Cathepsin B regulates hepatic lipid metabolism by cleaving liver fatty acid-binding protein.

Synthesis and secretion of hepatic triglycerides (TAG) associated with very-low-density lipoprotein (VLDL) play a major role in maintaining overall lipid homeostasis. This study aims to identify factors affecting synthesis and secretion of VLDL-TAG using the growth hormone-deficient Ames dwarf mouse model, which has reduced serum TAG. Proteomic analysis coupled with a bioinformatics-driven approach revealed that these mice express greater amounts of hepatic cathepsin B and lower amounts of liver fatty acid-binding protein (LFABP) than their wildtype littermates. siRNA-mediated knockdown of cathepsin B in McA-RH7777 cells resulted in a 39% increase in [3H]TAG associated with VLDL secretion. Cathepsin B knockdown was accompanied by a 74% increase in cellular LFABP protein levels, but only when cells were exposed to 0.4 mm oleic acid (OA) complexed to BSA. The cathepsin B knockdown and 24-h treatment with OA resulted in increased CD36 expression alone and additively. Co-localization of LFABP and cathepsin B was observed in a distinct Golgi apparatus-like pattern, which required a 1-h OA treatment. Moreover, we observed co-localization of LFABP and apoB, independent of the OA treatment. Overexpression of cathepsin B resulted in decreased OA uptake and VLDL secretion. Co-expression of cathepsin B and cathepsin B-resistant mutant LFABP in McA-RH7777 cells resulted in an increased TAG secretion as compared with cells co-expressing cathepsin B and wildtype LFABP. Together, these data indicate that cathepsin B regulates VLDL secretion and free fatty acid uptake via cleavage of LFABP, which occurs in response to oleic acid exposure.

Reticulon 3 regulates very low density lipoprotein secretion by controlling very low density lipoprotein transport vesicle biogenesis.

Secretion of very low density lipoprotein (VLDL) by the liver is an important physiological process; however, the rate of VLDL secretion is determined by its transport from the endoplasmic reticulum (ER) to the Golgi. This transport event is facilitated by a specialized ER-derived vesicle, the VLDL transport vesicle (VTV). We have reported earlier a detailed VTV proteome, which revealed that reticulon 3 (RTN3) is uniquely present in the VTV. Our immunoblotting and electron microscopic data demonstrate that RTN3 is enriched in the VTV; however, other ER-derived vesicles do not contain RTN3. Co-immunoprecipitation data coupled with confocal microscopic analyses strongly suggest that RTN3 interacts with VLDL core protein, apoB100, at the ER level. Our data show that either blocking of RTN3 using specific antibodies or RTN3 knockdown resulted in significant reduction in VTV biogenesis from hepatic ER membranes. Additionally, VLDL secretion from hepatocytes was significantly decreased when RTN3 was silenced by RTN3 siRNA. We conclude that RTN3 regulates VLDL secretion by controlling VTV-mediated ER-to-Golgi transport of nascent VLDL.

Silencing of Small Valosin-containing Protein-interacting Protein (SVIP) Reduces Very Low Density Lipoprotein (VLDL) Secretion from Rat Hepatocytes by Disrupting Its Endoplasmic Reticulum (ER)-to-Golgi Trafficking.

The transport of nascent very low density lipoprotein (VLDL) particles from the endoplasmic reticulum (ER) to the Golgi determines their secretion by the liver and is mediated by a specialized ER-derived vesicle, the VLDL transport vesicle (VTV). Our previous studies have shown that the formation of ER-derived VTV requires proteins in addition to coat complex II proteins. The VTV proteome revealed that a 9-kDa protein, small valosin-containing protein-interacting protein (SVIP), is uniquely present in these specialized vesicles. Our biochemical and morphological data indicate that the VTV contains SVIP. Using confocal microscopy and co-immunoprecipitation assays, we show that SVIP co-localizes with apolipoprotein B-100 (apoB100) and specifically interacts with VLDL apoB100 and coat complex II proteins. Treatment of ER membranes with myristic acid in the presence of cytosol increases SVIP recruitment to the ER in a concentration-dependent manner. Furthermore, we show that myristic acid treatment of hepatocytes increases both VTV budding and VLDL secretion. To determine the role of SVIP in VTV formation, we either blocked the SVIP protein using specific antibodies or silenced SVIP by siRNA in hepatocytes. Our results show that both blocking and silencing of SVIP lead to significant reduction in VTV formation. Additionally, we show that silencing of SVIP reduces VLDL secretion, suggesting a physiological role of SVIP in intracellular VLDL trafficking and secretion. We conclude that SVIP acts as a novel regulator of VTV formation by interacting with its cargo and coat proteins and has significant implications in VLDL secretion by hepatocytes.

