Adiponectin and resistin gene polymorphisms in patients with anorexia nervosa and obesity and its influence on metabolic phenotype.

Genes for adiponectin and resistin are candidate genes of insulin resistance and type 2 diabetes mellitus. The aim of our study was to determine the frequency of single nucleotide polymorphisms (SNP) 45T>G and 276G>T of the adiponectin gene and 62G>A and -180C>G of the resistin gene in patients with obesity (OB), anorexia nervosa (AN) and in control healthy normal-weight women (NW) and to study the influence of particular genotypes on serum concentrations of these hormones and on insulin sensitivity. Serum adiponectin, resistin, tumor necrosis factor alpha (TNF-alpha), insulin, cholesterol, glycated hemoglobin (HbA1c) and blood glucose levels were measured in 77 patients with OB, 28 with AN and 38 NW. DNA analysis was carried out by polymerase chain reaction with restriction analysis of PCR product. The presence of SNP ADP+276 G>T allele was accompanied by higher cholesterol levels in AN patients, higher adiponectin concentrations in OB patients and lower HbA1c levels in NW. SNP of the resistin gene 62G>A was associated with lower HbA1c in NW and higher cholesterol concentrations in OB group. The carriers of the minor G allele in the position -180 of the resistin gene within AN group had significantly higher BMI relative to non-carriers. We conclude that polymorphisms in adiponectin and resistin genes can contribute to metabolic phenotype of patients with obesity and anorexia nervosa.

[The influence of obesity on the gene expression of adiponectin and its receptor in subcutaneous adipose tissue]. / Vliv obezity na genovou expresi adiponektinu a jeho receptoru v subkutánní tukové tkáni.

UNLABELLED: The objective of the study was to measure the concentration of adiponectin in plasma, its mRNA expression and the expression of the adiponectin receptors AdipoR1 and AdipoR2 in subcutaneous adpipose tissue (ST) of women with various levels of fat in their organism. A further objective of the study was to determine to what extent the stated parameters correlate with obesity as defined by BMI (body mass index) and how it can be affected by a very low calorie diet (VLCD). PATIENT SAMPLE: The sample of 70 women was divided into groups by BMI: patients with class 3 obesity (BMI > 40 kg.m(-2), n = 25), patients with class 1 and 2 obesity (BMI 30-40 kg.m(-2), n = 15), overweight patients (BMI 25-30 kg.m(-2), n = 10) and a normal healthy control group (BMI 20-25 kg.m(-2), n = 20). In the case of 14 women with class 3 obesity, the parameters were measured before and after a three-week diet with an energy content of 2200 kJ (550 kcal)/day (VLCD). METHOD: Plasma concentrations of adiponectin were measured using an ELISA kit (LINCO Research, USA). Subcutaneous adipose tissue was taken using biopsy. RNA was isolated using a MagNA Pure Compact RNA Isolation Kit (Tissue) (Roche, SRN). The gene expression of adiponectin, AdipoR1 and AdipoR2 was determined using the real-time PCR (RT-PCR) method on a ABI Real-Time PCR 7500 instrument (Applied Biosystems, USA) with TaqMan Gene Expression Assays hydrolisation probes. beta-2-mikroglobulin (beta2M) was used as an endogenous control, to which the data was normalised. RESULTS: The circulatory concentration of adiponectin, its mRNA expression and the mRNA expression of AdipoR1 in ST correlate negatively with BMI (r = -0.524, p < 0.001; r = -0.460, p < 0 001; p = -0.354, p = 0.004). The expression of AdipoR2 in ST did not correlate with BMI. The VLCD reduced weight by 9% but did not affect the plasma concentration of adiponectin or the rate of its expression, or the expression of AdipoR1 and AdipoR2. CONCLUSION: Our results show that not only the circulatory concentration of adiponectin but also its mRNA expression and the expression of AdipoR1 in ST are significantly lower in obese women compared to a control group and may contribute to the development of metabolic complications in obesity.

