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1.
Molecules ; 25(19)2020 Oct 01.
Article in English | MEDLINE | ID: mdl-33019656

ABSTRACT

An increased understanding of low-density lipoprotein receptor (LDLR) and its regulation may facilitate drug development for the treatment of hypercholesterolemia. Triciribine (TCN), which is a highly selective AKT inhibitor, increases the stability of LDLR mRNA downstream of extracellular signal-regulated kinase (ERK) in human hepatoma cells (HepG2). Here, a candidate approach was used in order to determine whether the RNA-binding proteins (RBPs) ZFP36 ring finger protein like 1 (ZFP36L1) and Hu antigen R (HuR) play a role in TCN-mediated stabilization of LDLR mRNA. The depletion of HuR led to a reduction of LDLR mRNA stability, an event that was more pronounced in TCN-treated cells. TCN was found to induce the translocation of nuclear HuR to cytoplasm in an ERK-dependent manner. ZFP36L1 depletion increased the stability of LDLR mRNA consistent with its destabilizing role. However, in contrast to HuR, TCN had no effect on LDLR mRNA turnover in ZFP36L1-depleted cells. TCN induced the phosphorylation of ZFP36L1 in an ERK/RSK-dependent manner and promoted its dissociation from the CCR4-NOT complex. In sum, these data suggest that TCN utilizes ERK signaling to increase the activity of HuR and inhibit ZFP36L1 to stabilize LDLR mRNA in HepG2 cells.


Subject(s)
ELAV-Like Protein 1/metabolism , RNA Stability/genetics , Receptors, LDL/genetics , Ribonucleosides/pharmacology , Butyrate Response Factor 1/metabolism , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Exoribonucleases/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Hep G2 Cells , Humans , Phosphorylation/drug effects , Protein Transport/drug effects , RNA Stability/drug effects , Repressor Proteins/metabolism , Ribosomal Protein S6 Kinases, 90-kDa/metabolism
2.
Atherosclerosis ; 276: 28-38, 2018 09.
Article in English | MEDLINE | ID: mdl-30025252

ABSTRACT

BACKGROUND AND AIMS: Induction of low-density lipoprotein receptor (LDLR) plays a significant role in reduction of plasma LDL-cholesterol (LDL-C) levels. Therefore, strategies that enhance the protein level of LDLR provide an attractive therapeutic target for the treatment of hypercholesterolemia. With this aim in mind, we concentrated our effort on studying the role of AKT kinase in regulation of LDLR levels and proceeded to examine the effect of MK-2206, an allosteric and highly selective AKT inhibitor, on LDLR expression. METHODS: Cultured human hepatoma cells were used to examine the effect of MK-2206 on the proteolytic processing of sterol regulatory element-binding protein-2 (SREBP-2), the expression of LDLR and cellular internalization of LDL. We also examined the effect of MK-2206 on LDLR levels in primary human hepatocytes. RESULTS: MK-2206 induced the proteolytic processing of SREBP-2, upregulated LDLR expression and stimulated LDL uptake. In contrast to statins, induction of LDLR levels by MK-2206 did not rely on 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) inhibition. As a result, cotreatment of cells with MK-2206 and mevastatin potentiated the impact of mevastatin on LDLR. Importantly, MK-2206 stimulated the expression of LDLR by primary human hepatocytes. CONCLUSIONS: MK-2206 is a novel LDLR-inducing agent that, either alone or in combination with statins, exerts a stimulating effect on cellular LDL uptake.


Subject(s)
Anticholesteremic Agents/pharmacology , Cholesterol, LDL/metabolism , Hepatocytes/drug effects , Heterocyclic Compounds, 3-Ring/pharmacology , Hypercholesterolemia/drug therapy , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Receptors, LDL/metabolism , Biological Transport , HeLa Cells , Hep G2 Cells , Hepatocytes/enzymology , Humans , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Hypercholesterolemia/enzymology , Lovastatin/analogs & derivatives , Lovastatin/pharmacology , Proteolysis , Proto-Oncogene Proteins c-akt/metabolism , Receptors, LDL/genetics , Signal Transduction/drug effects , Sterol Regulatory Element Binding Protein 2/genetics , Sterol Regulatory Element Binding Protein 2/metabolism
3.
Cell Cycle ; 15(5): 628-43, 2016.
Article in English | MEDLINE | ID: mdl-27104745

