ regulate the nucleoside transporter hENT1

Human equilibrative nucleoside transporter 1 (hENT1) is an important determinant for nucleoside analog based chemotherapy success. Preliminary data suggest hENT1 regulation by PPARs. Using A2780 cells, we investigated the role of PPARs on hENT1 expression and activity. PPARα and PPARγ agonists, Wy14,643 and RGZ, increased hENT1 expression, but only PPARα activation or overexpression resulted in higher hENT1 transport activity. On the other hand, promoter analysis showed two putative PPRE in hENT1 promoter and luciferase-coupled promoter constructs were generated and analyzed. Our results suggest that PPARα-but not PPARγ-mediated expression regulation of hENT1 is PPRE-dependent. In conclusion, PPARα and PPARγ activation modulate hENT1 expression.     

PPAR�� isoforms differentially regulate metabolic networks to mediate mouse prostatic epithelial differentiation

Recent observations indicate prostatic diseases are comorbidities of systemic metabolic dysfunction. These discoveries revealed fundamental questions regarding the nature of prostate metabolism. We previously showed that prostate-specific ablation of PPARγ in mice resulted in tumorigenesis and active autophagy. Here, we demonstrate control of overlapping and distinct aspects of prostate epithelial metabolism by ectopic expression of individual PPARγ isoforms in PPARγ knockout prostate epithelial cells. Expression and activation of either PPARγ 1 or 2 reduced de novo lipogenesis and oxidative stress and mediated a switch from glucose to fatty acid oxidation through regulation of genes including Pdk4, Fabp4, Lpl, Acot1 and Cd36. Differential effects of PPARγ isoforms included decreased basal cell differentiation, Scd1 expression and triglyceride fatty acid desaturation and increased tumorigenicity by PPARγ1. In contrast, PPARγ2 expression significantly increased basal cell differentiation, Scd1 expression and AR expression and responsiveness. Finally, in confirmation of in vitro data, a PPARγ agonist versus high-fat diet (HFD) regimen in vivo confirmed that PPARγ agonization increased prostatic differentiation markers, whereas HFD downregulated PPARγ-regulated genes and decreased prostate differentiation. These data provide a rationale for pursuing a fundamental metabolic understanding of changes to glucose and fatty acid metabolism in benign and malignant prostatic diseases associated with systemic metabolic stress.     

CaMKII-γ mediates phosphorylation of BAD at Ser170 to regulate cytokine-dependent survival and proliferation

Phosphorylation of the BH3 (Bcl-2 homology domain 3)-only protein BAD (Bcl-2/Bcl-X(L)-antagonist, causing cell death) can either directly disrupt its association with the pro-survival proteins Bcl-X(L) and/or Bcl-2, or cause association of BAD with 14-3-3 proteins. In the present study, we further characterize phosphorylation of BAD at Ser170, a unique site with unclear function. We provide further evidence that mutation of Ser170 to a phospho-mimetic aspartic acid residue (S170D) can have a profound inhibitory effect on the pro-apoptosis function of BAD. Furthermore, mutated BAD with an alanine substitution inhibited cell proliferation, slowing progression specifically through S-phase. We identify the kinase responsible for phosphorylation at this site as CaMKII-γ (γ isoform of Ca2+/calmodulin-dependent kinase II), but not the other three isoforms of CaMKII, revealing an extraordinary specificity among these closely related kinases. Furthermore, cytokine treatment increased BAD-Ser170-directed CaMKII-γ activity and phosphorylation of CaMKII-γ at an activating site, and CaMKII activity directed to the BAD-Ser170 site was elevated during S-phase. Treating cells with a selective inhibitor of CaMKII caused apoptosis in cells expressing BAD, but not in cells expressing the BAD-S170D mutant. The present study provides support for BAD-Ser170 phosphorylation playing a key role not only in regulating BAD's pro-apoptotic activity, but also in cell proliferation.     