[Polymorphisms of adiponectin and resistin genes in patients with obesity and anorexia nervosa]. / Polymorfismy genu pro adiponectin a rezistin u pacientek s mentální anorexií a obezitou--pilotní studie.

BACKGROUND: Adiponectin and resistin are hormones that may represent a link between obesity and insulin resistance. Genes for these hormones are new candidate genes of insulin resistance and type 2 diabetes mellitus. The aim of our study was to determine the frequency of single nucleotide polymorphisms 45T > G and 276T > G of adiponectin gene and 62G > A and -180C > G of resistin gene in patients with obesity, anorexia nervosa and in lean women and to study the influence of particular genotypes on serum concentrations of these hormones. METHODS AND RESULTS: Serum adiponectin, resistin, TNF-alfa and insulin levels were measured in 51 patients with obesity, 17 with anorexia nervosa and 17 lean women. DNA analysis was carried out by means of polymerase chain reaction (PCR) with restriction analysis of PCR product (RFLP). Adiponectin levels were lowest in obese women and highest in anorexia nervosa patients. Resistin concentrations were lowest in anorexia nervosa and highest in obese patients. Genotype analysis within respective groups showed no differences in assessed parameters when comparing different adiponectin and resistin polymorphisms. The only difference detected was significantly higher BMI in G/G genotype relative to T allele carries in 276 position of ADP gene in control group (23.48 +/- 0.85 vs. 19,7 +/- 0.95, p < 0.05). In anorexia nervosa patients, frequency of G allele in RETN -180 polymorphism was significantly higher relative to control group (p < 0.05). CONCLUSIONS: Polymorphisms 45T > G a 276T > G of ADP gene and 62G>A and -180C > G RETN gene did not influence serum ADP and RETN concentrations. BMI was influenced by T allele presence in 276 position of ADP gene in control group only. Anorexia nervosa patients had higher frequency of G allele of RETN -180 polymorphism compared to healthy women.

Age-dependent changes in proteoglycan biosynthesis in human intervertebral discs.

Proteoglycans from annulus fibrosus and nucleus pulposus of human intervertebral disc were investigated by electrophoresis in a composite agarose-polyacrylamide gel and immunohistochemically using various monoclonal antibodies against components of extracellular matrix. There were at least five different populations of proteoglycans in both annulus fibrosus and nucleus pulposus. Proteoglycans represented by individual electrophoretic bands differed from each other in hydrodynamic size but all of them contained epitopes present in keratan sulphate. Chondroitin sulphate could be detected in populations with molecular weight above 200,000 daltons. We could postulate that the cleavage of proteoglycan chains starts at the C-terminal end. This is supported by the finding that the globular region G2 on the protein core was detected in the same populations as keratan sulphate. We could confirm these results using tissue cultures of nucleus pulposus, inner and external part of annulus fibrosus. Of interest is the finding that there is de novo synthesis of globular domain G1 only in structures of intervertebral disc from a 16-year-old male and not in the tissue of a 69-year-old male. This might contribute to an explanation of decreased aggregation of proteoglycans during the aging process.

Electrophoretic separation of large proteoglycans in large-pore polyacrylamide gradient gels (1.32-10.0% T) and a one-step procedure for simultaneous staining of proteins and proteoglycans.

A procedure is described for the preparation of 1.32-10% polyacrylamide gradient gels. Loose polyacrylamide gel on the top side of the gradient was stabilized with a layer of 0.4% agarose gel which also formed sample wells. The upper limit of separation achieved in these gels was estimated to be approximately 2 X 10(6) using globular protein standards. However, large aggregating proteoglycans from cartilage which have a molecular weight range of 1-4 X 10(6) penetrate and separate in these gels. A simple one-step procedure is also described for simultaneous staining of proteins and large proteoglycans in polyacrylamide gels.

[Indicators of articular cartilage degeneration in synovial fluid]. / Ukazatele degradace kloubní chrupavky v synoviální tekutine.