ABSTRACT

Cell division is strictly regulated by a diversity of proteins and lipids to ensure proper duplication and segregation of genetic material and organelles. Here we report a novel role of the putative lipid transporter ACAT-related protein required for viability 1 (Arv1) during telophase. We observed that the subcellular localization of Arv1 changes according to cell cycle progression and that Arv1 is recruited to the cleavage furrow in early telophase by epithelial protein lost in neoplasm (EPLIN). At the cleavage furrow Arv1 recruits myosin heavy chain 9 (MYH9) and myosin light chain 9 (MYL9) by interacting with IQ-motif-containing GTPase-activating protein (IQGAP1). Consequently the lack of Arv1 delayed telophase-progression, and a strongly increased incidence of furrow regression and formation of multinuclear cells was observed both in human cells in culture and in follicle epithelial cells of egg chambers of Drosophila melanogaster in vivo. Interestingly, the cholesterol-status at the cleavage furrow did not affect the recruitment of either IQGAP1, MYH9 or MYL. These results identify a novel function for Arv1 in regulation of cell division through promotion of the contractile actomyosin ring, which is independent of its lipid transporter activity.


Subject(s)
Carrier Proteins/physiology , Membrane Proteins/physiology , Myosin Light Chains/metabolism , ras GTPase-Activating Proteins/metabolism , Animals , Cell Membrane Structures/metabolism , Cell Proliferation , Cholesterol/metabolism , Cytoskeletal Proteins/metabolism , Drosophila melanogaster , HeLa Cells , Hep G2 Cells , Humans , Protein Transport , Telophase
4.
Gene ; 543(2): 268-74, 2014 Jun 15.
Article in English | MEDLINE | ID: mdl-24583166

ABSTRACT

In mammals, two carotenoid cleaving oxygenases are known; beta-carotene 15,15'-monooxygenase (BCMO1) and beta-carotene 9',10'-oxygenase (BCO2). BCMO1 is a key enzyme in vitamin A synthesis by symmetrically cleaving beta-carotene into 2 molecules of all-trans-retinal, while BCO2 is responsible for asymmetric cleavage of a broader range of carotenoids. Here, we show that the Atlantic salmon beta-carotene oxygenase (bco) gene family contains 5 members, three bco2 and two bcmo1 paralogs. Using public sequence databases, multiple bco genes were also found in several additional teleost species. Phylogenetic analysis indicates that bco2a and bco2b originate from the teleost fish specific genome duplication (FSGD or 3R), while the third and more distant paralog, bco2 like, might stem from a prior duplication event in the teleost lineage. The two bcmo1 paralogs (bcmo1 and bcmo1 like) appear to be the result of an ancient duplication event that took place before the divergence of ray-finned (Actinopterygii) and lobe-finned fish (Sarcopterygii), with subsequent nonfunctionalization and loss of one Sarcopterygii paralog. Gene expression analysis of the bcmo1 and bco2 paralogs in Atlantic salmon reveals regulatory divergence with tissue specific expression profiles, suggesting that the beta-carotene oxygenase subtypes have evolved functional divergences. We suggest that teleost fish have evolved and maintained an extended repertoire of beta-carotene oxygenases compared to the investigated Sarcopterygii species, and hypothesize that the main driver behind this functional divergence is the exposure to a diverse set of carotenoids in the aquatic environment.


