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1.
Cell Death Discov ; 2: 16009, 2016.
Article in English | MEDLINE | ID: mdl-27551503

ABSTRACT

Gender-specific differences are commonly found in metabolic pathways and in response to nutritional manipulation. Previously, we identified a role for caspase-2 in age-related glucose homeostasis and lipid metabolism using male caspase-2-deficient (Casp2 (-/-) ) mice. Here we show that the resistance to age-induced glucose tolerance does not occur in female Casp2 (-/-) mice and it appears to be independent of insulin sensitivity in males. Using fasting (18 h) as a means to further investigate the role of caspase-2 in energy and lipid metabolism, we identified sex-specific differences in the fasting response and lipid mobilization. In aged (18-22 months) male Casp2 (-/-) mice, a significant decrease in fasting liver mass, but not total body weight, was observed while in females, total body weight, but not liver mass, was reduced when compared with wild-type (WT) animals. Fasting-induced lipolysis of adipose tissue was enhanced in male Casp2 (-/-) mice as indicated by a significant reduction in white adipocyte cell size, and increased serum-free fatty acids. In females, white adipocyte cell size was significantly smaller in both fed and fasted Casp2 (-/-) mice. No difference in fasting-induced hepatosteatosis was observed in the absence of caspase-2. Further analysis of white adipose tissue (WAT) indicated that female Casp2 (-/-) mice may have enhanced fatty acid recycling and metabolism with expression of genes involved in glyceroneogenesis and fatty acid oxidation increased. Loss of Casp2 also increased fasting-induced autophagy in both male and female liver and in female skeletal muscle. Our observations suggest that caspase-2 can regulate glucose homeostasis and lipid metabolism in a tissue and sex-specific manner.

2.
Biochim Biophys Acta ; 1811(7-8): 468-75, 2011.
Article in English | MEDLINE | ID: mdl-21570480

ABSTRACT

AIMS: The study aims to determine the effect of long-chain saturated and polyunsaturated (PUFA) fatty acids, specifically palmitic acid (PA; 16:0), docosahexaenoic acid (DHA; 22:6n-3) and linoleic acid (LA; 18:2n-6), and their interactions with factors from adipose tissue, on insulin sensitivity and lipid metabolism in skeletal muscle. METHODS: L6 myotubes were cultured with PA, DHA or LA (0.4mmol/l), with or without conditioned media from human subcutaneous (SC) and visceral (IAB) fat. Insulin-stimulated glucose uptake, lipid content, mRNA expression of key genes involved in nutrient utilization and protein expression of inhibitor protein inhibitor kappa B (IκB)-α and mammalian target of rapamycin (mTOR) were measured. RESULTS: PA and IAB fat reduced insulin-stimulated glucose uptake and their combined effect was similar to that of PA alone. PA-induced insulin resistance was ameliorated by inhibiting the de novo synthesis of ceramide, IκBα degradation or mTOR activation. The PA effect was also partially reversed by DHA and completely by LA in the presence of SC fat. PA increased diacylglycerol content, which was reduced by LA and to a greater extent when either IAB or SC fat was also present. PA increased SCD1 whereas DHA and LA increased AMPKα2 mRNA. In the presence of SC or IAB fat, the combination of PA with either DHA or LA decreased SCD1 and increased AMPKα2 mRNA. CONCLUSIONS: PA-induced insulin resistance in skeletal muscle involves inflammatory (nuclear factor kappa B/mTOR) and nutrient (ceramide) pathways. PUFAs promote pathways, at a transcriptional level, that increase fat oxidation and synergize with factors from SC fat to abrogate PA-induced insulin resistance.


Subject(s)
Fatty Acids/pharmacology , Glucose/metabolism , Insulin/pharmacology , Muscle Fibers, Skeletal/drug effects , Muscle Fibers, Skeletal/metabolism , Animals , Biological Transport, Active/drug effects , Cell Line , Ceramides/metabolism , Culture Media, Conditioned , Docosahexaenoic Acids/pharmacology , Energy Metabolism/drug effects , Gene Expression/drug effects , Humans , Insulin Resistance , Intra-Abdominal Fat/metabolism , Linoleic Acid/pharmacology , NF-kappa B/metabolism , Palmitic Acid/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Signal Transduction/drug effects , Subcutaneous Fat/metabolism , TOR Serine-Threonine Kinases/metabolism
3.
Mol Cell Endocrinol ; 284(1-2): 1-10, 2008 Mar 12.
Article in English | MEDLINE | ID: mdl-18255222