Detection of proteoglycans in biological fluids is a perspective method for the evaluation of the degree of catabolic processes in articular cartilage. The demand of accuracy and specificity of detection of substructures of the degradation products of the cartilaginous matrix, with the perspective of routine large scale examinations, restricts available possible methods practically only to the use of immunochemical methods. In the present investigation in the inhibitory ELISA test polyclonal antibodies with a double specificity in relation to basic structures of the cartilage--proteoglycans--were used. The highest concentrations of degradation products of proteoglycans in punctates of synovial fluid were found in diseases where the clinical picture is dominated by repeated attacks of the joints with longer remission periods, and where the attack is stimulated, usually by stimuli of an intermittent systemic or exogenous character (gout, reactive arthritis incl. Reiter's syndrome, Lyme disease).

Proteoglycans of human articular cartilage. Identification of several populations of large and small proteoglycans and of hyaluronic acid-binding proteins in successive cartilage extracts.

Two specimens of human articulage were successively extracted with solutions of phosphate-buffered saline (PBS), 7 M-urea and 4 M-guanidine hydrochloride (Gdn-HCl). Proteoglycans from individual extracts were fractionated by DEAE-Sephacel chromatography and gel chromatography on Sephacryl S-400. The presence of three populations of large proteoglycans was demonstrated in all three extracts by composite agarose/polyacrylamide-gel electrophoresis (CAPAGE). The population corresponding to the fastest CAPAGE band of aggregating proteoglycans was shown to be extremely polydisperse, having Mr (as estimated by SDS/PAGE) decreasing continuously from more than 300,000 to the size corresponding to 'free' hyaluronic acid-binding region (HABR) (about 70,000). A rather polydisperse set of HABR-containing fragments which spanned a broad range of sizes, and also differed in their keratan sulphate contents, was isolated from both 7 M-urea and 4 M-Gdn-HCl extracts. PBS and 7 M-urea extracts, but not the Gdn-HCl extract, further contained small proteoglycans, identified as fast-migrating bands on CAPAGE electrophoretograms. One of those small species was recognized with an antibody against the small proteoglycan PG II; the other two remain to be positively identified. However, the glycosaminoglycan of the small species which was present exclusively in the PBS extract was identified as keratan sulphate; this species may thus belong to the family of small keratan sulphate-containing proteolygans.

[Changes in proteoglycans in human articular cartilage in relation to age]. / Zmeny proteoglykanu lidské kloubní chrupavky v závislosti na veku.

The authors investigated in articular cartilage the presence and amount of the free fragment of the protein nucleus of the proteoglycan monomer containing the area of the bond with hyaluronic acid (HABR) and the presence of the small dermatan sulfate proteoglycan in relation to age. The articular cartilage of older subjects contains a much higher ratio of the protein fragment with functional HABR, as compared with young adult tissue. The more ready extractability of this fragment suggests an impaired bond between the proteoglycan monomer and hyaluronic acid. The ratio of the small dermatan sulfate proteoglycan is significantly reduced in the articular cartilage of older subjects. In the articular cartilage of a very old person (87 years) this proteoglycan was not detected or its content is at the borderline of detection of the methods used.

[The effect of nonsteroidal antirheumatic agents on articular cartilage]. / Vliv nesteroidních antirevmatik na chrupavku kloubní.

The authors submit a review on the influence of non-steroid anti-rheumatic drugs on the articular cartilage. First they review the general literature and then discuss the effect of different NSA on cartilage, taking into account their classification into six groups with regard to their chemical composition. Experience is based practically only on laboratory and experimental work and the results therefore cannot be mechanically applied in clinical medicine. However, NSA which do not have a negative impact on the cartilage of experimental animals are probably also safer in clinical practice. In this country the following NSA are available: diclofenac, piroxicam and thia profenic acid.

