Adiponectin stimulates glucose uptake in mouse blastocysts and embryonic carcinoma cells.

Preimplantation embryos are sensitive to maternal hormones affecting embryonic signal transduction and metabolic functions. We examined whether adiponectin, the most abundantly secreted adipokine, can influence glucose transport in mouse embryonic cells. In mouse blastocysts full-length adiponectin stimulated glucose uptake, while no effect of globular adiponectin was found. Full-length adiponectin stimulated translocation of GLUT8 glucose transporter to the cell membrane; we did not detect significant changes in the intracellular localization of GLUT4 glucose transporter in adiponectin-treated blastocysts. To study adiponectin signaling in detail, we used embryoid bodies formed from mouse embryonic carcinoma cell (ECC) line P19. We confirmed the expression of adiponectin receptors in these cells. Similar to mouse blastocysts, full-length adiponectin, but not globular adiponectin, stimulated glucose uptake in ECC P19 embryoid bodies. Moreover, full-length adiponectin stimulated AMPK and p38 MAPK phosphorylation. These results indicate that besides AMPK, p38 MAPK is a potential target of adiponectin in mouse embryonic cells. AMPK inhibitor did not influence the adiponectin-stimulated p38 MAPK phosphorylation, indicating independent action of these two signaling pathways. In mouse embryos adiponectin acts as a hormonal regulator of glucose uptake, which becomes especially important in phases with reduced levels of circulating insulin. Our results suggest that adiponectin maintains the glucose supply for early embryos under hypoinsulinaemic conditions, for example, in mothers suffering from type 1 diabetes mellitus.

Novel ageing-biomarker discovery using data-intensive technologies.

Ageing is accompanied by many visible characteristics. Other biological and physiological markers are also well-described e.g. loss of circulating sex hormones and increased inflammatory cytokines. Biomarkers for healthy ageing studies are presently predicated on existing knowledge of ageing traits. The increasing availability of data-intensive methods enables deep-analysis of biological samples for novel biomarkers. We have adopted two discrete approaches in MARK-AGE Work Package 7 for biomarker discovery; (1) microarray analyses and/or proteomics in cell systems e.g. endothelial progenitor cells or T cell ageing including a stress model; and (2) investigation of cellular material and plasma directly from tightly-defined proband subsets of different ages using proteomic, transcriptomic and miR array. The first approach provided longitudinal insight into endothelial progenitor and T cell ageing. This review describes the strategy and use of hypothesis-free, data-intensive approaches to explore cellular proteins, miR, mRNA and plasma proteins as healthy ageing biomarkers, using ageing models and directly within samples from adults of different ages. It considers the challenges associated with integrating multiple models and pilot studies as rational biomarkers for a large cohort study. From this approach, a number of high-throughput methods were developed to evaluate novel, putative biomarkers of ageing in the MARK-AGE cohort.

Relationship between cardiac tissue glycation and skin autofluorescence in patients with coronary artery disease.

AIM: During ageing, advanced glycation end-products (AGEs) accumulate in extracellular matrix proteins like collagen and contribute to a decline in organ function. As skin autofluorescence (sAF) can assess subcutaneous accumulation of fluorescent AGEs, this study aimed to investigate the relationship between AGE-modified cardiac tissue collagen and AGE-related sAF in coronary artery bypass graft (CABG) surgery patients. METHODS: Between January 2011 and January 2012, data from 72 consecutive male patients undergoing isolated CABG were prospectively recorded. Collagen fractions were isolated from the right atrial appendages of these patients by proteolysis and collagenase digestion. Collagen was quantified by hydroxyproline assay, and AGEs by AGE-related intrinsic fluorescence; sAF was measured using an autofluorescence reader. RESULTS: Biochemical analysis showed that the insoluble cardiac collagen fraction contained the highest amounts of accumulated AGEs; the AGE-related intrinsic fluorescence of this fraction increased with age (P=0.0001), blood glucose (P=0.002), HbA1c (P=0.01) and sAF (P=0.008). CONCLUSION: This study demonstrated for the first time a relationship between cardiac tissue glycation and AGE-related sAF. In addition, cardiac tissue glycation was associated with age, blood glucose and long-term glucose values in patients with coronary artery disease.

The effect of an AGE-rich dietary extract on the activation of NF-κB depends on the cell model used.