Subject(s)
Evolution, Molecular , Fish Proteins/genetics , Mixed Function Oxygenases/physiology , Salmo salar/genetics , Animals , Fish Proteins/classification , Gene Expression Regulation, Enzymologic , Mixed Function Oxygenases/classification , Mixed Function Oxygenases/genetics , Phylogeny
5.
BMC Genet ; 12: 52, 2011 May 30.
Article in English | MEDLINE | ID: mdl-21619714

ABSTRACT

BACKGROUND: Red flesh colour is a unique trait found in some salmonid genera. Carotenoid pigments are not synthesized de novo in the fish, but are provided by dietary uptake. A better understanding of the molecular mechanisms underlying the cellular uptake and deposition of carotenoids could potentially be used to improve the low muscle deposition rate that is typically found in farmed Atlantic salmon. In addition, from an evolutionary point of view, the establishment and maintenance of this trait is still poorly understood. It has been demonstrated in several species that scavenger receptor class B, member 1 (SCARB1) is involved in intestinal absorption of carotenoids, which makes this gene a possible source of genetic variation in salmonid flesh pigmentation. RESULTS: In this study, a novel paralog of SCARB1 (SCARB1-2) was detected through screening for genetic variation in Atlantic salmon SCARB1. Full length SCARB1-2 encodes a protein with 89% identity to Atlantic salmon SCARB1, except for the C-terminal cytoplasmic tail that shows only 12% identity. The most prominent site of SCARB1 mRNA expression was in the mid gut, while a five-fold lower level was detected in Atlantic salmon skeletal muscle and liver. The SCARB1-2 mRNA was equally expressed in liver, muscle and mid gut, and at a lower level than SCARB1 mRNA. A total of seven different SCARB1-2 alleles comprising repetitive enhancer of zeste motifs (EZH2) were identified in the founding parents of a resource Atlantic salmon population. We mapped the SCARB1-2 paralog to a region on Atlantic salmon chromosome 1, containing a putative QTL for flesh colour. Addition of the SCARB1-2 marker increased the significance of this QTL, however the large confidence interval surrounding the QTL precludes confirmation of SCARB1-2 as a causative gene underlying variation in this trait. CONCLUSION: We have characterised a novel paralog of SCARB1 (SCARB1-2), have mapped it to Atlantic salmon chromosome 1 and have described its expression in various tissues. Mapping with SCARB1-2 alleles added further evidence for a QTL affecting flesh colour on this chromosome, however further studies are needed to confirm a functional role for this gene in flesh colour pigmentation.


Subject(s)
Receptors, Scavenger/genetics , Salmo salar/genetics , Animals , Base Sequence , Chromosome Mapping , Female , Genetic Variation , Molecular Sequence Data , Quantitative Trait Loci , RNA, Messenger/analysis , Sequence Alignment
6.
Fish Shellfish Immunol ; 28(2): 394-400, 2010 Feb.
Article in English | MEDLINE | ID: mdl-20004720

ABSTRACT

Bacterial and viral diseases are major problems in Atlantic salmon aquaculture, but may be challenged through selection of brood stock with enhanced survival to diseases. Today's selection strategy is based on controlled challenge tests using siblings of the breeding candidates, and is thus indirect. Direct trait records on breeding candidates can potentially be provided through identification of genetic variation linked to the susceptibility to the disease. Peroxisome proliferator-activated receptor gamma (PPARG) is a lipid-sensing transcription factor primarily known for inducing fat-accumulation in adipocytes, but also in lipid-accumulating macrophages, in mammalian species. Here we report a novel allele of PPARG, pparg-2, in Atlantic salmon. pparg-2 has an insertion of sixty nucleotides that encodes two additional copies of the almost perfect decapeptide motif, (F/C/Y)NHSPDR(S/N)HS, compared to the previously described pparg-1. pparg-1 contains six copies of this repeat unit whereas eight copies are present in the novel pparg-2 allele. pparg-2 mRNA was detectable in kidney and spleen of random Atlantic salmon samples. Here, we studied the effect of pparg-1 and pparg-2 on survival upon challenge to a highly virulent bacterium, Aeromonas salmonicida, causing furunculosis, and the virus causing infectious salmon anaemia (ISA), respectively, in a Norwegian aquaculture population of Atlantic salmon. ppar alleles were found to be significantly associated with survival upon challenge to A. salmonicida, but not to ISA. pparg-2 was the better allele in terms of survival in the challenge test for furunculosis, survival rates being 0.32, 0.40 and 0.42 for animals with the pparg-1,-1, pparg-1, -2 and pparg-2, -2 genotypes, respectively. We conclude that pparg-2 is in linkage disequilibrium (LD) with, or identical to, a locus contributing to different susceptibility to furunculosis in Atlantic salmon. PPARG was mapped to linkage group eight (LG8) but could only be positioned on the male linkage map since all the informative parents in the mapping families were males. This is the first report showing an association between pparg alleles and an enhanced immune response in fish.


