Slowly digestible carbohydrate sources can be used to attenuate the postprandial glycemic response to the ingestion of diabetes-specific enteral formulas.

PURPOSE: The purpose of this study is to compare the glycemic and insulinemic responses following the ingestion of recently developed diabetes-specific enteral formulas versus a standard and a high-fat formula. METHODS: Fifteen type 2 diabetes patients were selected to participate in a randomized, double-blind, crossover study. Two enteral formulas (47 energy percent [En%] carbohydrate, 34En% fat, and 4 g fiber/200 mL) were defined with either isomaltulose (formula 1) or sucromalt (formula 2) as the main carbohydrate source. For comparison, an isoenergetic diabetes-specific, high-fat (33En% carbohydrate, 50En% fat, 2.9 g fiber/200 mL) and a standard formula (55En% carbohydrate, 30En% fat, 2.8 g fiber/200 mL) were tested. RESULTS: Ingestion of formulas 1 and 2 and the high-fat formula resulted in an attenuated blood glucose response when compared with the standard formula (P < .05). In accordance, peak plasma glucose concentrations were significantly lower when compared with the standard formula (189 +/- 3.6 mg/dL [10.5 +/- 0.2 mmol/L], 196.2 +/- 3.6 mg/dL [10.9 +/- 0.2 mmol/L], 187.2 +/- 3.6 mg/dL [10.4 +/- 0.2 mmol/L], and 237.6 +/- 3.6 mg/dL [13.2 +/- 0.2 mmol/L], respectively). Plasma insulin responses were lower after consumption of the newly developed and high-fat formulas. Ingestion of the high-fat formula resulted in a greater postprandial triglyceride response (P < .05). CONCLUSIONS: Diabetes-specific enteral formulas rich in slowly digestible carbohydrate sources can be equally effective in attenuating the postprandial blood glucose response as low-carbohydrate, high-fat enteral formulas without elevating the plasma triglyceride response.

Effect of oral contraceptives on haemostasis variables.

Combined oral contraceptives (COCs) have effects on a large number of haemostasis variables. We have summarised literature data on effects of COCs containing 30-35 micrograms ethinyl estradiol for the third generation of progestogens (PGs): desogestrel, gestodene and norgestimate. It is concluded that about 15 variables show a shift in distribution of the order of magnitude of their interindividual variation coefficient. When comparing the third generation of PGs with the second one (norgestrel, levonorgestrel) stronger increases are noted for the former for some haemostatic variables. Also differences between desogestrel and gestodene for factor VII were apparent. It indicates that the role of PGs in the effects of COCs is significant and their design may in addition to reduction of oestrogen dosage be important in reducing haemostatic complications. The survey on molecular and cellular mechanisms by which the sex steroids might operate showed a great lack of knowledge. Only for factor XII has a functional oestrogen response element in the DNA definitely been identified. The study of molecular markers of coagulation and fibrinolysis have shown a distinct increased activation of coagulation (F 1 + 2, FPA) and fibrinolysis (PAP), and an increased fibrin turn-over (increased FDPs); platelet products are not found increased (beta TG, PF-4). The increase in fibrinolysis represent a counterforce, but individual changes in variables in coagulation and fibrinolysis do not correlate indicating independent effects and no evidence for a individually regulated balance. A first step in further research might be in understanding the increase in coagulation activation (F 1 + 2) which has so far not been satisfactorily related to changes in blood concentrations of haemostatic factors and possibly local factors.

The effects of sex steroids on plasma levels of marker proteins of endothelial cell functioning.

We studied thirteen male-to-female (M-->F) and ten female-to-male (F-->M) transsexuals who, for four months, received cross-sex treatment with, respectively, ethinylestradiol and cyproterone acetate, and with testosterone esters. We assessed the effects of treatment on plasma levels of tissue-type plasminogen activator (tPA), von Willebrand factor (vWF), vWF-propeptide (vWF:AgII) and big-endothelin-1 (big-ET-1), four proteins that are markers of endothelial cell functioning. We also measured urokinase-type PA (uPA) and plasminogen activator inhibitor-type 1 (PAI-1), which may not be endothelium-derived but share major clearance pathways with tPA. In M-->F transsexuals, mean plasma levels of tPA (minus 4.4 ng/ml), big-ET-1 (minus 0.8 pg/ml), uPA (minus 0.5 ng/ml) and PAI-1 (minus 26 ng/ml) decreased (all Ps < or =0.02). The level of vWF increased (plus 24%; P = 0.005), while vWF: AgII did not change (P = 0.49). In F-->M transsexuals, levels of big-ET-1 increased (plus 0.4 pg/ml; P = 0.02), while tPA, uPA and PAI-1 did not change (all Ps >0.25). In this group vWF decreased (minus 14%; P = 0.06), but vWF:AgII did not change (P = 0.38). Estrogens and androgens have clear effects on plasma levels of endothelial marker proteins. The mechanisms behind these effects are complex and appear to involve both altered secretion (big-ET-1) and processing and/or clearance (vWF and possibly tPA). Therefore, effects of hormones on the levels of endothelial marker proteins do not necessarily reflect changes in endothelial cell functioning, at least with regard to changes in vWF level associated with the oral administration of high doses of ethinylestradiol and cyproterone acetate to healthy men and the parenteral administration of testosterone to healthy women.

