Maternal body mass index and placental weight: a role for fetal insulin, maternal insulin and leptin.

PURPOSE: Placental weight (PW) has been found to mediate the main effect of maternal BMI on fetal size. Still, the BMI-PW association is poorly understood. Therefore, we aimed to explore potential explanatory variables, including gestational weight gain (GWG), early- and late-pregnancy circulating levels of maternal glucose, insulin, leptin, adiponectin, triglycerides, LDL-C, and HDL-C, and fetal insulin. METHODS: We included two studies of pregnant women from Oslo University Hospital, Norway: the prospective STORK (n = 263) and the cross-sectional 4-vessel method study (4-vessel; n = 165). We used multiple linear regression for data analyses. A non-linear BMI-PW association was observed, which leveled off from BMI25. Therefore, BMI <25 and ≥25 were analyzed separately (n = 170/122 and 93/43 for STORK/4-vessel). Confounding variables included maternal age, parity, and gestational age. RESULTS: PW increased significantly per kg m-2 only among BMI <25 (univariate model's std.ß[p] = 0.233 [0.002] vs. 0.074[0.48]/0.296[0.001] vs. -0.030[0.85] for BMI <25 vs. ≥25 in STORK/4-vessel). Maternal early- but not late-pregnancy insulin and term fetal insulin were associated with PW. The estimated effect of early pregnancy insulin was similar between the BMI groups but statistically significant only among BMI <25 (std.ß[p] = 0.182[0.016] vs. 0.203[0.07] for BMI <25 vs. ≥25). Late pregnancy leptin was inversely associated with PW with a 1.3/1.7-fold greater effect among BMI ≥25 than BMI <25 in the STORK/4-vessel. CONCLUSIONS: The BMI-PW association was non-linear: an association was observed for BMI <25 but not for BMI ≥25. Leptin may be involved in the non-linear association through a placental-adipose tissue interplay. Maternal early pregnancy insulin and fetal insulin at term were associated with PW.

Differences in bone mineral density, markers of bone turnover and extracellular matrix and daily life muscular activity among patients with recent motor-incomplete versus motor-complete spinal cord injury.

Spinal cord injury (SCI) leads to severe bone loss, but the associated mechanisms are poorly described in incomplete SCI individuals. The purpose of the study is to compare alterations in bone mineral density (BMD) and serum biomarkers of bone turnover in recent motor-incomplete to -complete SCI men, as well as to describe their physical activity and spasticity. We studied 31 men with acute SCI. Whole-body DXA scans, serum biomarkers and self-reported activity and spasticity were examined 1 and/or 3 and 12 months after the injury. We observed a decrease in proximal femur BMD (p < 0.02) in both the groups. Serum phosphate and carboxy-terminal-collagen crosslinks were significantly lower in motor-incomplete versus complete SCI men, whereas albumin-corrected Ca(2+) (p = 0.02) were lower only 3 months after injury. When data from all 31 SCI participants were pooled, we observed increased serum matrix metalloproteinase-2 (MMP-2) and tissue inhibitors of MMP-2 (TIMP-2) (p < 0.02) whereas TIMP-1 decreased (p = 0.03). BMD correlated positively with self-reported activity (r = 0.59, p = 0.04) and negatively with spasticity (r = 0.74, p = 0.02) 12 months after injury. As a summary, men with motor-incomplete SCI developed significant proximal femur bone loss 12 months after injury and exhibited increased bone resorption throughout the first year after the injury. Compared with complete SCI men, incomplete SCI men show attenuated bone resorption. Our pooled data show increased turnover of extracellular matrix after injury and that increased exercise before and after injury correlated with reduced bone loss.

Diabetic nephropathy and extracellular matrix.