Subject(s)
Aeromonas salmonicida/physiology , Alleles , Fish Diseases/genetics , Gram-Negative Bacterial Infections/genetics , Immunity, Innate/genetics , PPAR gamma/genetics , Salmo salar , Animals , Female , Fish Diseases/mortality , Genotype , Gram-Negative Bacterial Infections/mortality , Male , RNA, Messenger/metabolism
7.
Article in English | MEDLINE | ID: mdl-19416703

ABSTRACT

Pigs are recognised as suitable biomedical models to study obesity and obesity-related diseases; however, little is known about adipose tissue development and adipogenesis in pigs. In this study, the temporal expression of key genes involved in lipid metabolism was investigated during porcine adipogenesis and the metabolic fate of exogenously administered palmitic acid (16:0) was examined in differentiating preadipocytes. The expression of genes encoding elongases and desaturases increased simultaneously with those involved in fatty acid and triacylglycerol synthesis during porcine adipogenesis, and a high biosynthesis of monounsaturated fatty acids was measured prior to storage in differentiating preadipocytes. Although the total fatty acid oxidation in differentiating preadipocytes was low, differentiating cells showed increased expression of hormone-sensitive lipase and mitochondrial and peroxisomal genes. These data provide new insight into the temporal expression of genes involved in lipid metabolism during porcine adipogenesis and suggest a possible role of elongation and desaturation events prior to lipid accumulation in porcine adipocytes.


Subject(s)
Adipocytes/metabolism , Adipogenesis/genetics , Gene Expression Profiling , Lipid Metabolism/genetics , Acetyltransferases/genetics , Adipocytes/cytology , Adipose Tissue/cytology , Animals , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cells, Cultured , Cluster Analysis , Fatty Acid Desaturases/genetics , Fatty Acid Elongases , Palmitic Acid/metabolism , Palmitic Acid/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , Swine
8.
Comp Biochem Physiol B Biochem Mol Biol ; 151(2): 147-52, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18621139

ABSTRACT

Four different subtypes of fatty acid binding proteins i.e. liver-type FABP1, heart/muscle-type FABP3, adipocyte-type FABP4 and epithelial/epidermal-type FABP5 are expressed in adipose tissue. However, only the regulatory role of FABP4 in adipogenesis has been thoroughly investigated. To increase the knowledge on possible roles of these FABP subtypes in preadipocyte differentiation, gene expression patterns were examined during adipogenesis in pig (Sus scrofa). FABP1 expression was induced in proliferating cells, whereas FABP3, FABP4 and FABP5 expression increased throughout preadipocyte differentiation. Interestingly, the FABP4 and FABP5 expression increased early in the differentiation, followed by FABP3 later in the differentiation process. This indicates a role of FABP4 and FABP5 in intracellular fatty acid transport during initiation of differentiation, whereas, FABP3 likely is involved in the transport of fatty acids during intermediate stages of adipogenesis. In this study we demonstrate that FABP3, FABP4 and FABP5 expression is correlated with that of the peroxisome proliferator-activated receptors alpha and gamma (PPARA and PPARG). Altogether, this suggests a role of FABP1 during cell proliferation, whereas a coordinated expression of FABP3, FABP4 and FABP5 together with that of PPARA, PPARG1 and PPARG2 might be critical for the metabolic regulation during porcine adipogenesis.