The onset of rheumatoid arthritis in relation to pregnancy and childbirth.

It has been suggested that the onset of RA symptoms is reduced during pregnancy and increased in the postpartum period. In the present study symptom onset in relation to the pregnancy prior to disease onset was compared between 135 young RA patients with definite RA and 378 controls with soft tissue rheumatism or osteoarthritis. Two RA patients developed symptoms during pregnancy versus 9 controls (odds ratio OR = 0.64). In the 3 months postpartum 5 RA patients and 5 controls developed symptoms (OR = 3.37). These results show the same trend as those of previous studies, i.e. a decrease in the onset of RA during pregnancy and an increased onset of RA after delivery. These findings might be explained by a delayed clinical onset of RA that started during pregnancy, analogous with the ameliorating effect of pregnancy on the course of existing RA and the flare-up of disease activity in the postpartum period.

Stimulation of tissue-type plasminogen activator expression by retinoic acid in human endothelial cells requires retinoic acid receptor beta 2 induction.

We previously showed the involvement of retinoic acid receptor alpha (RAR alpha) in the induction of tissue-type plasminogen activator (t-PA) synthesis by RA in human umbilical vein endothelial cells (HUVECs). However, the rather slow onset of this induction of t-PA synthesis suggested an indirect role of RAR alpha. Here, we show that the protein synthesis inhibitor, cycloheximide completely blocks the induction of t-PA by RA, which points to the need of an intermediary protein in t-PA stimulation. This intermediary protein is likely to be RAR beta 2 on the basis of the following findings: (1) the induction of RAR beta by RA exactly precedes that of t-PA; (2) HUVECs with elevated RAR beta mRNA levels show an undelayed t-PA induction on stimulation with RA, and this response can be almost completely inhibited with an RAR antagonist; and (3) an antisense oligodeoxynucleotide against the translation initiation site of RAR beta 2 mRNA greatly reduces the t-PA induction by RA. Thus, induction of t-PA by RA in HUVECs involves a 2-step mechanism requiring induction of RAR beta 2 via RAR alpha, followed by induction of t-PA synthesis via RAR beta 2. Each of these steps is shown to have a different activation profile with RA and 9 cis RA.

Effect of steroid hormones and retinoids on the formation of capillary-like tubular structures of human microvascular endothelial cells in fibrin matrices is related to urokinase expression.

Angiogenesis, the formation of new capillary blood vessels, is a feature of a variety of pathological processes. To study the effects of a specific group of hormones (all ligands of the steroid/retinoid/thyroid hormone receptor superfamily) on the angiogenic process in humans, we have used a model system in which human microvascular endothelial cells from foreskin (hMVEC) are cultured on top of a human fibrin matrix in the presence of basic fibroblast growth factor and tumor necrosis factor-alpha. This model mimics the in vivo situation where fibrin appears to be a common component of the matrix present at sites of chronic inflammation and tumor stroma. Our results show that testosterone and dexamethasone are strong inhibitors and all-trans retinoic acid (at-RA) and 9-cis retinoic acid (9-cis RA) are potent stimulators of the formation of capillary-like tubular structures. These effects are mediated by their respective nuclear hormone receptors as demonstrated by the use of specific synthetic receptor agonists and antagonists. 17beta-estradiol, progesterone, and 1,25-dihydroxyvitamin D3 did not affect or only weakly affected in vitro angiogenesis, which may be related to the lack of significant nuclear receptor expression. Although hMVEC express both thyroid hormone receptors alpha and beta, no effect of thyroid hormone on tube formation was found. The effects of testosterone, dexamethasone, at-RA, and 9-cis RA on tube formation were accompanied by parallel changes in urokinase-type plasminogen activator (u-PA) expression, at both mRNA and antigen levels. Exogenous suppletion of the medium with single chain u-PA enhances tube formation in our in vitro model, whereas quenching of u-PA activity (but not of tissue-type plasminogen activator activity) or of u-PA binding to u-PA receptor by specific antibodies suppressed basal and retinoid-stimulated tube formation. Moreover, addition of scu-PA to testosterone- or dexamethasone-treated hMVEC restored the suppressed angiogenic activity for a substantial part. Aprotinin, an inhibitor of plasmin activity, completely inhibited tube formation, indicating that the proteolytic properties of the u-PA/u-PA receptor complex are crucial in this process. Our results show that steroid hormones (testosterone and dexamethasone) and retinoids have strong, but opposite effects on tube formation in a human in vitro model reflecting pathological angiogenesis in the presence of fibrin and inflammatory mediators. These effects can be explained by hormone-receptor-mediated changes in u-PA expression, resulting in enhanced local proteolytic capacity of the u-PA/u-PA receptor complex.