Diabetic nephropathy (DN) is a serious complication in diabetes. Major typical morphological changes are the result of changes in the extracellular matrix (ECM). Thus, basement membranes are thickened and the glomerular mesangial matrix and the tubulointerstitial space are expanded, due to increased amounts of ECM. One important ECM component, the proteoglycans (PGs), shows a more complex pattern of changes in DN. PGs in basement membranes are decreased but increased in the mesangium and the tubulointerstitial space. The amounts and structures of heparan sulfate chains are changed, and such changes affect levels of growth factors regulating cell proliferation and ECM synthesis, with cell attachment affecting endothelial cells and podocytes. Enzymes modulating heparan sulfate structures, such as heparanase and sulfatases, are implicated in DN. Other enzyme classes also modulate ECM proteins and PGs, such as matrix metalloproteinases (MMPs) and serine proteases, such as plasminogen activator, as well as their corresponding inhibitors. The levels of these enzymes and inhibitors are changed in plasma and in the kidneys in DN. Several growth factors, signaling pathways, and hyperglycemia per se affect ECM synthesis and turnover in DN. Whether ECM components can be used as markers for early kidney changes is an important research topic, whereas at present, the clinical use remains to be established.

Type of carbohydrate in feed affects the expression of small leucine-rich proteoglycans (SLRPs), glycosaminoglycans (GAGs) and interleukins in skeletal muscle of Atlantic cod (Gadus morhua L.).

Aquaculture requires feed that ensures rapid growth and healthy fish. Higher inclusion of plant ingredients is desirable, as marine resources are limited. In this study we investigated the effects of higher starch inclusion in feed on muscular extracellular matrix and interleukin expression in farmed cod. Starch was replaced by complex fibers in the low-starch diet to keep total carbohydrate inclusion similar. Blood glucose and fructosamine levels were elevated in the high-starch group. The group fed a high-starch diet showed up-regulation on mRNA level of proteoglycans biglycan and decorin. ELISA confirmed the real-time PCR results on protein level for biglycan and also showed increase of lumican. For decorin the protein levels were decreased in the high-starch group, in contrast to real-time PCR results. Disaccharide analyses using HPLC showed reduction of glycosaminoglycans. Further, there was up-regulation of interleukin-1ß and -10 on mRNA level in muscle. This study shows that the muscular extracellular matrix composition is affected by diet, and that a high-starch diet results in increased expression of pro-inflammatory genes similar to diabetes in humans.

Proteases in Plasma and Kidney of db/db Mice as Markers of Diabetes-Induced Nephropathy.

Db/db mice are overweight, dyslipidemic and develop diabetic complications, relevant for similar complications in human type 2 diabetes. We have used db/db and db/+ control mice to investigate alterations in proteinase expression and activity in circulation and kidneys by SDS-PAGE zymography, electron microscopy, immunohistochemistry, Western blotting, and in situ zymography. Plasma from db/db mice contained larger amounts of serine proteinases compared to db/+ mice. Kidneys from the db/db mice had a significantly larger glomerular surface area and somewhat thicker glomerular basement membranes compared to the db/+ mice. Furthermore, kidney extracts from db/+ mice contained metalloproteinases with M(r) of approximately 92000, compatible with MMP-9, not observed in db/db mice. These results indicate that higher levels of serine proteinases in plasma may serve as potential markers for kidney changes in db/db mice, whereas a decrease in MMP-9 in the kidney may be related to the glomerular changes.

Biocompatibility and pathways of initial complement pathway activation with Phisio- and PMEA-coated cardiopulmonary bypass circuits during open-heart surgery.

A randomized open-heart surgery study comprising 30 patients was undertaken to compare the biocompatibility of Phisio-(phosphorylcholine) and PMEA-(poly-2-methoxyethyl acrylate) coated cardiopulmonary bypass (CPB) circuits and to assess the initial complement pathway activation during open-heart surgery. Blood samples were obtained at five time points, from the start of surgery to 24 hours postoperatively. The following analyses were performed: haemoglobin, lactate dehydrogenase, leukocyte and platelet counts, myeloperoxidase and neutrophil-activating peptide-2, thrombin-anti-thrombin complexes, syndecan-1 and the complement activation products C1rs-C1-inhibitor complexes, C4bc, C3bc, C3bBbP and the terminal complement complex (TCC). No significant inter-group difference was found in any parameters, except for the concentration of TCC which was moderately lower in the PMEA group at termination of CPB. Complement activation during open-heart surgery was mainly mediated through the alternative pathway. In conclusion, PMEA- and Phisio-coated circuits displayed similar biocompatibility with respect to inflammatory and haemostatic responses during and after open-heart surgery.

Reducing added sugar intake in Norway by replacing sugar sweetened beverages with beverages containing intense sweeteners - a risk benefit assessment.