MCP-1-induced protein promotes endothelial-like and angiogenic properties in human bone marrow monocytic cells.

Monocytic cells enhance neovascularization by releasing proangiogenic mediators and/or by transdifferentiating into endothelial-like cells. However, the mechanisms that govern this transdifferentiation process are largely unknown. Recently, monocyte chemotactic protein-1 (MCP-1)-induced protein (MCPIP) has been identified as a novel CCCH-type zinc-finger protein expressed primarily in monocytic cells. Here, we analyzed whether MCPIP might exert angiogenic effects by promoting differentiation of monocytic cells into endothelial cell (EC)-like phenotype. The expression of MCPIP increased during MCP-1-induced transdifferentiation in human bone marrow mononuclear cells (BMNCs). Knockdown of MCPIP with small interfering RNA (siRNA) abolished MCP-1-induced expression of EC markers Flk-1 and Tie-2 in human BMNCs. BMNCs transfected with MCPIP expression vector displayed EC-like morphology accompanied by downregulation of monocytic markers CD14 and CD11b, upregulation of EC markers Flk-1 and Tie-2, induction of cadherin (cdh)-12 and -19, activation of endoplasmic reticulum (ER) stress, and autophagy. Knockdown of cdh-12 or cdh-19 markedly inhibited MCPIP-induced enhancement of cell attachment and EC-marker expression. Inhibition of ER stress by tauroursodeoxycholate abolished MCPIP-induced expression of EC markers. Inhibition of autophagy by knockdown of Beclin-1 with siRNA or by an autophagy inhibitor 3'-methyladenine inhibited MCPIP-induced expression of EC markers. Expression of MCPIP in BMNCs enhanced uptake of acetylated low-density lipoprotein (acLDL), formation of EC-colony, incorporation of cells into capillary-like structure on Matrigel, and exhibited increased neovascularization in the ischemic hindlimb in mice. These results demonstrate that MCPIP may be an important regulator of inflammatory angiogenesis and provide novel mechanistic insights into the link between MCP-1 and cardiovascular diseases.

Peroxisome proliferator-activated receptor {delta} is an essential transcriptional regulator for mitochondrial protection and biogenesis in adult heart.

RATIONALE: Peroxisome proliferator-activated receptors (PPARs) (alpha, gamma, and delta/beta) are nuclear hormone receptors and ligand-activated transcription factors that serve as key determinants of myocardial fatty acid metabolism. Long-term cardiomyocyte-restricted PPARdelta deficiency in mice leads to depressed myocardial fatty acid oxidation, bioenergetics, and premature death with lipotoxic cardiomyopathy. OBJECTIVE: To explore the essential role of PPARdelta in the adult heart. METHODS AND RESULTS: We investigated the consequences of inducible short-term PPARdelta knockout in the adult mouse heart. In addition to a substantial transcriptional downregulation of lipid metabolic proteins, short-term PPARdelta knockout in the adult mouse heart attenuated cardiac expression of both Cu/Zn superoxide dismutase and manganese superoxide dismutase, leading to increased oxidative damage to the heart. Moreover, expression of key mitochondrial biogenesis determinants such as PPARgamma coactivator-1 were substantially decreased in the short-term PPARdelta deficient heart, concomitant with a decreased mitochondrial DNA copy number. Rates of palmitate and glucose oxidation were markedly depressed in cardiomyocytes of PPARdelta knockout hearts. Consequently, PPARdelta deficiency in the adult heart led to depressed cardiac performance and cardiac hypertrophy. CONCLUSIONS: PPARdelta is an essential regulator of cardiac mitochondrial protection and biogenesis and PPARdelta activation can be a potential therapeutic target for cardiac disorders.

Transgenic cardiac-targeted overexpression of human thymidylate kinase.