ABSTRACT

Leptin stimulates fatty acid oxidation via the phosphorylation of AMPK (AMP-activated protein kinase) and ACC (acetyl-CoA carboxylase). Obesity is associated with resistance to the effects of leptin. We determined the action of leptin on AMPKalpha and ACCbeta phosphorylation and lipid metabolism in soleus (SOL) and extensor digitorum longus (EDL) muscles from lean and obese Wistar rats after 1 and 100 nM leptin. Both leptin doses stimulated phosphorylation of AMPKalpha and ACCbeta (P

Subject(s)
Acetyl-CoA Carboxylase/metabolism , Fatty Acids/metabolism , Leptin/metabolism , Multienzyme Complexes/metabolism , Muscle, Skeletal/metabolism , Obesity/metabolism , Protein Serine-Threonine Kinases/metabolism , AMP-Activated Protein Kinases , Acyl Coenzyme A/metabolism , Adipose Tissue/pathology , Animals , Body Weight , Dietary Fats/administration & dosage , Disease Models, Animal , Dose-Response Relationship, Drug , Energy Metabolism , Enzyme Activation , Glycolysis , Humans , Insulin/blood , Leptin/blood , Leptin/pharmacology , Male , Malonyl Coenzyme A/metabolism , Muscle Fibers, Skeletal/enzymology , Muscle Fibers, Skeletal/metabolism , Muscle, Skeletal/drug effects , Muscle, Skeletal/enzymology , Obesity/enzymology , Obesity/etiology , Obesity/pathology , Oxidation-Reduction , Phosphorylation , Rats , Rats, Wistar
4.
Mol Cell Endocrinol ; 267(1-2): 63-9, 2007 Mar 15.
Article in English | MEDLINE | ID: mdl-17270342

ABSTRACT

The endocannabinoids, a recently discovered endogenous, lipid derived, signaling system regulating energy metabolism, have effects on central and peripheral energy metabolism predominantly via the cannabinoid receptor type 1 (CB1). CB1 is expressed centrally in the hypothalamus and nucleus accumbens and peripherally in adipocytes and skeletal muscle. This study determined the effect of endocannabinoids on the expression of genes regulating energy metabolism in human skeletal muscle. Primary cultures of myotubes (lean and obese; n=3/group) were treated with the cannabinoid receptor agonist, anandamide (AEA) (0.2 and 5microM) and the CB1 specific antagonist AM251 (0.2 and 5microM) separately and in combination for 24h. The expression of mRNA for AMP-activated protein kinase (AMPK) alpha 1 (alpha1) and alpha 2 (alpha2), pyruvate dehydrogenase kinase 4 (PDK4) and peroxisome proliferator-activated receptor-gamma co-activator-1alpha (PGC-1alpha) were determined using 'Real Time' RT-PCR. AMPKalpha1 mRNA increased in lean and obese myotubes in response to AM251 (P<0.05). AEA inhibited the effect of AM251 on AMPKalpha1 mRNA levels in myotubes from lean and obese subjects (P<0.05); the dose-response curve was shifted to the left in the obese. In response to AM251, irrespective of the presence of AEA, PDK4 expression was decreased in lean and obese myotubes (P<0.05). Taken together these data suggest that endocannabinoids regulate pathways affecting skeletal muscle oxidation, effects particularly evident in myotubes from obese individuals.


Subject(s)
Muscle, Skeletal/metabolism , Receptor, Cannabinoid, CB1/metabolism , AMP-Activated Protein Kinases , Adult , Arachidonic Acids/pharmacology , Cells, Cultured , Dose-Response Relationship, Drug , Endocannabinoids , Female , Gene Expression Regulation/drug effects , Gene Expression Regulation, Enzymologic/drug effects , Heat-Shock Proteins/genetics , Heat-Shock Proteins/metabolism , Humans , Male , Multienzyme Complexes/genetics , Multienzyme Complexes/metabolism , Muscle Fibers, Skeletal/drug effects , Muscle Fibers, Skeletal/enzymology , Muscle, Skeletal/enzymology , Obesity/enzymology , Oxidation-Reduction/drug effects , Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha , Piperidines/pharmacology , Polyunsaturated Alkamides/pharmacology , Protein Kinases/genetics , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Pyrazoles/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptor, Cannabinoid, CB1/agonists , Receptor, Cannabinoid, CB1/antagonists & inhibitors , Receptor, Cannabinoid, CB1/genetics , Thinness/enzymology , Transcription Factors/genetics , Transcription Factors/metabolism
5.
Bone ; 27(1): 61-7, 2000 Jul.
Article in English | MEDLINE | ID: mdl-10865210