Subject(s)
Adipogenesis/genetics , Fatty Acid-Binding Proteins/genetics , Adipocytes/cytology , Adipocytes/metabolism , Animals , Base Sequence , Cells, Cultured , DNA Primers/genetics , Fatty Acid-Binding Proteins/classification , Gene Expression Regulation, Developmental , Molecular Sequence Data , Peroxisome Proliferator-Activated Receptors/classification , Peroxisome Proliferator-Activated Receptors/genetics , Sus scrofa
9.
Biochim Biophys Acta ; 1781(6-7): 326-35, 2008.
Article in English | MEDLINE | ID: mdl-18503782

ABSTRACT

Atlantic salmon (Salmo salar) preadipocytes, isolated from visceral adipose tissue, differentiate from an unspecialized fibroblast like cell type to mature adipocytes filled with lipid droplets in culture. The expression of the adipogenic gene markers peroxisome proliferated activated receptor (PPAR) alpha, lipoprotein lipase (LPL), microsomal triglyceride transfer protein (MTP), fatty acid transport protein (FATP) 1 and fatty acid binding protein (FABP) 3 increased during differentiation. In addition, we describe a novel alternatively spliced form of PPARgamma (PPARgamma short), the expression of which increased during differentiation. Eicosapentaenoic acid (20:5n-3, EPA) and docosahexaenoic acid (22:6n-3, DHA) lowered the triacylglycerol (TAG) accumulation in mature salmon adipocytes compared to oleic acid (18:1n-9, OA). This finding indicates that a reduced level of highly unsaturated n-3 fatty acids (HUFAs) in fish diets, when the traditional marine oil is exchanged for n-9 fatty acids (FAs) rich vegetable oils (VOs), may influence visceral fat deposition in salmonids. Moreover, major differences in the metabolism of EPA, DHA and OA at different stages during differentiation of adipocytes occur. Most of the EPA and DHA were oxidized in preadipocytes, while they were mainly stored in TAGs in mature adipocytes in contrast to OA which was primarily stored in TAGs at all stages of differentiation.


Subject(s)
Adipocytes/cytology , Cell Differentiation , Fatty Acids/metabolism , Salmon/metabolism , Adipocytes/metabolism , Adipocytes/ultrastructure , Animals , Base Sequence , Cells, Cultured , DNA Primers , Fatty Acids/classification , Microscopy, Electron , Oxidation-Reduction , Polymerase Chain Reaction
10.
Cell Biol Int ; 32(5): 525-31, 2008 May.
Article in English | MEDLINE | ID: mdl-18299211

ABSTRACT

Recently a role of adipose tissue as an endocrine organ secreting factors involved in the regulation of whole-body energy homeostasis has emerged. Preadipocytes in different fat depots have distinct adipogenic potential and the metabolic activity differs between mature adipocytes of different depot origins. Here we describe the proliferation and differentiation of stromal-vascular cells derived from subcutaneous and visceral fat depots of adult pigs. We demonstrate that subcutaneous porcine preadipocytes proliferate more actively and that individual subcutaneous adipocytes have a more rapid accumulation of triacylglycerols than visceral cells. During differentiation, subcutaneous and visceral preadipocytes showed similar gene expression patterns with increased expression of adiponectin (APM1), adipocyte-specific fatty acid binding protein (FABP4), catalase (CAT), and peroxisome proliferator-activated receptor gamma 2 (PPARG2). Furthermore, initial data showing depot-originated effects on the expression of CAT, carnitine palmitoyl transferase 1B (CPT1B) and FABP4 suggest possible depot specific differences in the function and metabolism of mature porcine adipocytes.


Subject(s)
Adipogenesis , Adipose Tissue/cytology , Adipose Tissue/metabolism , Stromal Cells/cytology , Stromal Cells/metabolism , Adipocytes/chemistry , Adipocytes/cytology , Adipocytes/metabolism , Animals , Cell Proliferation , Cells, Cultured , Gene Expression Regulation , Swine , Triglycerides/metabolism
11.
Comp Biochem Physiol B Biochem Mol Biol ; 145(2): 239-48, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16971150