A risk benefit assessment in Norway on the intake of added sugar, intense sweeteners and benzoic acid from beverages, and the influence of changing from sugar sweetened to diet beverages was performed. National dietary surveys were used in the exposure assessment, and the content of added sugar and food additives were calculated based on actual contents used in beverages and sales volumes provided by the manufactures. The daily intake of sugar, intense sweeteners and benzoic acid were estimated for children (1- to 13-years-old) and adults according to the current intake level and a substitution scenario where it was assumed that all consumed beverages contained intense sweeteners. The change from sugar sweetened to diet beverages reduced the total intake of added sugar for all age groups but especially for adolescent. This change did not result in intake of intense sweeteners from beverages above the respective ADIs. However, the intake of acesulfame K approached ADI for small children and the total intake of benzoic acid was increased to above ADI for most age groups. The highest intake of benzoic acid was observed for 1- to 2-year-old children, and benzoic acid intake in Norwegian children is therefore considered to be of special concern.

Serglycin--structure and biology.

Serglycin is a proteoglycan found in hematopoietic cells and endothelial cells. It has important functions related to formation of several types of storage granules. In connective tissue mast cells the covalently attached glycosaminoglycan is heparin, whereas mucosal mast cells and activated macrophages contain oversulfated chondroitin sulfate (type E). In mast cells, serglycin interact with histamine, chymase, tryptase and carboxypeptidase, in neutrophils with elastase, in cytotoxic T cells with granzyme B, in endothelial cells with tissue-type plasminogen activator and in macrophages with tumor necrosis factor-alpha. Serglycin is important for the retention of key inflammatory mediators inside storage granules and secretory vesicles. Serglycin can further modulate the activities of partner molecules in different ways after secretion from activated immune cells, through protection, transport, activation and interactions with substrates or target cells. Serglycin is a proteoglycan with important roles in inflammatory reactions.

Decreasing the metastatic potential in cancers--targeting the heparan sulfate proteoglycans.

The heterogeneity of proteoglycans (PG)s contributes to their functional diversity. Many functions depend on their ability to bind and modulate the activity of components of the extracellular matrix (ECM). The ability of PGs to interact with other molecules, such as growth factors, is largely determined by the fine structure of the glycosaminoglycan (GAG) chains. Tumorigenesis is associated with changes in the PG synthesis. Heparan sulfate (HS) PGs are involved in several aspects of cancer biology including tumor progression, angiogenesis, and metastasis. PGs can have both tumor promoting and tumor suppressing activities depending on the protein core, the GAG attached, molecules they associate with, localization, the tumor subtype, stages, and degree of tumor differentiation. Perlecan is an angiogenic factor involved in tumor invasiveness. The C-terminal domain V of perlecan, named endorepellin, has however been shown to inhibit angiogenesis. Another angiogenic factor is endostatin, the COOH-terminal domain of the part-time PG collagen XVIII. Glypicans and syndecans may promote local cancer cell growth in some cancer tissues, but inhibit tissue invasion and metastasis in others. The GAG hyaluronan (HA) promotes cancer growth by providing a loose matrix for migrating tumor cells and mediates adhesion of cancer cells. HSPG degrading enzymes like heparanase, heparitinase, and other enzymes such as hyaluronidase and MMP are also important in tumor metastasis. Several different treatment strategies that target PGs have been developed. They have the potential to be effective in reducing tumor growth and inhibit the formation of metastases. PGs are also valuable tumor markers in several cancers.

8-isoprostane increases scavenger receptor A and matrix metalloproteinase activity in THP-1 macrophages, resulting in long-lived foam cells.