Exercise-associated generation of PPARγ ligands activates PPARγ signaling events and upregulates genes related to lipid metabolism

The aim of the present study was to test the hypotheses that exercise is associated with generation of peroxisome proliferator-activated receptor-γ (PPARγ) ligands in the plasma and that this may activate PPARγ signaling within circulating monocytes, thus providing a mechanism to underpin the exercise-induced antiatherogenic benefits observed in previous studies. A cohort of healthy individuals undertook an 8-wk exercise-training program; samples were obtained before (Pre) and after (Post) standardized submaximal exercise bouts (45 min of cycling at 70% of maximal O(2) uptake, determined at baseline) at weeks 0, 4, and 8. Addition of plasma samples to PPARγ response element (PPRE)-luciferase reporter gene assays showed increased PPARγ activity following standardized exercise bouts (Post/Pre = 1.23 ± 0.10 at week 0, P < 0.05), suggesting that PPARγ ligands were generated during exercise. However, increases in PPARγ/PPRE-luciferase activity in response to the same standardized exercise bout were blunted during the training program (Post/Pre = 1.18 ± 0.14 and 1.10 ± 0.10 at weeks 4 and 8, respectively, P > 0.05 for both), suggesting that the relative intensity of the exercise may affect PPARγ ligand generation. In untrained individuals, specific transient increases in monocyte expression of PPARγ-regulated genes were observed within 1.5-3 h of exercise (1.7 ± 0.4, 2.6 ± 0.4, and 1.4 ± 0.1 fold for CD36, liver X receptor-α, and ATP-binding cassette subfamily A member 1, respectively, P < 0.05), with expression returning to basal levels within 24 h. In contrast, by the end of the exercise program, expression at the protein level of PPARγ target genes had undergone sustained increases that were not associated with an individual exercise bout (e.g., week 8 Pre/week 0 Pre = 2.79 ± 0.61 for CD36, P < 0.05). Exercise is known to upregulate PPARγ-controlled genes to induce beneficial effects in skeletal muscle (e.g., mitochondrial biogenesis and aerobic respiration). We suggest that parallel exercise-induced benefits may occur in monocytes, as monocyte PPARγ activation has been linked to beneficial antidiabetic effects (e.g., exercise-induced upregulation of monocytic PPARγ-controlled genes is associated with reverse cholesterol transport and anti-inflammatory effects). Thus, exercise-triggered monocyte PPARγ activation may constitute an additional rationale for prescribing exercise to type 2 diabetes patients.     

The marine-derived furanone reduces intracellular lipid accumulation in vitro by targeting LXRα and PPARα

Recent studies have demonstrated that commercially available lipid-lowering drugs cause various side effects; therefore, searching for anti-hyperlipidaemic compounds with lower toxicity is a research hotspot. This study was designed to investigate whether the marine-derived compound, 5-hydroxy-3-methoxy-5-methyl-4-butylfuran-2(5H)-one, has an anti-hyperlipidaemic activity, and the potential underlying mechanism in vitro. Results showed that the furanone had weaker cytotoxicity compared to positive control drugs. In RAW 264.7 cells, the furanone significantly lowered ox-LDL-induced lipid accumulation (~50%), and its triglyceride (TG)-lowering effect was greater than that of liver X receptor (LXR) agonist T0901317. In addition, it significantly elevated the protein levels of peroxisome proliferator-activated receptors (PPARα) and ATP-binding cassette (ABC) transporters, which could be partially inhibited by LXR antagonists, GSK2033 and SR9243. In HepG2 cells, it significantly decreased oleic acid-induced lipid accumulation, enhanced the protein levels of low-density lipoprotein receptor (LDLR), ABCG5, ABCG8 and PPARα, and reduced the expression of sterol regulatory element-binding protein 2 (~32%). PPARα antagonists, GW6471 and MK886, could significantly inhibit the furanone-induced lipid-lowering effect. Furthermore, the furanone showed a significantly lower activity on the activation of the expression of lipogenic genes compared to T0901317. Taken together, the furanone exhibited a weak cytotoxicity but had powerful TC- and TG-lowering effects most likely through targeting LXRα and PPARα, respectively. These findings indicate that the furanone has a potential application for the treatment of dyslipidaemia.     