Advanced glycation end products (AGEs) are the results of a chemical reaction of reactive aldehydes, such as sugars, with amino acid side chains. AGEs can be formed by the heating process of the food and taken up with the diet. They are thought to be at least in part responsible for major complications in age-related diseases. The activation of the transcription factor NF-κB plays a prominent role in AGE-induced cell signaling. This study aimed to elucidate the effect of exogenous AGEs on NF-κB activation in different cell models. Therefore a bread crust extract commonly found in a Western diet was chosen as an AGE-rich sample. Using RP-HPLC, 23 fractions from the bread crust extract were obtained. The immunodetection with specific antibodies for N-carboxymethyllysine arg-pyrimidine, pentosidine and 3-deoxyglucosone-imidazolone showed that the majority of the AGEs were located in the late fractions. Three different NF-κB reporter cell lines including NF-κB/293/GFP-Luc™, NF-κB/Jurkat/GFP™ and RAW/NF-κB/SEAPorter™ were stimulated with the 23 fractions. There was no direct correlation between the AGE content in the fractions and the cell activation. Whereas in Jurkat-T-cells, the stimulation seems to correlate at least in part with the AGE content, in HEK-293 epithelial cell nearly all fractions can stimulate NF-κB. In macrophages few fractions stimulate NF-κB whereas some fractions even inhibit the p38 MAP kinase. The highest expression of the AGE receptors like RAGE, AGER-1, AGER-2 and AGER-3 was detected in the macrophage RAW cell line. In conclusion the present study showed a new approach to study bioactive compounds in bread crust extract. The identification of the bioactive compounds is still ongoing.

[Protein glycation as a pathological mechanism in diabetes]. / Proteinglykierung als pathophysiologischer Mechanismus bei Diabetes.

The incidence of diabetes has increased in the recent years. Diabetes is characterized by increased sugar concentrations in the blood. Due to this dysregulation, more carbohydrate-induced modification of proteins - so-called advanced glycation end products (AGEs) - are formed endogenously by non-enzymatic reactions. These are discussed to be at least in part responsible for diabetes-associated diseases. The accumulation of AGEs in the tissue can be used as a biomarker for patient outcome. In contrast, the effects of the uptake of AGEs from nutrition are still unclear.

Role of cathepsins in blastocyst hatching in the golden hamster.

The mammalian embryo is encased in a glycoproteinaceous coat, the zona pellucida (ZP) during preimplantation development. Prior to implantation, the blastocyst must undergo 'hatching' or ZP escape. In hamsters, there is a thinning of the ZP followed by a focal lysis and a complete dissolution of the ZP during blastocyst hatching. Earlier studies from our laboratory have indicated a role for cysteine proteases in the hatching phenomenon. In this study, we tested the effect of specific inhibitors of the three classes of cysteine protease on blastocyst hatching. Cystatin, an endogenous cathepsin inhibitor, blocked blastocyst hatching. Similarly, Fmoc-Tyr-Ala-diazomethane, a synthetic cathepsin inhibitor, blocked hatching. Both showed dose-dependent and temporal inhibition of hatching. However, Z-Val-Ala-Asp-fluoromethylketone, a synthetic caspase inhibitor, and calpastatin, an endogenous calpain inhibitor, had no effect on hatching. The cathepsins were localized to blastocyst cells. Exogenous addition of cathepsins L, P or B to cultured 8-cell embryos caused a complete ZP dissolution. The expression of mRNA and protein of cathepsins L and P was observed in peri-hatching blastocysts. Cathepsins L and P were detected in trophectodermal projections and in the ZP of peri-hatching blastocysts. These data provide the first evidence that blastocyst-derived cathepsins are functionally involved as zonalytic factors in the hatching of blastocysts in the golden hamster.

Advanced glycation end products, diabetes and ageing.

Advanced glycation end products (AGEs) are formed in vivo by a non-enzymatic reaction of proteins with carbohydrates and accumulate in many tissues during ageing. They are discussed as being responsible for many age- and diabetes-related diseases. On the other hand, AGEs are formed by the heating of food and are taken up by the nutrition. The contribution of endogenously formed versus exogenous intake of AGEs to age-related diseases is still under discussion.

Differential expression of oxygen-regulated genes in bovine blastocysts.

Low oxygen conditions (2%) during post-compaction culture of bovine blastocysts improve embryo quality, which is associated with a small yet significant increase in the expression of glucose transporter 1 (GLUT-1), suggesting a role of oxygen in embryo development mediated through oxygen-sensitive gene expression. However, bovine embryos to at least the blastocyst stage lack a key regulator of oxygen-sensitive gene expression, hypoxia-inducible factor 1alpha (HIF1alpha). A second, less well-characterized protein (HIF2alpha) is, however, detectable from the 8-cell stage of development. Here we use differential display to determine additional gene targets in bovine embryos in response to low oxygen conditions. While development to the blastocyst stage was unaffected by the oxygen concentration used during post-compaction culture, differential display identified oxygen-regulation of myotrophin and anaphase promoting complex 1 expression, with significantly lower levels observed following culture under 20% oxygen than 2% oxygen. These results further support the hypothesis that the level of gene expression of specific transcripts by bovine embryos alters in response to changes in the oxygen environment post-compaction. Specifically, we have identified two oxygen-sensitive genes that are potentially regulated by HIF2 in the bovine blastocyst.