ABSTRACT

In this study, the effects of in vivo administration of 3-thia fatty acids (FAs) on lipid metabolism in muscle and liver of Atlantic salmon were investigated. Prior to analysis, the fish were kept in tanks supplied with 5 degrees C seawater for 20 weeks. The fish were fed fish meal and fish oil (FO)-based diets supplemented with either nothing (FO), or 0.3% and 0.6% of the 3-thia FAs dodecylthioacetic acid (DTA) and tetradecylthioacetic acid (TTA) respectively. The fish grew from an initial weight of 110 g to 220 g in the FO group and to approximately 160 g in the 3-thia FA groups. There was a significant higher mortality (66%) in fish fed 0.6% TTA than in fish fed the 0.3% DTA (15%) and FO diets (15%). None of the 3-thia FA diets affected the lipid content of the salmon muscle. The liver index, however, was significantly higher and the total liver fat content lower in the TTA group than in the FO group. Both DTA and TTA were incorporated into the lipid fraction of muscle and liver (0.4% to 0.9%). There were no major differences in the total FA composition of liver and muscle between the dietary groups; except for a small increase of n-3 polyunsaturated FAs (PUFAs) in liver of the DTA group. The mRNA expression of peroxisome proliferator-activated receptor (PPAR) alpha, apolipoprotein AI (ApoAI), apolipoprotein CII (ApoCII) and low-density lipoprotein receptor (LDL-R) was down-regulated in liver of the salmon fed 0.3% DTA. PPARalpha and ApoAI transcripts were also reduced in liver of salmon fed 0.6% TTA. Additionally, the hepatic lipoprotein lipase (LPL) mRNA level was 3.8 fold increased in TTA fish relative to the FO group. In muscle there were no significant changes in gene expression pattern of any of the genes investigated. This is the first report on the effects of 3-thia FAs on gene expression in Atlantic salmon.


Subject(s)
Dietary Fats/pharmacology , Fish Proteins/genetics , Salmo salar/genetics , Sulfides/pharmacology , Acyl-CoA Oxidase/metabolism , Animals , Apolipoprotein A-I/genetics , Apolipoprotein A-I/metabolism , Apolipoprotein C-II/genetics , Apolipoprotein C-II/metabolism , Dietary Fats/administration & dosage , Fish Proteins/metabolism , Liver/enzymology , Liver/metabolism , Muscles/metabolism , Peroxisome Proliferator-Activated Receptors/genetics , Peroxisome Proliferator-Activated Receptors/metabolism , Receptors, LDL/genetics , Receptors, LDL/metabolism , Salmo salar/metabolism , Sulfides/administration & dosage
12.
Lipids ; 38(3): 289-96, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12784870

ABSTRACT

The aim of the present study was to develop a cell culture system for studying the proliferation and differentiation of preadipocytes isolated from Atlantic salmon adipose tissue. The expression of proliferating cell nuclear antigen (PCNA) was used as a marker for cell proliferation. The cells started to proliferate within 48 h after seeding and continued to proliferate throughout the culture period of 2 wk. Undifferentiated preadipocytes showed a fibroblast-like morphology with a homogeneous cytoplasm devoid of lipid droplets. At confluence, an exogenous lipid mixture was added to the cell cultures. The preadipocytes became larger and rounder during the subsequent days, and the cytoplasm gradually filled with lipid-rich droplets. These droplets were revealed by oil red O staining. Immunocytochemical staining showed that differentiated adipocytes expressed detectable levels of the three regulatory proteins associated with adipocyte differentiation: peroxisome proliferator-activated receptor gamma (PPARgamma), CCAAT/enhancer binding protein alpha (C/EBPalpha), and leptin. The cells also showed activity of glycerol-3-phosphate dehydrogenase (GPDH) (EC 1.1.1.8), a biochemical marker of adipocyte differentiation. The morphological and biochemical data presented here show that fish preadipocytes have properties that are similar to those of preadipocytes in mammals. We conclude therefore that salmon adipose tissue contains a sizable population of preadipocytes. Exogenous lipids promote the activation of adipose-related genes and induce the differentiation of fish preadipocytes in vitro.


Subject(s)
Adipocytes/cytology , Cell Culture Techniques/methods , Cell Differentiation/physiology , Salmo salar , Animals , CCAAT-Enhancer-Binding Protein-alpha/metabolism , Cell Division/physiology , Cells, Cultured , Glycerolphosphate Dehydrogenase/metabolism , Leptin/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Transcription Factors/metabolism
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