Adipocyte NCoR knockout decreases PPARγ phosphorylation and enhances PPARγ activity and insulin sensitivity

Insulin resistance, tissue inflammation, and adipose tissue dysfunction are features of obesity and Type 2 diabetes. We generated adipocyte-specific Nuclear Receptor Corepressor (NCoR) knockout (AKO) mice to investigate the function of NCoR in adipocyte biology, glucose and insulin homeostasis. Despite increased obesity, glucose tolerance was improved in AKO mice, and clamp studies demonstrated enhanced insulin sensitivity in liver, muscle, and fat. Adipose tissue macrophage infiltration and inflammation were also decreased. PPARγ response genes were upregulated in adipose tissue from AKO mice and CDK5-mediated PPARγ ser-273 phosphorylation was reduced, creating a constitutively active PPARγ state. This identifies NCoR as an adaptor protein that enhances the ability of CDK5 to associate with and phosphorylate PPARγ. The dominant function of adipocyte NCoR is to transrepress PPARγ and promote PPARγ ser-273 phosphorylation, such that NCoR deletion leads to adipogenesis, reduced inflammation, and enhanced systemic insulin sensitivity, phenocopying the TZD-treated state.     

Neuregulin/ErbB regulate neuromuscular junction development by phosphorylation of α-dystrobrevin

Neuregulin (NRG)/ErbB signaling is involved in numerous developmental processes in the nervous system, including synapse formation and function in the central nervous system. Although intensively investigated, its role at the neuromuscular synapse has remained elusive. Here, we demonstrate that loss of neuromuscular NRG/ErbB signaling destabilized anchoring of acetylcholine receptors (AChRs) in the postsynaptic muscle membrane and that this effect was caused by dephosphorylation of α-dystrobrevin1, a component of the postsynaptic scaffold. Specifically, in mice in which NRG signaling to muscle was genetically or pharmacologically abolished, postsynaptic AChRs moved rapidly from the synaptic to the perisynaptic membrane, and the subsynaptic scaffold that anchors the AChRs was impaired. These defects combined compromised synaptic transmission. We further show that blockade of NRG/ErbB signaling abolished tyrosine phosphorylation of α-dystrobrevin1, which reduced the stability of receptors in agrin-induced AChR clusters in cultured myotubes. Our data indicate that NRG/ErbB signaling maintains high efficacy of synaptic transmission by stabilizing the postsynaptic apparatus via phosphorylation of α-dystrobrevin1.     

Can phosphorylation of phosphatidate phosphohydrolase by a cyclic AMP-dependent mechanism regulate its activity and subcellular distribution and control hepatic glycerolipid synthesis?

Incubating the particle-free supernatant of rat liver with alkaline phosphatase decreased the activity of phosphatidate phosphohydrolase by 21-29%. When the particle-free supernatant was incubated with various combinations of Mg2+, ATP, cyclic AMP and cyclic AMP-dependent protein kinase this failed to alter significantly phosphatidate phosphohydrolase activity under the conditions employed. The incubation of hepatocytes in monolayer culture with 0.5 mM-8-(4-chlorophenylthio)adenosine 3',5'-monophosphate increased the total activity of phosphatidate phosphohydrolase as measured in vitro. This also decreased the proportion of the phosphohydrolase that was associated with the membrane fraction of the cells and increased that in the cytosolic fraction. Adding 1 mM-oleate to the hepatocytes promoted the translocation of phosphatidate phosphohydrolase from the cytosol to the membrane-associated compartment. Oleate overcame the effect of the cyclic AMP analogue in favouring the cytosolic distribution of the phosphohydrolase. These results are discussed in relation to the interaction of hormonal balance and substrate supply in controlling the synthesis of phosphatidylcholine and triacylglycerol in the liver in stress and in diabetes. It is proposed that the cytosolic phosphatidate phosphohydrolase activity represents a reservoir of potential activity that becomes expressed when the enzyme translocates to the membranes on which the synthesis of glycerolipids occurs.     

Relationship between signal transduction and PPARα-regulated genes of lipid metabolism in rat hepatic-derived Fao cells

The goal of this study was to characterize phosphorylated proteins and to evaluate the changes in their phosphorylation level under the influence of a peroxisome proliferator (PP) with hypolipidemic activity of the fibrate family. The incubation of rat hepatic derived Fao cells with ciprofibrate leads to an overphosphorylation of proteins, especially one of 85 kDa, indicating that kinase (or phosphatase) activities are modified. Moreover, immunoprecipitation of ³²P-labeled cell lysates shows that the nuclear receptor, PP-activated receptor, α isoform, can exist in a phosphorylated form, and its phosphorylation is increased by ciprofibrate. This study shows that PP acts at different steps of cell signaling. These steps can modulate gene expression of enzymes involved in fatty acid metabolism and lipid homeostasis, as well as in detoxication processes.     