BACKGROUND: Oxidative stress is a key factor in atherogenesis, in which it is closely associated with the inflammation and formation of bioactive lipids. Although 8-isoprostane is regarded as a reliable marker of oxidative stress in vivo, the pathogenic role of this F(2)-isoprostane in atherogenesis is far from clear. Based on the important role of foam cells in the initiation and progression of atherosclerosis we hereby examined the ability of 8-isoprostane to modulate oxidized (ox)LDL-induced foam cell formation and the function of these cells, particularly focusing on the effect on matrix degradation. METHODS AND RESULTS: 8-isoprostane (10 micro M) augmented the oxLDL-induced (20 micro g mL(-1)) lipid accumulation of THP-1 macrophages evaluated by Oil-Red-O staining and lipid mass quantification (colourimetric assay). Additionally, 8-isoprostane induced the expression of the scavenger receptor A type 1 (MSR-1) [mRNA and protein level], assessed by RT-PCR and Western blotting, respectively. Moreover, 8-isoprostane counteracted the oxLDL-induced apoptosis of these cells, involving both mitochondrial-protective and caspase-suppressive mechanisms. Along with these changes, 8-isoprostane increased the oxLDL-induced gene expression of matrix metalloproteinase (MMP)-9 and its endogenous inhibitor [i.e. tissue inhibitor of MMP (TIMP)-1] accompanied by enhanced total MMP activity. CONCLUSIONS: We show that 8-isoprostane increases foam cell formation at least partly by enhancing MSR-1 expression and by inhibiting apoptosis of these cells, inducing long-lived foam cells with enhanced matrix degrading capacity. Our findings further support a role for 8-isoprostane not only as a marker of oxidative stress in patients with atherosclerotic disorders, but also as a mediator in atherogenesis and plaque destabilization.

Serglycin expression in CD2+ and CD14+ cells from patients with various rheumatic diseases.

The objective of the study was to look at the in vivo expression of serglycin in cells taken from patients with an inflammatory disease. The mRNA expression of the small proteoglycan serglycin was investigated in macrophages/monocytes and T-cells derived from the synovial fluid and blood of six patients with various rheumatic diseases and from the blood of two control subjects. Our results demonstrate higher Levels of expression in CD14+ cetts taken from patients with chronic inflammatory diseases than in control subjects. This suggests that serglycin may play a role during the inflammatory process.

The AGE product N epsilon-(carboxymethyl)lysine serum albumin is a modulator of proteoglycan expression in polarized cultured kidney epithelial cells.

AIMS/HYPOTHESIS: Changes in kidney function in diabetes could be due to changes in the kidney basement membranes. Proteoglycans are important constituents of this kidney extracellular matrix. This study explored the possibility that advanced glycation end products affect proteoglycan synthesis in cultured kidney epithelial cells. METHODS: Madin Darby Canine Kidney (MDCK) epithelial cells were cultured with either low glucose (5 mmol/l), low glucose with 10 micrograms/ml of N epsilon-(carboxymethyl)lysine bovine serum albumin (CML-BSA) or high glucose (25 mmol/l). From day 7-8 cells were labelled with either [35S]sulphate or [3H]glucosamine for 24 h. Labelled macromolecules were purified by gel and ion exchange chromatography, and isolated proteoglycans analysed by gel chromatography and electrophoresis. RESULTS: The CML-BSA treatment reduced the proteoglycan synthesis in MDCK cells. Neither the type of glycosaminoglycan chains made nor the molecular size of the chains was affected. CONCLUSION/INTERPRETATION: At concentrations found in the plasma of diabetes patients CML-BSA, decreases proteoglycan expression in kidney epithelial cells. Advanced glycation end products could, accordingly, promote pathological changes in kidneys of diabetics.

Proteoglycan synthesis is increased in cells with impaired clathrin-dependent endocytosis.

Overexpression of a GTPase deficient dynamin mutant in HeLa dynK44A cells causes a block in clathrin-dependent endocytosis. When endocytosis is inhibited, these cells incorporate higher levels of [(35)S]sulfate into both cellular and secreted macromolecules and larger amounts of proteoglycans such as syndecan and perlecan are immunoprecipitated from [(35)S]sulfate-labelled lysates. Gel filtration and ion-exchange chromatography revealed that the increased [(35)S]sulfate incorporation into proteoglycans was not due to significant differences in size or density of negative charge of glycosaminoglycan chains attached to proteoglycan core proteins. On the other hand, measurements of the syndecan-1 mRNA level and of [(3)H]leucine-labelled perlecan after immunoprecipitation supported the idea that the increased [(35)S]sulfate incorporation into proteoglycans was due to a selective increase in the synthesis of proteoglycan core proteins. Interestingly, the activity of protein kinase C was increased in cells expressing mutant dynamin and inhibition of protein kinase C with BIM reduced the differences in [(35)S]sulfate incorporation between cells with normal and impaired clathrin-dependent endocytosis. Thus, the activation of protein kinase C observed upon inhibition of clathrin-dependent endocytosis may be responsible for the increased synthesis of proteoglycans.

