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1.
Diabetologia ; 53(9): 1971-5, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20461355

ABSTRACT

AIMS/HYPOTHESIS: The pseudokinase tribbles homologue 3 (Drosophila) (TRIB3) negatively interferes with insulin-mediated phosphorylation and activation of v-akt murine thymoma viral oncogene homologue 1 (AKT1, also known as protein kinase B). Animal studies have shown that Trib3 expression was higher in the fasting state and in animal models of diabetes, promoting hyperglycaemia presumably by increasing glucose production in the liver. Less is known about the role of TRIB3 in insulin resistance in humans, although a gain-of-function mutation associated with abnormalities related to insulin resistance has been described in TRIB3. METHODS: We determined hepatic mRNA expression of TRIB3 and selected genes encoding enzymes, transcription factors and coactivators involved in glucose homeostasis. We also determined biochemical variables of intermediary metabolism in obese patients with varying degrees of insulin resistance. RESULTS: In our study population hepatic TRIB3 mRNA expression was associated with surrogate markers of insulin resistance. TRIB3 expression was significantly increased in a subgroup with high HOMA of insulin resistance (HOMA-IR) compared with a low HOMA-IR group (p = 0.0033). TRIB3 transcript levels were correlated with PEPCK (also known as PCK2) mRNA expression (p = 0.0014) and mRNA expression of PPARGC1A (p = 0.0020), PPARGC1B (p < 0.0001), USF1 (p = 0.0017), FOXO1 (p = 0.0003) and SREBP-1c (also known as SREBF1; p = 0.0360). Furthermore ligands of peroxisome proliferator-activated receptor alpha/retinoid X receptor and overexpression of its coactivator PPARGC1A as well as overexpression of SREBP-1c and its coactivator PPARGC1B increased TRIB3 promoter activity in HepG2 cells. CONCLUSIONS/INTERPRETATION: We have found evidence for a role of aberrant hepatic TRIB3 transcript levels in insulin resistance in obese humans and identified potential transcriptional pathways involved in regulation of TRIB3 gene expression in the liver.


Subject(s)
Cell Cycle Proteins/genetics , Insulin Resistance/genetics , Liver/metabolism , Obesity/genetics , Protein Serine-Threonine Kinases/genetics , Repressor Proteins/genetics , Carrier Proteins/genetics , Carrier Proteins/physiology , Cell Cycle Proteins/physiology , Gene Expression/drug effects , Heat-Shock Proteins/genetics , Heat-Shock Proteins/physiology , Hep G2 Cells , Humans , PPAR alpha/genetics , PPAR alpha/physiology , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha , Protein Serine-Threonine Kinases/physiology , Pyrimidines/pharmacology , RNA, Messenger , RNA-Binding Proteins , Repressor Proteins/physiology , Sterol Regulatory Element Binding Protein 1/genetics , Sterol Regulatory Element Binding Protein 1/physiology , Transcription Factors/genetics , Transcription Factors/physiology , Tretinoin/pharmacology
2.
Int J Obes (Lond) ; 34(5): 846-51, 2010 May.
Article in English | MEDLINE | ID: mdl-20125105

ABSTRACT

OBJECTIVE: Adiponectin signalling attenuates insulin resistance (IR) and steatosis hepatis in animal models. As adiponectin receptor (ADIPOR)1 and ADIPOR2 are critical components in the adiponectin signalling cascade, we studied hepatic ADIPOR1/2 mRNA levels in humans and their relation to IR. DESIGN: We determined metabolic risk factors and levels of hepatic mRNA transcribed from ADIPOR1, ADIPOR2 and FOXO1, a putative up-stream regulator, in 43 and 34 obese subjects with low and high homeostasis model assessment-IR, respectively. RESULTS: Plasma adiponectin and metabolic risk factors showed associations with IR as expected. Both hepatic ADIPOR1 and ADIPOR2 mRNA expression levels were higher in insulin-resistant subjects (P<0.0035). ADIPOR1 mRNA correlated with FOXO1 mRNA in obese insulin resistant (P=0.0034), but not insulin-sensitive subjects, while no correlations of ADIPOR2 with FOXO1 mRNA were noted. FOXO1 enhanced transcription from the ADIPOR1, but not the ADIPOR2 promoter in HepG2 cells. CONCLUSION: Increased hepatic ADIPOR1 and ADIPOR2 mRNA in insulin-resistant obese subjects may, at least in part, reflect a compensatory mechanism for reduced plasma adiponectin. FOXO1 may contribute to enhanced ADIPOR1, but not ADIPOR2 transcription in IR.


