George Hatzinikolas

Bovine latent transforming growth factor beta 1-binding protein 2: molecular cloning, identification of tissue isoforms, and immunolocalization to elastin-associated microfibrils.

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.

Further Characterization of Proteins Associated with Elastic Fiber Microfibrils Including the Molecular Cloning of MAGP-2 (MP25)

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 βig-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.

Expression of fibrillins and other microfibril-associated proteins in human bone and osteoblast-like cells

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.

Insulin-stimulated glucose uptake and pathways regulating energy metabolism in skeletal muscle cells: The effects of subcutaneous and visceral fat, and long-chain saturated, n-3 and n-6 polyunsaturated fatty acids

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.

AMPK and ACC phosphorylation : Effect of leptin, muscle fibre type and obesity

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<or=0.05) only in EDL muscles from lean animals. Malonyl-CoA levels were decreased in EDL muscles from lean animals after 1 and 100 nM leptin and significantly after 100 nM leptin in obese animals (P<or=0.05). Long-chain fatty acyl-CoA concentrations were decreased in EDL muscles from both phenotypes after 100 nM leptin. AMPK activation by leptin occurred independently of energy-related metabolites. These data demonstrate that the leptin effect on AMPKalpha and ACCbeta is muscle fibre type dependent and fails in diet-induced obesity.

The effect of dietary fat content on phospholipid fatty acid profile is muscle fiber type dependent

A high-saturated-fat diet (HFD) induces obesity and insulin resistance (IR). IR has been linked to alterations and increased saturation in the phospholipid composition of skeletal muscles. We aimed to determine whether HFD feeding affects fatty acid (FA) membrane profile in a muscle fiber type-specific manner. We measured phospholipid FAs and expression of FA synthesis genes in oxidative soleus (SOL) and glycolytic extensor digitorum longus (EDL) muscles from rats fed either standard chow (standard laboratory diet, SLD) or a HFD. The HFD increased fat mass, plasma insulin, and leptin levels. Compared with EDL, SOL muscles preferentially accumulated C18 over C16 FAs and n-6 over n-3 polyunsaturated FAs (PUFAs) on either diet. With the HFD, SOL muscles contained more n-9 monounsaturated FAs (MUFAs) and n-6 PUFAs and less n-7 MUFAs and n-3 PUFAs than EDL muscles and had lower unsaturation index, a pattern known to be associated with IR. Stearoyl-CoA desaturase-1 expression was approximately 13-fold greater in EDL than in SOL muscles but did not change with the HFD in either muscle. The expression of Elongase-5 was higher, and that of Elongase-6 (Elovl6) was lower in EDL compared with SOL muscles with both diets. In EDL muscles, the expression of Elovl6 was lower in the HFD than in the SLD. The pattern of FA uptake, expression, and diet-induced changes in FA desaturating and elongating enzymes maintained higher FA unsaturation in EDL muscles. Accordingly, the fiber type composition of skeletal muscles and their distribution may be important in the development and progression of obesity and IR.

TRPV1 Channels and Gastric Vagal Afferent Signalling in Lean and High Fat Diet Induced Obese Mice

Aim Within the gastrointestinal tract vagal afferents play a role in control of food intake and satiety signalling. Activation of mechanosensitive gastric vagal afferents induces satiety. However, gastric vagal afferent responses to mechanical stretch are reduced in high fat diet mice. Transient receptor potential vanilloid 1 channels (TRPV1) are expressed in vagal afferents and knockout of TRPV1 reduces gastro-oesophageal vagal afferent responses to stretch. We aimed to determine the role of TRPV1 on gastric vagal afferent mechanosensitivity and food intake in lean and HFD-induced obese mice. Methods TRPV1+/+ and -/- mice were fed either a standard laboratory diet or high fat diet for 20wks. Gastric emptying of a solid meal and gastric vagal afferent mechanosensitivity was determined. Results Gastric emptying was delayed in high fat diet mice but there was no difference between TRPV1+/+ and -/- mice on either diet. TRPV1 mRNA expression in whole nodose ganglia of TRPV1+/+ mice was similar in both dietary groups. The TRPV1 agonist N-oleoyldopamine potentiated the response of tension receptors in standard laboratory diet but not high fat diet mice. Food intake was greater in the standard laboratory diet TRPV1-/- compared to TRPV1+/+ mice. This was associated with reduced response of tension receptors to stretch in standard laboratory diet TRPV1-/- mice. Tension receptor responses to stretch were decreased in high fat diet compared to standard laboratory diet TRPV1+/+ mice; an effect not observed in TRPV1-/- mice. Disruption of TRPV1 had no effect on the response of mucosal receptors to mucosal stroking in mice on either diet. Conclusion TRPV1 channels selectively modulate gastric vagal afferent tension receptor mechanosensitivity and may mediate the reduction in gastric vagal afferent mechanosensitivity in high fat diet-induced obesity.

The Exon Structure of the Human MAGP-2 Gene SIMILARITY WITH THE MAGP-1 GENE IS CONFINED TO TWO EXONS ENCODING A CYSTEINE-RICH REGION

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.

Sex-specific alterations in glucose homeostasis and metabolic parameters during ageing of caspase-2-deficient mice

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.

Caspase-2 deficiency enhances whole-body carbohydrate utilisation and prevents high-fat diet-induced obesity.

Caspase-2 has been shown to be involved in metabolic homeostasis. Here, we show that caspase-2 deficiency alters basal energy metabolism by shifting the balance in fuel choice from fatty acid to carbohydrate usage. At 4 weeks of age, whole-body carbohydrate utilisation was increased in Casp2−/− mice and was maintained into adulthood. By 17 weeks of age, Casp2−/− mice had reduced white adipose mass, smaller white adipocytes decreased fasting blood glucose and plasma triglycerides but maintained normal insulin levels. When placed on a 12-week high-fat diet (HFD), Casp2−/− mice resisted the development of obesity, fatty liver, hyperinsulinemia and insulin resistance. In addition, HFD-fed Casp2−/− mice had reduced white adipocyte hypertrophy, apoptosis and expansion of both subcutaneous and visceral adipose depots. Increased expression of UCP1 and the maintenance of adiponectin levels in white adipose tissue of HFD-fed Casp2−/− mice indicated increased browning and adipocyte hyperplasia. We found that while the preference for whole-body carbohydrate utilisation was maintained, HFD-fed Casp2−/− mice were not impaired in their ability to switch to utilising fats as a fuel source. Our findings suggest that caspase-2 impacts basal energy metabolism by regulating adipocyte biology and fat expansion, most likely via a non-apoptotic function. Furthermore, we show that caspase-2 deficiency shifts the balance in fuel choice towards increased carbohydrate utilisation and propose that this is due to mild energy stress. As a consequence, Casp2−/− mice show an adaptive remodelling of adipose tissue that protects from HFD-induced obesity and improves glucose homeostasis while paradoxically increasing their susceptibility to oxidative stress induced damage and premature ageing.