ABSTRACT

Fibrillin-containing microfibrils are structural components of extracellular matrices of a diverse range of tissues, including bone. Their importance in bone biology is illustrated by the skeletal abnormalities manifest in the congenital disorder, Marfan syndrome, which results from mutations in the fibrillin-1 gene. We investigated the expression of fibrillins and other microfibril-associated proteins in human bone and bone-derived osteoblasts. Analysis of RNA extracted from cancellous bone showed expression of mRNAs encoding fibrillin-1 and -2, MAGP-1 and -2, LTBP-2, and MP78/70 (Big-h3). In demineralized normal mature bone, fibrillin-1 was immunolocalized to fibrils within the bone matrix and pericellularly to cells lining the endosteal surfaces of trabecular bone, some osteocytes, and cells associated with blood vessels. LTBP-2 was also identified at the endosteal surface and within the bone matrix in a lamellar fashion. In addition, primary osteoblast-like cells cultured from human trabecular bone (obtained from patients at joint replacement surgery) were found to express abundant mRNA for fibrillins and associated glycoproteins. Moreover, using western blot analysis, fibrillin-1 protein was shown to be secreted into the medium and to be deposited into the cell layer. Immunofluorescence staining of the cell layer visualized fibrillin-1 in the matrix as a three-dimensional network of fine filaments. Expression of fibrillin-1 by osteoblast-like cells was constitutive, and a number of skeletally active agents had little effect on mRNA or protein levels. These results show that human osteoblasts from mature bone express fibrillins and other microfibril-associated proteins, and suggest a role for these molecules in adult human bone.


Subject(s)
Extracellular Matrix Proteins/biosynthesis , Microfilament Proteins/biosynthesis , Osteoblasts/metabolism , Adult , Bone and Bones/metabolism , Cells, Cultured , Contractile Proteins/biosynthesis , Fibrillin-1 , Fibrillins , Humans , Marfan Syndrome/metabolism , RNA Splicing Factors , RNA, Messenger/biosynthesis
6.
Int J Biochem Cell Biol ; 31(5): 613-26, 1999 May.
Article in English | MEDLINE | ID: mdl-10399321

ABSTRACT

Subtractive hybridisation was used to select for genes which are differentially expressed between a highly metastatic human colon carcinoma cell line, KM12SM, and the isogenetic non-metastatic cell line, KM12C. This led to the isolation of cDNA clones for a novel human adenosine 5'-phosphosulphate kinase/ATP sulphurylase (PAPS synthetase). Northern hybridisation revealed a single 4.2 kb mRNA species which showed an approximately 20-fold higher level of expression in the non-metastatic cell line than in the metastatic cell line. The overlapping cDNA clones together covered 3,774 bp including the entire coding region of 1,842 bp encoding a protein of 614 amino acids (calculated molecular mass of 69,496 Da). The protein contains consensus sequences for APS kinase and ATP sulphurylase, in its amino- and carboxy-terminal regions, respectively, as well as other sequences that are highly conserved amongst ATP sulphurylases and APS kinases. Interestingly, consensus sequences for GTPase activity were also identified, indicating that enzyme activity may be regulated by an intrinsic GTPase mechanism. Overall the new protein is 78% homologous with a previously described human PAPS synthetase (PAPSS1) indicating that we have identified the second member of a gene family which we have provisionally named PAPSS2. The gene locus for PAPSS2 was identified on chromosome 10 at 10q23.1-q23.2. This locus has synteny with the mouse brachymorphic gene recently identified as a PAPS synthetase (SK2). PAPSS2 appears to be the human homologue of this gene and thus PAPSS2 is likely to be important in human skeletogenesis.


