FXR controls insulin content by regulating Foxa2-mediated insulin transcription.

Farnesoid X receptor (FXR) is a nuclear ligand-activated receptor of bile acids that plays a role in the modulation of insulin content. However, the underlying molecular mechanisms remain unclear. Forkhead box a2 (Foxa2) is an important nuclear transcription factor in pancreatic ß-cells and is involved in ß-cell function. We aimed to explore the signaling mechanism downstream of FXR to regulate insulin content and underscore its association with Foxa2 and insulin gene (Ins) transcription. All experiments were conducted on FXR transgenic mice, INS-1 823/13 cells, and diabetic Goto-Kakizaki (GK) rats undergoing sham or Roux-en-Y gastric bypass (RYGB) surgery. Islets from FXR knockout mice and INS-1823/13 cells with FXR knockdown exhibited substantially lower insulin levels than that of controls. This was accompanied by decreased Foxa2 expression and Ins transcription. Conversely, FXR overexpression increased insulin content, concomitant with enhanced Foxa2 expression and Ins transcription in INS-1 823/13 cells. Moreover, FXR knockdown reduced FXR recruitment and H3K27 trimethylation in the Foxa2 promoter. Importantly, Foxa2 overexpression abrogated the adverse effects of FXR knockdown on Ins transcription and insulin content in INS-1 823/13 cells. Notably, RYGB surgery led to improved insulin content in diabetic GK rats, which was accompanied by upregulated FXR and Foxa2 expression and Ins transcription. Collectively, these data suggest that Foxa2 serves as the target gene of FXR in ß-cells and mediates FXR-enhanced Ins transcription. Additionally, the upregulated FXR/Foxa2 signaling cascade could contribute to the enhanced insulin content in diabetic GK rats after RYGB.

FXR-mediated epigenetic regulation of GLP-1R expression contributes to enhanced incretin effect in diabetes after RYGB.

In this study, we investigated how Roux-en-Y gastric bypass (RYGB) enhances glucagon-like peptide 1 (GLP-1) response in GK rats and explored the potential link between RYGB-stimulated BAs/FXR signalling and GLP-1R-linked signalling in ß-cells, a key pathway that regulates glucose-stimulated insulin secretion (GSIS). Here we show that RYGB restores GLP-1R expression in GK rat islets. This involves increased total BAs as well as chenodeoxycholic acid (CDCA), leading to FXR activation, increasing FXR binding to the promoter of Glp-1r and enhancing occupancy of histone acetyltransferase steroid receptor coactivator-1 (SRC1), thus increasing histone H3 acetylation at the promoter. These coordinated events bring about increased GLP-1R expression, resulting in greater GLP-1 response in ß-cells. Moreover, ablation of FXR suppressed the stimulatory effects of GLP-1. Thus, this study unravels the crucial role of the BAs/FXR/SRC1 axis-controlled GLP-1R expression in ß-cells, which results in enhanced incretin effect and normalized blood glucose of GK rats after RYGB.

Roux-en-Y gastric bypass enhances insulin secretion in type 2 diabetes via FXR-mediated TRPA1 expression.

OBJECTIVE: Roux-en-Y gastric bypass surgery (RYGB) improves the first phase of glucose-stimulated insulin secretion (GSIS) in patients with type 2 diabetes. How it does so remains unclear. Farnesoid X receptor (FXR), the nuclear receptor of bile acids (BAs), is implicated in bariatric surgery. Moreover, the transient receptor potential ankyrin 1 (TRPA1) channel is expressed in pancreatic ß-cells and involved in insulin secretion. We aimed to explore the role of BAs/FXR and TRPA1 in improved GSIS in diabetic rats after RYGB. METHODS: RYGB or sham surgery was conducted in spontaneous diabetic Goto-Kakizaki (GK) rats, or FXR or TRPA1 transgenic mice. Gene and protein expression of islets were assessed by qPCR and western blotting. Electrophysiological properties of single ß-cells were studied using patch-clamp technique. Binding of FXR and histone acetyltransferase steroid receptor coactivator-1 (SRC1) to the TRPA1 promoter, acetylated histone H3 (ACH3) levels at the TRPA1 promoter were determined using ChIP assays. GSIS was measured using enzyme-linked immunosorbent assays or intravenous glucose tolerance test (IVGTT). RESULTS: RYGB increases GSIS, particularly the first-phase of GSIS in both intact islets and GK rats in vivo, and ameliorates hyperglycemia of GK rats. Importantly, the effects of RYGB were attenuated in TRPA1-deficient mice. Moreover, GK ß-cells displayed significantly decreased TRPA1 expression and current. Patch-clamp recording revealed that TRPA1-/- ß-cells displayed a marked hyperpolarization and decreased glucose-evoked action potential firing, which was associated with impaired GSIS. RYGB restored TRPA1 expression and current in GK ß-cells. This was accompanied by improved glucose-evoked electrical activity and insulin secretion. Additionally, RYGB-induced TRPA1 expression involved BAs/FXR-mediated recruitment of SRC1, promoting ACH3 at the promoter of TRPA1. CONCLUSIONS: The BAs/FXR/SRC1 axis-mediated restoration of TRPA1 expression plays a critical role in the enhanced GSIS and remission of diabetes in GK rats after RYGB.