Involvement of retinoic acid receptor alpha in the stimulation of tissue-type plasminogen-activator gene expression in human endothelial cells.

Retinoids stimulate tissue-type plasminogen-activator (t-PA) gene expression in human endothelial cells, and are likely to do so by binding to one or more nuclear retinoid receptors. The present study was initiated to identify the retinoid receptor(s) involved in this process. Expression and regulation of retinoic acid receptors (RARs) and retinoid X receptors (RXRs) were analyzed by Northern-blot analysis of total or poly(A)-rich RNA prepared from cultured human umbilical vein endothelial cells (HUVEC). Prior to any exposure to retinoids, HUVEC express two transcripts for RAR-alpha (3.6 kb and 2.8 kb), and low levels of transcripts for RAR-beta (3.4 kb and 3.2 kb) and RAR-gamma (3.3 kb and 3.1 kb). Two RXR subtypes were identified, RXR-alpha (4.8 kb) and, at a much lower concentration, RXR-beta (2.4 kb); no evidence for the presence of RXR-gamma was found. Furthermore, HUVEC express cellular retinol-binding protein I (CRBP-I) and cellular retinoic-acid-binding protein I (CRABP-I) mRNA. Exposure of HUVEC to 1 microM retinoic acid or the retinobenzoic acid, Ch55, led to the induction of the two RAR-beta mRNAs, RXR-alpha mRNA and CRBP-I mRNA, whereas the expression of the other receptor and CRABP-I transcripts did not change appreciably. Using retinoid analogues that bind preferentially to one of the RAR or RXR subtypes, we found evidence that RAR-alpha is involved in the retinoid-induced t-PA expression in HUVEC. This conclusion was strengthened by experiments in which blocking of RAR-alpha with a specific RAR-alpha antagonist, Ro 41-5253, was demonstrated to suppress the induction of t-PA by retinoids.

Stimulation of tissue-type plasminogen activator gene expression by sodium butyrate and trichostatin A in human endothelial cells involves histone acetylation.

We have previously shown that the pleiotropic agent sodium butyrate strongly stimulates tissue-type plasminogen activator (t-PA) expression in human umbilical vein endothelial cells (HUVEC). Here we provide the following evidence that the butyrate-induced t-PA expression in HUVEC involves histone H4 acetylation. (1) t-PA induction by butyrate occurs at the transcriptional level and does not require new protein synthesis, indicating a direct effect. (2) t-PA induction by butyrate can be fully mimicked by a specific, structurally unrelated, histone deacetylase inhibitor, trichostatin A. (3) At optimally stimulatory conditions, a combination of butyrate and trichostatin A does not enhance t-PA production more than each of the compounds alone, indicating that both compounds act through a common regulatory mechanism. (4) Induction of t-PA transcription by butyrate and trichostatin A was found to be preceded by histone H4 acetylation; at suboptimal inducing concentrations of butyrate and trichostatin A, the degree of acetylation of histone H4 caused by each agent was similarly reduced. These results are consistent with a role for histone H4 acetylation in t-PA induction by butyrate in HUVEC.

Cell-type specific DNA-protein interactions at the tissue-type plasminogen activator promoter in human endothelial and HeLa cells in vivo and in vitro.