Subject(s)
Colonic Neoplasms/genetics , Gene Expression Regulation, Neoplastic , Multienzyme Complexes/genetics , Sulfate Adenylyltransferase/genetics , Amino Acid Sequence , Base Sequence , Chromosomes, Human, Pair 10 , Cloning, Molecular , Gene Library , Humans , Molecular Sequence Data , Multienzyme Complexes/metabolism , Neoplasm Metastasis , Sequence Homology, Amino Acid , Sulfate Adenylyltransferase/metabolism , Tissue Distribution
7.
J Biol Chem ; 273(45): 29309-14, 1998 Nov 06.
Article in English | MEDLINE | ID: mdl-9792630

ABSTRACT

A cDNA for human microfibril-associated glycoprotein-2 (MAGP-2) was used to screen a human leukocyte genomic DNA library in EMBL-3 vector. One clone, clone H (10 kilobase pairs (kbp)), was isolated that contained most of the MAGP-2 gene. The remainder of the 3' end of the gene was obtained by direct polymerase chain reaction amplification of genomic DNA. The human MAGP-2 gene was found to be about 11 kbp in size and to contain 10 evenly distributed exons. The internal exons range in size from 30 base pairs (bp) to 88 bp with exons 4 and 6 the only exons of equal size (45 bp). All internal intron:exon junctions are defined by canonical splice donor and acceptor sites. Each junction has a 1/2 codon split with the exception of the exon 8/9 junction, which has a 2/1 split. The translation initiation codon is in exon 2, and the final exon contains 110 bp of coding sequence, including 2 cysteine codons. Primer extension experiments identified only one major transcription initiation site, 213 bases upstream of the ATG site. Rapid analysis of cDNA ends-polymerase chain reaction analysis of the 5' end of MAGP-2 mRNA from placenta confirmed this result and did not detect any alternative splicing of transcripts. The putative promoter region of the MAGP-2 gene was found to be AT-rich and it lacked a TATA box and other common regulatory elements. However the sequence surrounding the transcription start site CTCA(+1)TTCC was similar to the consensus CTCA(+1)NTCT (N is any nucleoside) for an initiator element found in terminal deoxynucleotidyltransferase and a number of other highly regulated genes. Comparison with the previously characterized human MAGP-1 gene showed that structural similarity was largely confined to the exact size, sequence, and junction alignment of the two penultimate exons which encode the first six of the seven cysteine residues that are precisely spaced in both proteins. The findings are consistent with the growing evidence that, although MAGP-1 and MAGP-2 are both intimately involved in the biology of fibrillin-containing microfibrils, the MAGPs are structurally, functionally, and developmentally diverse proteins which share one characteristic cysteine-rich motif.


Subject(s)
Contractile Proteins/genetics , Exons , Extracellular Matrix Proteins , Glycoproteins/genetics , Amino Acid Sequence , Base Sequence , DNA, Complementary , Humans , Intercellular Signaling Peptides and Proteins , Molecular Sequence Data , Promoter Regions, Genetic , Protein Biosynthesis , RNA Splicing Factors , Sequence Homology, Nucleic Acid
8.
J Biol Chem ; 271(2): 1096-103, 1996 Jan 12.
Article in English | MEDLINE | ID: mdl-8557636

ABSTRACT

Together with the 31-kDa microfibril-associated glycoprotein (MAGP), four polypeptides designated MP340 (340 kDa), MP78 (78 kDa), MP70 (70 kDa), and MP25 (25 kDa) have previously been identified in tissue extracts designed specifically to solubilize the microfibrillar component of elastic fibers. In the present study, both MP78 and MP70 were shown to be forms of a protein which is closely related to the human protein beta ig-h3, and MP340 was confirmed to be the bovine form of fibrillin-1. Peptide sequences from MP25 proved to be unique, and affinity-purified anti-MP25 antibodies were shown, by immunofluorescence and immunoelectron microscopy, to localize specifically to the elastin-associated microfibrils. This confirmed that MP25 was a distinct component of these structures. Expression screening of nuchal ligament cDNA libraries yielded a cDNA, cM10A (770 base pairs) which encodes amino acid sequences matching those of the MP25 peptides. Further library screening with cM10A identified cDNAs which encode the complete primary structures of bovine and human MP25. Bovine and human MP25 were found to be around 80% homologous and contain 170 and 173 amino acids, respectively. Data base searches revealed that MP25 had significant similarity of structure only with MAGP, indicating that the two proteins form a new family of microfibrillar proteins. In acknowledgment, MP25 has been formally renamed MAGP-2, and MAGP is referred to as MAGP-1. The close similarity between the two proteins (57%) is confined to a central region of 60 amino acids where there is precise alignment of 7 cysteine residues. Elsewhere the MAGP-2 molecule is rich in serine and threonine residues and contains an RGD motif. MAGP-2 lacks the proline-, glutamine-, and tyrosine-rich sequences and a hydrophobic carboxyl terminus, characteristic of MAGP-1. These structural differences suggest that MAGP-2 has some functions which are distinct from those of MAGP-1. The locus of the human MAGP-2 gene was identified on chromosome 12 in the region of 12p12.3-12p13.1.