Subject(s)
Insulin Resistance/genetics , Obesity/metabolism , Receptors, Adiponectin/metabolism , Adiponectin/blood , Adult , Body Mass Index , Female , Forkhead Box Protein O1 , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolism , Gene Expression , Humans , Male , Obesity/genetics , Obesity/physiopathology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Adiponectin/genetics , Risk Factors
3.
J Intern Med ; 264(5): 452-62, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18537870

ABSTRACT

OBJECTIVE: Apolipoprotein A-V (apoAV) contributes to the regulation of triglyceride metabolism, which plays a role in the pathogenesis of atherosclerotic diseases. We therefore ascertained determinants of hepatic APOA5 transcript and apoAV plasma levels in humans. DESIGN: We determined influences of anthropometric variables, biochemical factors related to lipid and glucose metabolism, hepatic mRNA levels transcribed from the APOA1/C3/A4/A5 cluster and transcription factor genes implicated in the regulation of APOA5 as well as common single nucleotide polymorphisms (SNPs) at the APOA5 locus on APOA5 expression in 89 obese patients and 22 non-obese controls. RESULTS: Mean, age and sex adjusted, hepatic APOA5 mRNA or apoAV plasma levels did not differ by obesity status, homoeostasis model assessment insulin resistance or inflammatory markers. In multivariate regression models, the c56C > G SNP, plasma apoCIII, plasma nonesterified fatty acids, hepatic APOA5 transcripts, sex and a weak association with obesity status explained 61% of the variance in apoAV plasma levels. Hepatic transcript levels of carnitine palmitoyltransferase 1 (CPT1A1) and peroxisome proliferator-activated receptor alpha (PPARA), plasma nonesterified fatty acids and the c56C > G SNP explained 48% of the variance in hepatic APOA5 transcript levels. CONCLUSION: Apolipoprotein A-V plasma levels are independently associated with plasma free fatty acid and hepatic APOA5 mRNA levels. Associations of APOA5 transcripts with PPARA and CPT1A1 transcripts suggest that APOA5 expression is intimately linked to hepatic lipid metabolism.


Subject(s)
Apolipoproteins A/blood , Apolipoproteins A/genetics , Obesity/metabolism , Polymorphism, Single Nucleotide , Adult , Apolipoprotein A-V , Body Composition , Carnitine O-Palmitoyltransferase/metabolism , Case-Control Studies , Fatty Acids, Nonesterified/blood , Female , Genotype , Humans , Insulin Resistance , Liver/metabolism , Male , Middle Aged , Multivariate Analysis , Obesity/blood , PPAR alpha/metabolism , Phenotype , RNA, Messenger/analysis
4.
Curr Biol ; 11(11): 891-5, 2001 Jun 05.
Article in English | MEDLINE | ID: mdl-11516653