Expression of the insulin-responsive glucose transporter isoform 4 in blastocysts of C57/BL6 mice.

Glucose is the most important energy substrate for mammalian blastocysts. In preimplantation embryos glucose uptake is mainly mediated by facilitative glucose transporter molecules (GLUT). Employing RT-PCR in 3.5-day-old mouse blastocysts of strain C57/BL6 we have investigated the expression of the GLUT isoforms 1-4 and 8. We could not only detect GLUT 1, 3 and 8 but, in contrast to earlier studies, also the insulin-responsive isoform 4. GLUT2 was not expressed. The specificity for GLUT4 amplification was verified by sequence analysis. GLUT4 protein was localized by immunohistochemistry with two GLUT4 antibodies. It was found in ICM and trophoblast cells in the cytoplasmic compartment with a strong perinuclear staining. This is the first report on the expression of the insulin-sensitive GLUT4 isoform in mouse preimplantation embryos.

Glucose transporter expression is developmentally regulated in in vitro derived bovine preimplantation embryos.

Glucose is readily been taken up and utilized by preimplantation embryos from different species. However, a comprehensive analysis of the glucose transporter expression throughout preimplantation development is still missing. Here, we have investigated the expression of facilitative glucose transporters (Glut1-5 and 8) and sodium-dependent-glucose transporter (SGLT-I) in bovine oocytes and preimplantation embryos up to d16 of development, using RT-PCR and immunohistochemistry. The embryos were produced in vitro by IVM-IVF. Glut1, Glut3, Glut8, and SGLT-I were expressed in all stages studied. Glut4 transcripts were first detected at the blastocyst stage. Glut2 expression was restricted to the period of blastocyst elongation at d14 and d16. Transcription of the fructose transporter Glut5 started at the 8-/16-cell stage. Our results show a distinct expression pattern for glucose transporters during bovine embryo development in vitro indicating specialized functions for these isoforms at different developmental stages in bovine embryos. Mol. Reprod. Dev. 60:370-376,

Expression of proto-oncogenes in bovine preimplantation blastocysts.

Proto-oncogenes are involved in the regulation of gene expression, for example after ligand binding to growth factor receptors. Expression of the proto-oncogenes c-fos, c-jun, c-ha-ras and c-myc was studied in in vivo grown and in vitro cultured bovine preimplantation blastocysts employing RT-PCR, ribonuclease protection assay and immunohistochemistry. Thirteen- and 14- day-old preimplantation blastocysts, i.e. stages before and during trophoblast elongation, were used. In in vivo-grown blastocysts c-fos, c-jun and c-ha-ras transcripts as well as c-Fos, c-Jun and c-Myc proteins were detected in all stages studied. Cultured blastocysts were treated with 10 nM epidermal growth factor and 10 nM transforming growth factor-alpha simultaneously. Epidermal growth factor and transforming growth factor-alpha treatment induced c-fos mRNA and c-Myc protein expression. The induction of downstream targets of the epidermal growth factor receptor by epidermal growth factor and transforming growth factor-alpha indicates a functional epidermal growth factor signal transduction pathway in elongating bovine blastocysts.

The insulin-dependent glucose transporter isoform 4 is expressed in bovine blastocysts.

We have investigated the expression of two glucose transporter isoforms, Glut1 and 4, in 14- and 16-day-old bovine blastocysts (d14, d16) using RT-PCR, competitive RT-PCR and in situ hybridization. The blastocysts were grown in vivo or had been produced in vitro. Glut1 mRNA was detected in all blastocysts studied, Glut4 in all d14 blastocysts, but only in a few d16 blastocysts. Glut4 mRNA was localized in trophoblast and endoderm cells. Glut1 mRNA increased from d14 to d16 while Glut4 transcription was down-regulated in d16 blastocysts. The mRNA amounts varied between 0.8 to 23 pg and 3.9 to 65 fg per 100 ng embryonic RNA for Glut1 and Glut4, respectively, displaying a 100- to 1500-fold lower expression of Glut4 compared with Glut1 during blastocyst elongation. This is the first report on the expression of the insulin-sensitive Glut4 isoform in mammalian preimplantation embryos.

Cell-cell contact of human T cells with fibroblasts changes lymphocytic mRNA expression: increased mRNA expression of interleukin-17 and interleukin-17 receptor.