Subject(s)
Elastic Tissue/metabolism , Extracellular Matrix Proteins , Glycoproteins/isolation & purification , Transforming Growth Factor beta , Amino Acid Sequence , Animals , Base Sequence , Cattle , Chromosome Mapping , Chromosomes, Human, Pair 12 , Cloning, Molecular , Elastic Tissue/embryology , Female , Fibrillin-1 , Fibrillins , Glycoproteins/genetics , Humans , Microfilament Proteins/genetics , Molecular Sequence Data , Neoplasm Proteins/genetics , Pregnancy , Sequence Alignment , Sequence Analysis
9.
Mol Cell Biol ; 15(12): 6932-42, 1995 Dec.
Article in English | MEDLINE | ID: mdl-8524260

ABSTRACT

Monoclonal antibodies to fibrillin 1 (MP340), a component of elastin-associated microfibrils, were used to screen cDNA libraries made from bovine nuchal ligament mRNA. One of the selected clones (cL9; 1.2 kb) hybridized on Northern (RNA) blotting with nuchal ligament mRNA to two abundant mRNAs of 9.0 and 7.5 kb, which were clearly distinct from fibrillin mRNA (10 kb). Further library screening and later reverse transcription PCR by the rapid amplification of cDNA ends (RACE) technique resulted in the isolation of additional overlapping cDNAs corresponding to about 6.7 kb of the mRNA. The encoded protein exhibited sequence similarity of around 80% with a recently identified human protein named latent transforming growth factor beta 1 (TGF-beta 1)-binding protein 2 (LTBP-2), indicating that the new protein was bovine LTBP-2. This was confirmed by the specific localization of bovine LTBP-2 cDNA probes to human chromosome 14q24.3, which is the locus of the human LTBP-2 gene. The domain structure of bovine LTBP-2 is very similar to that of the human LTBP-2, containing 20 examples of 6-cysteine epidermal growth factor-like repeats, 16 of which have the consensus sequence for calcium binding, together with 4 examples of 8-cysteine motifs characteristic of fibrillins and LTBP-1. A 4-cysteine sequence which is unique to bovine LTBP-2 and which has similarity to the 8-cysteine motifs was also present. Antibodies raised to two unique bovine LTBP-2 peptides specifically localized in tissue sections to the elastin-associated microfibrils, indicating that LTBP-2 is closely associated with these structures. Immunoblotting experiments identified putative LTBP-2 isoforms as a 260-kDa species released into the medium by cultured elastic tissue cells and as larger 290- and 310-kDa species in tissue extracts. A major proportion of tissue-derived LTBP-2 required treatment with 6 M guanidine for solubilization, indicating that the protein was strongly bound to the microfibrils. Most of the guanidine-solubilized LTBP-2 appeared to be monomeric, indicating that it was not involved in disulfide-bonded aggregation either with itself or with latent TGF-beta. Additional LTBP-2 was resistant to solubilization with 6 M guanidine but was readily extracted with a reductive saline solution. This treatment is relatively specific for solubilization of microfibrillar constituents including fibrillin 1 and microfibril-associated glycoprotein. Therefore, it can be inferred that some LTBP-2 is bound covalently to the microfibrils by reducible disulfide linkages. The evidence suggests that LTBP-2 has a direct role in elastic fiber structure and assembly which may be independent of its growth factor-binding properties. Thus, LTBP-2 appears to share functional characteristics with both LTBP-1 and fibrillins.


Subject(s)
Adaptor Proteins, Signal Transducing , Carrier Proteins/biosynthesis , Carrier Proteins/genetics , Elastin/analysis , Intracellular Signaling Peptides and Proteins , Microbodies/metabolism , Amino Acid Sequence , Animals , Base Sequence , Carrier Proteins/analysis , Cattle , Chromosome Mapping , Chromosomes, Human, Pair 14 , Cloning, Molecular , Cysteine , DNA Primers , Epidermal Growth Factor/genetics , Humans , In Situ Hybridization, Fluorescence , Latent TGF-beta Binding Proteins , Microbodies/ultrastructure , Molecular Sequence Data , Polymerase Chain Reaction , Sequence Homology, Amino Acid
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