Tissue-type plasminogen activator (t-PA) gene expression in human endothelial cells and HeLa cells is stimulated by the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) at the level of transcription. To study the mechanism of transcriptional regulation, we have characterized a segment of the t-PA gene extending from -135 to +100 by in vivo footprinting analysis [dimethyl sulphate (DMS) method] and gel mobility shift assay. In vivo footprinting analysis revealed changes in cleavage pattern in five distinct promoter elements in both endothelial cells and HeLa cells, including a PMA-responsive element (TRE), a CTF/NF-1 binding site and three GC-boxes, and an altered cleavage pattern of the TRE and CTF/NF-1 element after PMA treatment of HeLa cells. Although endothelial cells and HeLa cells differed in the exact G residues protected by nuclear proteins,in vitro bandshift analysis showed that nuclear protein binding to the t-PA promoter was qualitatively and quantitatively very similar in both cell types, except for the TRE. Protein binding to the TRE under non- stimulated conditions was much higher in human endothelial cells than in HeLa cells, and this TRE-bound protein showed a lower dissociation rate in the endothelial cells than in HeLa cells. In endothelial cells, the proteins bound to the TRE consisted mainly of the AP-1 family members JunD and Fra-2, while in HeLa cells predominantly JunD, FosB and Fra-2 were bound. The proteins bound to the other protected promoter elements were identified as SP-1 (GC-box II and III) and CTF/NF-1 (CTF/NF-1 binding site). After PMA treatment of the cells, AP-1 and SP-1 binding was increased two-fold in endothelial cell nuclear extracts and >20-fold in HeLa nuclear extracts. In the endothelial cells, all Jun and Fos forms (c-Jun, JunB, JunD, c-Fos, FosB, Fra-1 and Fra-2) were part of the AP-1 complex after PMA induction. In HeLa cells, the complex consisted predominantly of c-Jun and the Fos family members FosB and Fra-2. In the light of previous studies involving mutational analysis of the human and murine t-PA promoter our results underline an important role of the five identified promoter regions in basal and PMA-stimulated t-PA gene expression in intact human endothelial cells and HeLa cells. The small differences in DMS protection pattern and differences in the individual AP-1 components bound in endothelial cells and HeLa cells point to subtle cell-type specific differences in t-PA gene regulation.

Differences in metabolism and isomerization of all-trans-retinoic acid and 9-cis-retinoic acid between human endothelial cells and hepatocytes.

Retinoic acid stimulates the expression of tissue-type plasminogen activator (t-PA) in vascular endothelial cells in vitro and enhances t-PA levels in plasma and tissues in vivo. Compared with the in vivo situation, high retinoic acid concentrations are required to induce optimally t-PA expression in vitro. These findings led us to study retinoic acid metabolism in cultured human endothelial cells. For comparison, these studies were also performed in the human hepatoma cell line, HepG2, and key experiments were repeated with human primary hepatocytes. Both hepatocyte cultures gave very similar results. Human endothelial cells were shown to possess an active retinoic acid metabolizing capacity, which is quantitatively comparable to that of hepatocytes, but different from that of hepatocytes in several qualitative aspects. Our results demonstrate that all-trans-retinoic acid is quickly metabolized by both endothelial cells and hepatocytes. All-trans-retinoic acid induces its own metabolism in endothelial cells but not in hepatocytes. 9-cis-Retinoic acid is degraded slowly by endothelial cells, whereas hepatocytes metabolize 9-cis-retinoic acid very quickly. Furthermore, our data show that hepatocytes, but not endothelial cells, detectably isomerise all-trans-retinoic acid to 9-cis-retinoic acid and vice versa. In both endothelial cells and hepatocytes all-trans-retinoic acid metabolism was inhibitable by the cytochrome P-450 inhibitors liarozole (10 microM) and ketoconazole (10 microM), albeit to different extents and with different specificities. In the presence of the most potent retinoic acid metabolism inhibitor in endothelial cells, liarozole, at least 10-fold lower all-trans-retinoic acid concentrations were required than in the absence of the inhibitor to obtain the same induction of t-PA. In conclusion, our results clearly demonstrate that all-trans-retinoic acid and 9-cis retinoic acid are actively but differently metabolized and isomerised by human endothelial cells and hepatocytes. The rapid metabolism of retinoic acid explains the relatively high concentrations of retinoic acid required to induce t-PA in cultured endothelial cells.

Increased clearance explains lower plasma levels of tissue-type plasminogen activator by estradiol: evidence for potently enhanced mannose receptor expression in mice.