Internalization and stepwise degradation of heparan sulfate proteoglycans in rat hepatocytes.

Intracellular transport and degradation of membrane anchored heparan sulfate proteoglycans (HSPGs) were studied in cultured rat hepatocytes labeled with [35S]sulfate and [3H]glucosamine. Pulse chase experiments showed that membrane anchored HSPGs were constitutively transported to the cell surface after completion of polymerization and modification of the glycosaminoglycan chains in the Golgi apparatus. The intact HSPGs had a relatively short residence time at the cell surface and in non-degrading compartments (T(1/2) approximately 2-3 h), while [35S]sulfate labeled degradation products were found in lysosomes, and to a lesser extent in late endosomes. These degradation products which were free heparan sulfate chains with little or no protein covalently attached, were approximately half the size of the original glycosaminoglycan chains and were the only degradation intermediate found in the course of HSPG catabolism in these cells. In cells incubated in the presence of the microtubule perturbant vinblastine, or in the presence of the vacuolar ATPase inhibitor bafilomycin A1, and in cells incubated at 19 degrees C, the endocytosed HSPGs were retained in endosomes and no degradation products were detected. Disruption of lysosomes with glycyl-phenylalanine 2-naphthylamide (GPN) revealed a GPN resistant degradative compartment with both intact and partially degraded HSPGs. This compartment probably corresponds to late endosomes. Treatment of hepatocytes with the thiol protease inhibitor leupeptin inhibited the final degradation of the protein moiety of the HSPGs. The protein portion seems to be degraded completely before the glycosaminoglycan chains are cleaved. The degradation of the glycosaminoglycan chains is rapid and complete with one observable intermediate.

Macrophages secrete matrix metalloproteinase 9 covalently linked to the core protein of chondroitin sulphate proteoglycans.

Matrix metalloproteinases (MMPs) secreted from the leukemic macrophage cell-line THP-1 have been investigated. Under serum-free conditions, this cell-line synthesizes and secretes proMMP-9, which was detected in the culture medium as a monomer of 92 kDa, and in dimeric forms, including a homodimer of approximately 225 kDa. In addition, a new heterodimer complex is described, in which proMMP-9 is covalently linked to the core protein of chondroitin sulphate proteoglycan (CSPG) through one or more disulphide bridges. After SDS-PAGE electrophoresis, at least two forms of this complex were detected, a large form in the stacking gel and a smaller form with an estimated size of 300 kDa. When the CS chains were removed by chondroitin ABC lyase treatment, heterodimers of proMMP-9/CSPG core protein of approximately 145, 127 and 109 kDa were found, based on zymography and Western blots. Since as much as 10-15 % of the total proMMP-9 secreted from THP-1 cells was covalently linked to CSPG, this association may have important implications for transport, targetting and regulation of the enzyme activity.

Lithocholic acid and sulphated lithocholic acid differ in the ability to promote matrix metalloproteinase secretion in the human colon cancer cell line CaCo-2.

The human colon carcinoma cell line CaCo-2 has the ability to sulphate the secondary bile acid lithocholic acid (LA), whereas other primary or secondary bile acids were not sulphated [Halvorsen, Kase, Prydz, Gharagozlian, Andresen and Kolset (1999) Biochem. J. 343, 533--539]. To study the biological implications of this modification, CaCo-2 cells were incubated with either LA or sulphated lithocholic acid (3-sulpholithocholic acid, SLA), and in some experiments with taurine-conjugated lithocholic acid. Increased secretion of matrix metalloproteinases (MMPs) correlates with transformation of colon epithelial cells. When CaCo-2 cells were incubated with LA, the secretion of MMP-2 was found to increase approx. 60% when analysed by gelatin zymography, and 80% when analysed by Western blotting. SLA, in contrast, did not affect the level of MMP-2 secretion, and after zymography the level of enzyme activity was 78% of control values after 18 h incubation. The secretion of MMPs is linked to increased cellular invasion and, in tumours, to increased capacity for metastasis. The ability of CaCo-2 cells to invade in a chamber assay was stimulated after exposure to LA, whereas SLA-treated cells did not differ from control cells. LA therefore seems to induce a more invasive CaCo-2 cell phenotype, as judged by the two parameters tested, whereas the sulphated counterpart, SLA, did not have these effects. Sulphation of LA in the colon may be an important mechanism to decrease the potential LA has to promote a malignant epithelial phenotype.