ABSTRACT

Cell movement is mediated by the protrusion of cytoplasm in the form of sheet- and rod-like extensions, termed lamellipodia and filopodia. Protrusion is driven by actin polymerization, a process that is regulated by signaling complexes that are, as yet, poorly defined. Since actin assembly is controlled at the tips of lamellipodia and filopodia [1], these juxtamembrane sites are likely to harbor the protein complexes that control actin polymerization dynamics underlying cell motility. An understanding of the regulation of protrusion therefore requires the characterization of the molecular components recruited to these sites. The Abl interactor (Abi) proteins, targets of Abl tyrosine kinases [2-4], have been implicated in Rac-dependent cytoskeletal reorganization in response to growth factor stimulation [5]. Here, we describe the unique localization of Abi proteins in living, motile cells. We show that Abi-1 and Abi-2b fused to enhanced yellow fluorescent protein (EYFP) are recruited to the tips of lamellipodia and filopodia. We identify the targeting domain as the homologous N terminus of these two proteins. Our findings are the first to suggest a direct involvement of members of the Abi protein family in the control of actin polymerization in protrusion events, and establish the Abi proteins as potential regulators of motility.


Subject(s)
Actins/isolation & purification , Adaptor Proteins, Signal Transducing , Cytoskeletal Proteins , Homeodomain Proteins/isolation & purification , Pseudopodia/ultrastructure , Animals , Cell Compartmentation , Cell Membrane/metabolism , Homeodomain Proteins/metabolism , Melanoma, Experimental , Mice , Protein Binding , Protein Sorting Signals , Protein Transport , Proto-Oncogene Proteins c-abl/metabolism , Recombinant Fusion Proteins/metabolism
5.
FEBS Lett ; 492(3): 215-20, 2001 Mar 16.
Article in English | MEDLINE | ID: mdl-11257497

ABSTRACT

Cell motility entails the extension of cytoplasmic processes, termed lamellipodia and filopodia. Extension is driven by actin polymerisation at the tips of these processes via molecular complexes that remain to be characterised. We show here that a green fluorescent protein (GFP) fusion of the Wiskott-Aldrich syndrome protein family member Scar1/WAVE1 is specifically recruited to the tips of lamellipodia in living B16F1 melanoma cells. Scar1-GFP was recruited only to protruding lamellipodia and was absent from filopodia. The localisation of Scar was facilitated by the finding that the formerly described inhibition of lamellipodia formation by ectopical expression of Scar, could be overcome by the treatment of cells with aluminium fluoride. These findings show that Scar is strategically located at sites of actin polymerisation specifically engaged in the protrusion of lamellipodia.


Subject(s)
Cell Movement/physiology , Microfilament Proteins/metabolism , Pseudopodia/metabolism , Actins/physiology , Animals , Cytoskeleton/metabolism , Fluorescence , Mice , Microfilament Proteins/physiology , Pseudopodia/physiology , Transfection , Tumor Cells, Cultured , Wiskott-Aldrich Syndrome Protein Family
6.
Microsc Res Tech ; 47(1): 3-17, 1999 Oct 01.
Article in English | MEDLINE | ID: mdl-10506758

ABSTRACT

The actin cytoskeleton is a dynamic filamentous network whose formation and remodeling underlies the fundamental processes of cell motility and shape determination. To serve these roles, different compartments of the actin cytoskeleton engage in forming specific coupling sites between neighbouring cells and with the underlying matrix, which themselves serve signal transducing functions. In this review, we focus on methods used to visualise the actin cytoskeleton and its dynamics, embracing the use of proteins tagged with conventional fluorophores and green fluorescent protein. Included also is a comparison of cooled CCD technology, confocal and 2-photon fluorescence microscopy of living and fixed cells, as well as a critique of current procedures for electron microscopy.


Subject(s)
Actins/analysis , Cytoskeleton/chemistry , Microscopy/methods , Actins/chemistry , Actins/ultrastructure , Animals , Cells, Cultured , Chickens , Fishes , Fluorescent Dyes/metabolism , Freezing , Green Fluorescent Proteins , Immunohistochemistry/methods , Keratinocytes/chemistry , Keratinocytes/metabolism , Luminescent Proteins , Phalloidine/metabolism , Rhodamines/metabolism , Staining and Labeling , Structure-Activity Relationship , Tissue Fixation
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