Thymidylate kinase (TMPK) is a nucleoside monophosphate kinase that catalyzes phosphorylation of thymidine monophosphate to thymidine diphosphate. TMPK also mediates phosphorylation of monophosphates of thymidine nucleoside analog (NA) prodrugs on the pathway to their active triphosphate antiviral or antitumor moieties. Novel transgenic mice (TG) expressing human (h) TMPK were genetically engineered using the alpha-myosin heavy chain promoter to drive its cardiac-targeted overexpression. In '2 by 2' protocols, TMPK TGs and wild-type (WT) littermates were treated with the NA zidovudine (a deoxythymidine analog, 3'-azido-3'deoxythymidine (AZT)) or vehicle for 35 days. Alternatively, TGs and WTs were treated with a deoxycytidine NA (racivir, RCV) or vehicle. Changes in mitochondrial DNA (mtDNA) abundance and mitochondrial ultrastructure were defined quantitatively by real-time PCR and transmission electron microscopy, respectively. Cardiac performance was determined echocardiographically. Results showed TMPK TGs treated with either AZT or RCV exhibited decreased cardiac mtDNA abundance. Cardiac ultrastructural changes were seen only with AZT. AZT-treated TGs exhibited increased left ventricle (LV) mass. In contrast, LV mass in RCV-treated TGs and WTs remained unchanged. In all cohorts, LV end-diastolic dimension remained unchanged. This novel cardiac-targeted overexpression of hTMPK helps define the role of TMPK in mitochondrial toxicity of antiretrovirals.

Peroxisome proliferator-activated receptor delta regulates mitofusin 2 expression in the heart.

Mitofusin 2 (Mfn2) has been proposed as an important mitochondrial protein in maintaining mitochondrial network and bioenergetics. Mfn2 is highly expressed in the heart, but is downregulated in response to hypertrophic stimuli. However, little is known about how Mfn2's expression is regulated in cardiomyocytes. Here, we have investigated how Mfn2 expression in the heart responds to fasting condition and determined if Mfn2 is one of those PPARdelta-selective target genes that are involved in myocardial energy metabolism. Fasting for 48 h in mice led to a robust increase of Mfn2 expression in the heart. On the other hand, cardiomyocyte-restricted PPARdelta deficiency in mice led to substantially diminished cardiac expression of Mfn2 transcript and protein compared to that of controls. Fasting induced cardiac expression of Mfn2 was blunted in cardiomyocyte-restricted PPARdelta deficient hearts. Moreover, PPARdelta-selective ligand treatment in cultured cardiomyocytes induced elevated Mfn2 expression. A functional PPRE consensus sequence located at -837 to -817 bp upstream of the mouse Mfn2 promoter was identified and confirmed by Electrophoretic Mobility Shift Assays and Luciferase Promoter Reporter Assays. We conclude that Mfn2 is a PPARdelta-selective target, which may play an important role in regulating myocardial energy homeostasis.

Generation of an inducible, cardiomyocyte-specific transgenic mouse model with PPAR ß/δ overexpression.

Peroxisome proliferator-activated receptors (PPARs) consist of three subtypes, each displaying distinctive tissue distribution. In general, the three PPAR subtypes exert overlapping function in transcriptional regulation of lipid metabolism. However, each PPAR subtype possesses distinctive functions in different tissues dependent on their expression abundance, endogenous ligands, and the PPAR coregulators in a specific tissue. Transgenesis is an invaluable technique in defining the in vivo function of a particular gene and its protein. Cre/LoxP-mediated gene targeting has been extensively used to explore the tissue-specific function of PPARs. While this tissue-specific loss-of-function approach is extremely useful in determining the essential role of a PPAR, the tissue-specific gain-of-function approach is another important technique used to understand the effects of PPAR activation in a particular tissue. Transgenic overexpression of PPAR in a specific tissue has been used. However, this conventional technique requires generating the transgenic models individually for each target tissue. In this chapter, we describe the methodology for a more efficient generation of transgenic mouse models with a constitutively active form of PPARß/δ in different tissues.

Peroxisome proliferator-activated receptor ß/δ activation in adult hearts facilitates mitochondrial function and cardiac performance under pressure-overload condition.

Peroxisome proliferator-activated receptor ß/δ (PPARß/δ) is an essential transcription factor in myocardial metabolism. This study aims to investigate the effects of PPARß/δ activation in the adult heart on mitochondrial biology and oxidative metabolism under normal and pressure-overload conditions. We have investigated the effects of cardiac constitutively active PPARß/δ in adult mice using a tamoxifen-inducible transgenic approach with Cre-LoxP recombination. The expression of PPARß/δ mRNA and protein in cardiomyocytes of adult mice was substantially increased after short-term induction. In these mice, the cardiac expression of key factors involved in mitochondrial biogenesis, such as PPARγ coactivator-1, endogenous antioxidants Cu/Zn superoxide dismutase, and catalase, fatty acid, and glucose metabolism, such as carnitine palmitoyltransferase Ib, carnitine palmitoyltransferase II, and glucose transporter 4, were upregulated. Subsequently, myocardial oxidative metabolism was elevated concomitant with an increased mitochondrial DNA copy number and an enhanced cardiac performance. Moreover, activation of PPARß/δ in the adult heart improved cardiac function and resisted progression to pathological development in mechanical stress condition. We conclude that PPARß/δ activation in the adult heart will promote cardiac performance along with transcriptional upregulation of mitochondrial biogenesis and defense, as well as oxidative metabolism at basal and pressure-overload conditions.