畜禽PPARα基因研究进展

过氧化物酶体增殖物激活受体(Peroxisome proliferator-activated receptors,PPARs)是核激素受体家族中的配体激活受体,控制许多细胞内的代谢过程,PPARα作为过氧化物酶体增殖物激活受体家族重要成员之一,是调控机体脂质代谢的重要枢纽,在调控畜禽机体肝脏脂质代谢方面有重要作用。PPARα基因由四个结构域组成,多在机体肝脏和脂肪组织中表达,可作为细胞核受体被外源和内源的特异性配体结合并激活,进而结合靶基因发挥对肝脏脂质代谢的调控作用。就PPARα基因的结构特点及表达模式、PPARα基因对肝脏脂代谢的调控机制,以及现阶段PPARα在畜禽方面的研究进展进行阐述,旨在引起人们对PPARα基因调控脂质代谢的关注,并为畜禽肝脏脂质代谢过程的机理研究和相关疾病的治疗提供一些理论支持。     

β-Adrenergic agonists differentially regulate highly selective and nonselective epithelial sodium channels to promote alveolar fluid clearance in vivo

β-Adrenergic receptors (β-AR) increase epithelial sodium channel (ENaC) activity to promote lung fluid clearance. However, the effect of selective β-AR agonist on highly selective cation (HSC) channels or nonselective cation (NSC) channels in alveolar type 1 (T1) and type 2 (T2) cells is unknown. We hypothesized that stimulation with β(1)-AR agonist (denopamine) or β(2)-AR agonist (terbutaline) would increase HSC and/or NSC channel activity in alveolar epithelial cells. We performed single-channel measurements from T1 and T2 cells accessed from rat lung slices. Terbutaline (20 μM) increased HSC ENaC activity (open probability, NP(o)) in T1 (from 0.96 ± 0.61 to 1.25 ± 0.71, n = 5, P <0.05) and T2 cells (from 0.28 ± 0.14 to 1.0 ± 0.30, n = 8, P = 0.02). Denopamine (20 μM) increased NSC NP(o) in T1 cells (from 0.34 ± 0.09 to 0.63 ± 0.14, n = 7, P = 0.02) and in T2 cells (from 0.47 ± 0.09 to 0.68 ± 0.10, P = 0.004). In vivo X-ray imaging of lung fluid clearance and ICI 118,551 selective inhibition of β(2)-ARs confirmed patch-clamp findings. cAMP concentrations increased following treatment with denopamine or terbutaline (n = 3, P < 0.002). The effects of systemic (intraperitoneal, IP) and local (intratracheal, IT) modes of delivery on lung fluid clearance were assessed. IT delivery of denopamine promoted alveolar flooding, whereas IP delivery promoted delayed fluid clearance. In summary, β-AR agonists differentially regulate HSC and NSC in T1 and T2 cells to promote lung fluid clearance in vivo, and the mode of drug delivery is critical for maximizing β-AR agonist efficacy.     

N-NBD-l-α-Dilauroylphosphatidylethanolamine. A new fluorescent probe to study spontaneous lipid transfer

Migration of the fluorescent phospholipid $\text{N-(7-}\text{nitro-2,1,3-benzoxadiazol-4-yl)-}\text{l}\text{-α-dilauroylphosphatidyl-ethanolamine}$ between small sonicated egg phosphatidylcholine vesicles was studied by use of the fluorescence resonance energy transfer method. Contrary to the results of lipid transfer experiments reported for acyl chain NBD-labeled phospholipids (Nichols, J.W. and Pagano, R.E. (1982) Biochemistry 21, 1720–1728), the migration kinetics of N-NBD-DLPE had to be described by a sum of two exponential functions. The fast component $\text{(t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 38}\text{min}\text{)}$ was assigned to lipid transfer via soluble monomers and the slow component $\text{(t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≈ 400}\text{min}\text{)}$ to transbilayer motion. A reversible four-stage process is suggested as a kinetic model. Mathematical treatment of this scheme is given yielding an analytical expression for the time dependence of NBD emission intensity. The use of N-NBD-DLPE in the resonance energy transfer measurements offers the advantage of simple chemical synthesis of the fluorescent probe and leads to additional information on transbilayer motion which was not available with the NBD-labeled lipids used so far.