Selective effects of sodium chlorate treatment on the sulfation of heparan sulfate.

We have analyzed the effect of sodium chlorate treatment of Madin-Darby canine kidney cells on the structure of heparan sulfate (HS), to assess how the various sulfation reactions during HS biosynthesis are affected by decreased availability of the sulfate donor 3'-phosphoadenosine 5'-phosphosulfate. Metabolically [(3)H]glucosamine-labeled HS was isolated from chlorate-treated and untreated Madin-Darby canine kidney cells and subjected to low pH nitrous acid cleavage. Saccharides representing (i) the N-sulfated domains, (ii) the domains of alternating N-acetylated and N-sulfated disaccharide units, and (iii) the N-acetylated domains were recovered and subjected to compositional disaccharide analysis. Upon treatment with 50 mM chlorate, overall O-sulfation of HS was inhibited by approximately 70%, whereas N-sulfation remained essentially unchanged. Low chlorate concentrations (5 or 20 mM) selectively reduced the 6-O-sulfation of HS, whereas treatment with 50 mM chlorate reduced both 2-O- and 6-O-sulfation. Analysis of saccharides representing the different domain types indicated that 6-O-sulfation was preferentially inhibited in the alternating domains. These data suggest that reduced 3'-phosphoadenosine 5'-phosphosulfate availability has distinct effects on the N- and O-sulfation of HS and that O-sulfation is affected in a domain-specific fashion.

Sulfated glycosaminoglycans and collagen in two bovine muscles (M. Semitendinosus and M. Psoas major) differing in texture.

M. semitendinosus (ST) and M. psoas major (PM) were used as models for tough and tender meat to study a possible role of sulfated glycosaminoglycans (GAGs) for muscle tenderness. The difference in texture was confirmed by Warner Bratzler shear force measurements. No significant difference in total amount of GAGs in the muscles was found. In contrast, a significant difference in the ratio of GAG/collagen was found between the two muscles. After separation of the GAGs by density gradient ultracentrifugation and ion-exchange chromatography, dermatan sulfate (DS), keratan sulfate (KS), chondroitin sulfate (CS), and heparan sulfate (HS) were identified by cellulose acetate electrophoresis after use of specific enzymes and chemical methods. The content of DS was higher in the tougher muscle (ST) than in PM, and the difference in DS content was statistically significant. Furthermore, a significant difference in the GAG composition pattern of the two muscles was found. The yield of GAGs extracted from the muscles was 77% for ST and 87% for PM. The residue after extraction was further analyzed and found to contain mainly HS. Immunohistochemical studies using antibodies against CS/DS showed a staining pattern of the perimysium of ST different from that of PM.

Sulphation of lithocholic acid in the colon-carcinoma cell line CaCo-2.

High levels of bile acids in the colon may correlate with an increased risk of colon cancer, but the underlying mechanisms are not known. Proteoglycan structures have been shown to change when human colon cells differentiate in vitro. The expression of [(35)S]sulphated molecules was used as a phenotypic marker to study the effects of bile acids on the human-colon-carcinoma cell line CaCo-2. [(35)S]sulphated compounds were isolated from the medium of cell fractions of cells metabolically labelled with [(35)S]sulphate in the absence and presence of cholic acid, deoxycholic acid, chenodeoxycholic acid and lithocholic acid (LA). Labelled molecules were analysed by gel chromatography, HPLC and SDS/PAGE in combination with chemical and enzymic methods. The expression of (35)S-labelled proteoglycans was not affected by any of the bile acids tested. However, the level of sulphated metabolites increased 7-18-fold in different experiments during a 22 h labelling period in the presence of an LA concentration of 10 microg/ml (26.6 nmol/ml) compared with controls. Further analyses showed that this was due, at least in part, to the sulphation of LA itself. This sulphation of LA was a rapid process followed by secretion back to the medium. Brefeldin A did not reduce the sulphation of LA, indicating that this conversion takes place in the cytosol, rather than in the Golgi apparatus of the CaCo-2 cells. LA in colon may be sulphated efficiently by the colonocytes to reduce the toxic effects of this particular bile acid. Sulphation may possibly be an important protective mechanism in the colon.