Conditional PPARγ knockout from cardiomyocytes of adult mice impairs myocardial fatty acid utilization and cardiac function.

While the roles of PPARα and PPARδ (ß) in transcriptional regulation of myocardial lipid metabolisms are well established, an essential role of PPARγ in regulating lipid metabolisms in the adult heart remains unclear. In this study, we investigated whether PPARγ is required for normal myocardial lipid metabolism at basal condition in adult mice. We assessed the short-term cardiomyocyte-restricted PPARγ knockout mice with a Tamoxifen inducible Cre-LoxP mediated gene targeting strategy. The expression of PPARγ mRNA and protein in cardiomyocytes of adult mice was substantially reduced after short-term induction. Transcript and protein levels of important proteins in fatty acid uptake and oxidation, such as CD36, heart type-fatty acid binding protein (FABP), and carnitine palmitoyltransferase I (CPT-I) were reduced in the PPARγ deficient hearts. Myocardial fatty acid utilization and cardiac contraction were depressed in PPARγ deficient hearts. The PPARγ deficient hearts exhibited modest cardiac hypertrophy compared with controls. These results indicate that PPARγ is a transcription factor that is required for basal myocardial fatty acid utilization in the adult heart.

Liver X receptors are negative regulators of cardiac hypertrophy via suppressing NF-kappaB signalling.

AIMS: Nuclear factor-kappaB (NF-kappaB) plays a critical role in cell growth and inflammation during the progression of cardiac hypertrophy and heart failure. Several members of nuclear receptor superfamily, including liver X receptors (LXRalpha and LXRbeta), have been shown to suppress inflammatory responses, but little is known about their effects in cardiomyocytes. METHODS AND RESULTS: We investigated LXR expression patterns in pressure overload-induced hypertrophic hearts and the hypertrophic growth of the LXRalpha-deficient hearts from mice (C57/B6) in response to pressure overload. The underlying mechanisms were also explored using cultured myocytes. We found that cardiac expression of LXRalpha was upregulated in pressure overload-induced left ventricular hypertrophy in mice. Transverse aorta coarctation-induced left ventricular hypertrophy was exacerbated in LXRalpha-null mice relative to control mice. A synthetic LXR ligand, T1317, suppressed cardiomyocyte hypertrophy in response to angiotensin II and lipopolysaccharide treatments. In addition, LXR activation suppressed NF-kappaB signalling and the expression of associated inflammatory factors. Overexpression of constitutively active LXRalpha and beta in cultured myocytes suppressed NF-kappaB activity. CONCLUSION: LXRs are negative regulators of cardiac growth and inflammation via suppressing NF-kappaB signalling in cardiomyocytes. This should provide new insights into novel therapeutic targets for treating cardiac hypertrophy and heart failure.

Monocyte chemotactic protein (MCP)-1 promotes angiogenesis via a novel transcription factor, MCP-1-induced protein (MCPIP).

Monocyte chemotactic protein-1 (MCP-1) has been recognized as an angiogenic chemokine. The molecular mechanism of MCP-1-mediated angiogenesis remains unknown. We recently identified a novel transcription factor, designated MCP-1-induced protein (MCPIP), in human monocytes after treatment with MCP-1. We investigated whether MCP-1-induced angiogenesis is mediated via MCPIP. Treatment of human umbilical vein endothelial cells (HUVECs) with MCP-1 induced expression of MCPIP and capillary-like tube formation. Knockdown of MCPIP by small interfering RNA (siRNA) suppressed MCP-1-induced angiogenesis-related gene VEGF and HIF-1alpha expression as well as tube formation. Transfection of HUVECs with an MCPIP expression vector induced angiogenesis-related genes and tube formation. Chromatin immunoprecipitation analysis revealed that cadherin (cdh) 12 and cdh19 are in vivo targets of MCPIP. Transfection of HUVECs with MCPIP expression vector activated the expression of cdh12 and cdh19 genes. Knockdown of cdh12 or cdh19 expression markedly inhibited MCPIP-induced capillary-like tube formation. Moreover, knockdown of MCPIP also significantly suppressed MCP-1-induced cdh12 and cdh19 gene expression. Our data strongly suggest that MCP-1-induced angiogenesis is mediated via MCPIP, at least in part through transcriptional activation of cdh12 and cdh19.