Several clinical studies have demonstrated an inverse relationship between circulating levels of estrogen and tissue-type plasminogen activator (t-PA). The present study was designed to test the hypothesis that estrogens lower plasma levels of t-PA by increasing its clearance from the bloodstream. 17alpha-Ethinyl estradiol (EE) treatment resulted in a significant increase in the clearance rate of recombinant human t-PA in mice (0.46 mL/min in treated mice v 0. 32 mL/min in controls; P <.01). The clearance of endogenous, bradykinin-released t-PA in rats was also significantly increased after EE treatment (area under the curve [AUC], 24.9 ng/mL. min in treated animals v 31.9 ng/mL. min in controls; P <.05). Two distinct t-PA clearance systems exist in vivo: the low-density lipoprotein receptor-related protein (LRP) on liver parenchymal cells and the mannose receptor on mainly liver endothelial cells. Inhibition of LRP by intravenous injection of receptor-associated protein (RAP) as a recombinant fusion protein with Salmonella japonicum glutathione S-transferase (GST) significantly retarded t-PA clearance in control mice (from 0.41 to 0.25 mL/min; n = 5, P <.001) and EE-treated mice (from 0.66 to 0.35 mL/min; n = 5, P <.005), but did not eliminate the difference in clearance capacity between the 2 experimental groups. Similar results were obtained in mice in which LRP was inhibited via overexpression of the RAP gene in liver by adenoviral gene transduction. In contrast, administration of mannan, a mannose receptor antagonist, resulted in identical clearances (0.22 mL/min in controls and 0.24 mL/min in EE-treated mice). Northern blot analysis showed a 6-fold increase in mannose receptor mRNA expression in the nonparenchymal liver cells of EE-treated mice, whereas the parenchymal LRP mRNA levels remained unchanged. These findings were confirmed at the protein level by ligand blotting and Western blotting analysis. Our results demonstrate that EE treatment results in increased plasma clearance rate of t-PA via induction of the mannose receptor and could explain for the inverse relationship between estrogen status and plasma t-PA concentrations as observed in humans.

Binding of human urokinase-type plasminogen activator to its receptor: residues involved in species specificity and binding.

Urokinase-type plasminogen activator (UPA), particularly when bound to its receptor (UPAR), is thought to play a major role in local proteolytic processes, thus facilitating cell migration as may occur during angiogenesis, neointima and atherosclerotic plaque formation, and tumor cell invasion. To facilitate understanding of the need and function of the UPA/UPAR interaction in cell migration and vascular remodeling, we changed several amino acid residues in UPA so as to interfere with its interaction with its receptor. The receptor-binding domain of UPA has been localized to a region in the growth factor domain between residues 20 and 32. Since the binding of UPA to UPAR appears to be species specific, we used the differences in amino acid sequences in the growth factor domain of UPA between various species to construct a human UPA variant that does not bind to the human UPAR. We substituted Asn22 for its mouse equivalent Tyr by site-directed mutagenesis. This mutant UPA had similar plasminogen activator characteristics as wild-type UPA, including its specific activity and interaction with plasminogen activator inhibitor-1. However, no UPA/UPAR complexes could be observed in cross-linking experiments using DFP-treated 125I-labeled mutant UPA and lysates of various cells, including U937 histiocytic lymphoma cells, phorbol myristate acetate-treated human ECs, and mouse LB6 cells transfected with human UPAR cDNA. In direct binding experiments, DFP-treated 125I-labeled mutant UPA could not bind to phorbol myristate acetate-treated ECs, whereas wild-type UPA did bind. Furthermore, a 25-fold excess of wild-type UPA completely prevented the binding of DFP-treated 125I-labeled wild-type UPA to the human receptor on transfected LB6 cells, whereas an equal amount of mutant UPA had only a very small effect. In ligand blotting assays, very weak binding of mutant UPA to human UPAR could be observed. Changing Asn22 into the other amino acid residues alanine or glutamine had no effect on binding to UPAR on human ECs. The functional integrity of the growth factor domain in the non-receptor binding Asn22Tyr mutant is suggested by the fact that binding of this mutant to a murine UPAR can be restored after additional mutations in the growth factor domain, Asn27,His29,Trp30 to Arg27,Arg29,Arg30. We conclude that Asn22 and Asn27,His29,Trp30 in human UPA are key determinants in the species-specific binding of the enzyme to its receptor and that changing Asn22 into Tyr results in a UPA mutant with strongly reduced binding to UPAR.