[Levels of large proteoglycan aggregate binding proteins and the level of small proteoglycans in healthy human articular cartilage and in osteoarthrosis]. / Obsah vazebné bílkoviny velkého proteoglykanového agregátu a obsah malého proteoglykanu v lidské kloubní chrupavce zdravé a osteoartrotické.

The investigation was focused on the electronmicroscopic localization of one structure of the aggregate of the large proteoglycan--binding proteins and the small dermatan sulphate proteoglycan, using specific antibodies prepared against these structures and the complex colloid gold--protein A. The authors investigated the incidence and distribution of these structures in the intercellular area of ultrathin sections of the sound and arthritic human articular cartilage. In the arthritic cartilage, consistent with biochemical findings, the authors detected a loss of the binding protein or its disappearance and conversely an increase of the small dermatan sulphate proteoglycan.

[Degenerative changes in intervertebral disks]. / Regresívní zmeny v meziobratlových ploténkách.

A histotopographic study of lumbar spine was made in 27 deceased of the age of 2 to 85 years lacking of any spinal clinical symptomatology. Regressive changes were found in all the intervertebral disks except in infants. A central necrosis was present in 26 cases and pericentral striped necrosis in 19 cases. Other disks showed consequences of necrosis--vascularization, scarring, regenerative proliferation of chondrocytes, ganglion type cavities. A concept of disk lesion as a trigger of further spinal lesions was supported by frequent regressive changes of the disks. Schmorl's (internal) nodes were found in 35 cases, quite often in young persons without clinical symptomatology. The amount of glycosaminoglycans during the disk regression was detected by Safranin O-Fast Green stain with ambiguous results. A hypothetical spontaneous vanishing of the nucleus pulposus in accordance with the age was a topic of discussion. Protrusions of the disks into the spinal canal were not found. Initial osteophytes at ventral margins of vertebral bodies were identified in 49 cases, at dorsal margins in only 3 cases.

Urinary proteoglycan degradation product excretion in patients with rheumatoid arthritis and osteoarthritis.

The excretion of sugar components of glycosaminoglycans in the urine was investigated in 19 healthy controls, 27 patients with rheumatoid arthritis, and 24 with osteoarthritis. Both groups of patients excreted significantly more glucosamine, galactosamine, and mannose than the controls. The total uronic acid excretion, also, was higher in the two groups than in the healthy subjects. The possibility of using this method for the long term follow up of rheumatoid arthritis and osteoarthritis and the response to treatment is discussed.

Effect of tolfenamic acid on the metabolism of the main connective tissue components in rats.

Tolfenamic acid (Clotam) has been used in the therapy of rheumatic diseases for some years. Regarding its chemical structure it belongs to the group of fenamates. The effect of tolfenamic acid on the synthesis of collagen and proteoglycans in granulation tissue, skin or cartilage of rat weanlings was tested and compared with the action of mefenamic acid. According to the results obtained, tolfenamic acid is a potent inhibitor of collagen as well as proteoglycan syntheses. The concentrations of the constituents of proteoglycans, i.e. protein core, link protein as well as glycosaminoglycans were decreased in the tissue after treatment with tolfenamic acid. In comparison with mefenamic acid, if the same doses were used (50 mg and 100 mg/kg of body weight/day in in vivo experiments and 10 mg/g wet tissue in in vitro experiments), tolfenamic acid exhibits more distinct inhibitory effect. A general inhibitory effect of tolfenamic acid on proteosynthesis is suggested.

Effect of glycosaminoglycan polysulphate on the metabolism of cartilage ribonucleic acid.

The effect of glycosaminoglycan polysulphate (GAGPS, Arteparon) on the metabolism of cartilage ribonucleic acid (RNA), collagen, and proteoglycan was studied. GAGPS stimulated the synthesis of collagen and the both components of proteoglycan. The effect of GAGPS on RNA was compared with those of chondroitin sulphate and heparin. All three investigated substances enhanced the RNA synthesis, GAGPS, however, had a comparable effect in concentrations ten times smaller.