Using random arbitrarily primed-reverse transcribed-PCR and sequence analysis, we investigated changes in lymphocytic molecules after cell-cell contact with fibroblasts. An mRNA species which was upregulated in Jurkat T cells by cell-cell contact with MRHF cells (a human foreskin fibroblast line) was identified as coding for the human interleukin-17 receptor. This finding was confirmed by quantitative RT-PCR for the HUT78 and Jurkat T cell lines, for peripheral blood lymphocytes, and for tonsillar T cells. Furthermore, the interleukin-17 mRNA, coding for a proinflammatory cytokine, was also upregulated in peripheral blood lymphocytes and tonsillar T cells after cell-cell contact with fibroblasts. Supernatants obtained from cell-cell contact-stimulated peripheral blood lymphocytes enhance the production of interleukin-6 and interleukin-8 by fibroblast-like synoviocytes and this effect could be blocked by interleukin-17 antibodies. Changes in the mRNA levels of Jurkat T cells induced by cell-cell contact with adherent cells were also found for M-type pyruvate kinase, for tropomyosin TM30 and for the p54nrb gene product.

Aminopeptidase N/CD13 is associated with raft membrane microdomains in monocytes.

Ectopeptidases play important roles in cell activation, proliferation, and communication. Human monocytic cells express considerable amounts of aminopeptidase N/CD13, a transmembrane protein previously proposed to play a role in the regulation of neuropeptides and chemotactic mediators as well as in adhesion and cell-cell interactions. Here, we report for the first time that aminopeptidase N/CD13 in monocytes is partially localized in detergent-insoluble membrane microdomains enriched in cholesterol, glycolipids, and glycosylphosphoinositol-anchored proteins, referred to as "rafts." Raft fractions of monocytes were characterized by the presence of GM1 ganglioside as raft marker molecule and by the high level of tyrosine-phosphorylated proteins. Furthermore, similar to polarized cells, rafts in monocytic cells lack Na(+), K(+)-ATPase. Cholesterol depletion of monocytes by methyl-beta-cyclodextrin greatly reduces raft localization of aminopeptidase N/CD13 without affecting ala-p-nitroanilide cleaving activity of cells.

Regulation by transforming growth factor-beta1 of class II mRNA and protein expression in fibroblast-like synoviocytes from patients with rheumatoid arthritis.

Transforming growth factor (TGF)-beta1 is an immunosuppressive cytokine that modulates the expression of class II histocompatibility antigens on human cells. Aberrant HLA class II expression on synovial lining cells of rheumatoid arthritis synovial membrane has been described, and the extent and intensity of class II expression on the cells was claimed to be linked with the severity of the disease. In this study, the effects of TGF-beta1 on HLA class II antigen expression in fibroblast-like synoviocytes (SFC) from rheumatoid synovectomy tissues were determined by flow cytometric analysis and quantitative RT-PCR. We found that pre-incubation of cells with TGF-beta1 was able to down-regulate IFN-gamma-induced DR protein expression in SFC. TGF-beta1, additionally, down-regulated IFN-gamma-stimulated class II transactivator (CIITA) and DRB mRNA expression. The constitutive expression of CIITA mRNA was completely abolished and the constitutive expression of DRB mRNA was decreased after treatment of SFC with TGF-beta1 for 24 h. Addition of the TGF-beta inhibitor decorin to SFC for 24 h before TGF-beta1/IFN-gamma treatment was able to reduce the down-regulatory effect of TGF-beta1 on DR antigen expression induced by IFN-gamma. Using competitive RT-PCR, we found that SFC constitutively expressed decorin mRNA and that treatment of cells with TGF-beta1 for 24 h reduced the constitutive expression of decorin mRNA by 65%. Our results show that TGF-beta1 is able to reduce the expression of HLA class II mRNA and protein, and suggest a tight regulation between TGF-beta1 and decorin in SFC of the rheumatoid synovium.

Constitutive expression of HLA class II mRNA in synovial fibroblast-like cells from patients with rheumatoid arthritis .

We describe the quantification of the absolute amounts of HLA class II mRNA and class II transactivator (CIITA) mRNA by competitive reverse transcription polymerase chain reaction in cultured synovial fibroblast-like cells (SFC) of patients with rheumatoid arthritis. High basal levels of transcription of class II mRNA (10(7)-10(9) molecules/microgram total RNA) and CIITA mRNA were detected in cultured SFC, with DPB < DRB = DQB, although SFC only express small amounts of MHC class II proteins. In contrast to SFC, we did not detect class II mRNA nor CIITA mRNA in skin fibroblasts. After treatment with IFN-gamma, we observed a 3- to 28-fold increase in class II mRNA in SFC and an increase of DRB and DPB in skin fibroblasts from undetectable levels to 10(8)-10(9) molecules/microgram total RNA.