Autocrine effects of visfatin on hepatocyte sensitivity to insulin action.

Visfatin was originally described as an adipokine with insulin mimetic effects. Recently, it was found that visfatin is identical with the Nampt (nicotinamide phosphoribosyltransferase) gene that codes for an intra- and extracellular NAD biosynthetic enzyme and is predominantly expressed outside the adipose tissue. In the current study, we found strong protein and mRNA expression of visfatin in rat heart, liver, kidney, and muscle, while the expression of visfatin in visceral fat was significantly lower and undetectable in subcutaneous fat. The insulin-mimetic effects of visfatin (extracellular form of Nampt or eNampt) are controversial and even less is known about autocrine effects of visfatin (intracellular form of Nampt or iNampt). Since liver plays a major role in glucose metabolism, we studied visfatin effects on insulin-stimulated cellular glucose uptake in Fao rat hepatocytes using RNA interference (RNAi). RNAi-mediated downregulation of visfatin expression in Fao cells was associated with significantly reduced NAD biosynthesis (0.3+/-0.01 vs. 0.5+/-0.01 mmol/h/g, P<0.05) and with significantly decreased incremental glucose uptake after stimulation with insulin when compared to controls with normal expression of visfatin (0.6+/-0.2 vs. 2.2+/-0.5 nnmol/g/2 h, P=0.02). These results provide evidence that visfatin exhibits important autocrine effects on sensitivity of liver cells to insulin action possibly through its effects on NAD biosynthesis.

CD36 regulates fatty acid composition and sensitivity to insulin in 3T3-L1 adipocytes.

In the current study, we tested a hypothesis that CD36 fatty acid (FA) transporter might affect insulin sensitivity by indirect effects on FA composition of adipose tissue. We examined the effects of CD36 downregulation by RNA interference in 3T3-L1 adipocytes on FA transport and composition and on sensitivity to insulin action. Transfected 3T3-L1 adipocytes, without detectable CD36 protein, showed reduced neutral lipid levels and significant differences in FA composition when levels of essential FA and their metabolites were lower or could not be detected including gamma linolenic (C18:3 n6), eicosadienic (C20:2 n6), dihomo-gamma linolenic (C20:3 n6), eicosapentaenoic (EPA) (C20:5 n3), docosapentaenoic (DPA) (C22:5 n3), and docosahexaenoic (DHA) (C22:6 n3) FA. Transfected 3T3-L1 adipocytes exhibited a significantly higher n6/n3 FA ratio, reduced 5-desaturase and higher 9-desaturase activities. These lipid profiles were associated with a significantly reduced insulin-stimulated glucose uptake (4.02+/-0.1 vs. 8.42+/-0.26 pmol.10(-3) cells, P=0.001). These findings provide evidence that CD36 regulates FA composition thereby affecting sensitivity to insulin action in 3T3-L1 adipocytes.

[Age-related post-translational modification of proteins in human tissue and its use determination of age in forensic medicine. Review]. / Závislost nekterých posttranslacních modifikací proteinu lidských tkání na veku a jejich pouzití pro urcování veku v soudním lékarství. Prehled poznatku.

This article deals with some of posttranslational modification of proteins which are, or could be useful to work out objective and standard methods for age estimation in forensic medicine. From many posttranslational modifications other than racemization, the evaluation of pentosidine, one of the products of nonenzymatic glycosylation, seems to be promising. The enlargement of menu of methods for age estimation is important mainly for forensic sciences when determination of the age of an unknown dead body is necessary. Morphological methods are quite often subjective and charged with errors.

[Age determination using peptide mapping of non-collagenous proteins in human dentin]. / Moznosti stanovení veku peptidovým mapováním nekolagenních proteinu lidského dentinu.

The submitted paper deals with possibilities of assessment of human age based on findings of stereospecific breakdown of proteins containing D-forms of aspartic acid. The stereospecificity of enzymatic breakdown assumes that after enzymatic hydrolysis peptide breakdown products with different molecular weights--the so-called peptide map--will be formed, depending how many D-aspartyl residues the protein contains. The authors proved in the submitted preliminary study in subjects of different age the formation of breakdown products of different size in non-collagenous proteins of human dentin which was hydrolyzed by protease V8 from Staphylococcus aureus.

Polyclonal antibodies for immunochemical determination of human pepsinogens.

The human gastric mucosa contains two main groups of aspartic proteinases, pepsin (EC 3.4.23.1) and gastricsin (EC 3.4.23.3), which differ by their structural, kinetic and immunological characteristics. The ratios between human aspartic proteases are important from the diagnostic point of view. Rabbit polyclonal antisera against human pepsinogen A and pepsinogen C (progastricsin) were obtained and tested for clinical purposes. Immunoblotting procedure seems to be a simple and sufficiently sensitive method for qualitative determination of pepsinogens in human gastric mucosa.

High performance liquid chromatography enables fast determination of ratios between individual proteolytic enzymes in human gastric mucosa.

The human gastric mucosa contains five isozymogens of pepsinogen (pepsinogens PGA1-PGA5), two isozymogens of gastricsinogen (gastricsinogens PGC6-PGC7) and zymogens of cathepsins. Ratios between some individual pepsins or pepsinogens are very important from a diagnostic point of view. The ratio of pepsinogen 3 to pepsinogen 5 is significant marker of gastric cancer and gastric ulcer. High-performance ion-exchange chromatography is an easy and fast method for determination of ratios between individual proteolytic zymogens in human gastric mucosa and thus could serve for additional diagnosis of gastric diseases.

Carbohydrates as a tool for oriented immobilization of antigens and antibodies.

A biospecific sorbent for the isolation of ovalbumin antibodies was prepared by coupling of ovalbumin via its periodate-oxidized carbohydrate moiety to bead cellulose modified with adipic acid dihydrazide. The anti-ovalbumin IgG fraction isolated on this sorbent from immune rabbit serum contained only antibodies against protein determinants of ovalbumin. Thus, when these IgG were immobilized through their carbohydrate moieties to cellulose beads it became possible to prepare a biospecific sorbent for concanavalin A by oriented adsorption of ovalbumin. Ovalbumin was specifically adsorbed via its protein moiety and its carbohydrate part remained free for interaction with concanavalin A.