[Blood compatibility of two novel polyurethane coating materials].

Amphiphilic coupling-polymer of stearyl poly (ethylene oxide)-co-4, 4'-methylendiphenyl diisocyanate-co-stearyl poly(ethylene oxide), MSPEO, was specially designed as surface-modifying additives. The blends of MSPEO in both polyether urethane (PEU) and chitosan(Chi), as the coating materials for intravascular device were investigated. Two kinds of static clotting time tests, plasma recalcification time (PRT) and prothrombin time(PT), as well as the static platelet adhesion experiment were carried out. And the dynamic anti-coagulation experiment was performed with a closed-loop tubular system under a blood shear rate of 1,500 s-1. The results demonstrate that both blend coatings can improve the anti-coagulation of polyurethane greatly and will not lead to hemolysis, and that more platelets adhere to the surface modified by Chi-MSPEO blend coating as compared with those adhere to the surface modified by PEU-MSPEO blend coating. The surface modified by Chi-MSPEO has longer PRT, whereas the surface modified by PEU-MSPEO has longer PT.

Novel human endothelial cell-engineered polyurethane biomaterials for cardiovascular biomedical applications.

A tri-block coupling-polymer composed of 4,4'-methylenediphenyl diisocyanate and poly (ethylene oxide) (PEO), abbreviated MPEO, was used as the template surface-modifying additive (SMA), based on which selected amino acids (lysine, arginine, glycin, and aspartic acid) and RGD peptide were respectively conjugated as functional endgroups of the PEO spacer-arms through sulfonyl chloride-activation routes. After the immobilization of biofunctional factors, the SMA-MPEO derivatives were noncovalently introduced onto the biomedical poly(ether urethane) (PEU) surfaces by physical blending methods. The SMA synthesis and PEU surface modification were monitored and analyzed by nuclear magnetic resonance spectroscopy, attenuated total reflection-infrared spectroscopy, and X-ray photoelectron spectroscopy. The human umbilical vein endothelial cells (HUVECs) were collected and harvested manually by collagenase digestion. The cell culture was performed respectively on the MPEO derivative-modified PEU surfaces and also on the surfaces of the commercially available polystyrene cell-culture plates (TCPS) for control. The cell adhesion rates and cell proliferation rates of the in vitro cultivated HUVEC were measured using flow cytometry. The individual cell viability rates were determined with MTT assay. The cell morphologies of the living HUVECs were investigated by optical inverted microscopy, and more detailed information was acquired from scanning electrical microscopy. The results indicated that the efficacy of SMA functional endgroups was the dominant factor for HUVEC compatibility; the proper-sized PEO spacers (M(w) 2 k) could support and mobilize the functional endgroups, optimizing the surface (interface) environment for the cell growth. As the endgroups of the SMA-MPEO derivatives and the bio-functional factors, the basic amino acids (lysine and arginine) demonstrated similar performances to that of the widely acknowledged cell growth-promoter, RGD peptide, which were superior to TCPS. Therefore, these MPEO derivative-modified PEU materials are promising to serve as novel polymeric permanent implants or interventional devices for cardiovascular biomedical applications.

In situ immobilization of proteins and RGD peptide on polyurethane surfaces via poly(ethylene oxide) coupling polymers for human endothelial cell growth.

A "CBABC"-type pentablock coupling polymer, mesylMPEO, was designed and synthesized to promote human endothelial cell growth on the surfaces of polyurethane biomaterials. The polymer was composed of a central 4,4'-methylenediphenyl diisocyanate (MDI) coupling unit and poly(ethylene oxide) (PEO) spacer arms with methanesulfonyl (mesyl) end groups pendent on both ends. As the presurface modifying additive (pre-SMA), the mesylMPEO was noncovalently introduced onto the poly(ether urethane) (PEU) surfaces by dip coating, upon which the protein/peptide factors (gelatin, albumin, and arginine-glycine-aspartic acid tripeptide [RGD]) were covalently immobilized in situ by cleavage of the original mesyl end groups. The pre-SMA synthesis and PEU surface modification were characterized using nuclear magnetic resonance spectroscopy ((1)H NMR), attenuated total reflection infrared spectroscopy (ATR-FTIR), and X-ray photoelectron spectroscopy (XPS). Human umbilical vein endothelial cells (HUVEC) were harvested manually by collagenase digestion and seeded on the modified PEU surfaces. Cell adhesion ratios (CAR) and cell proliferation ratios (CPR) were measured using flow cytometry, and the individual cell viability (ICV) was determined by MTT assay. The cell morphologies were investigated by optical inverted microscopy (OIM) and scanning electrical microscopy (SEM). The gelatin- and RGD-modified surfaces were HUVEC-compatible and promoted HUVEC growth. The albumin-modified surfaces were compatible but inhibited cell adhesion. The results also indicated that, for HUVEC in vitro cultivation, the cell adhesion stage was of particular importance and had a significant impact on the cell responses to the modified surfaces.

Surface engineering of poly(D,L-lactic acid) by entrapment of chitosan-based derivatives for the promotion of chondrogenesis.

Chitosan and chitosan-amino acid derivatives were explored to engineer poly(D,L-lactic acid) (PDL-LA) as an extracellular matrix-like surface to promote cell adhesion and growth. Four kinds of chitosan-amino acid derivatives were prepared to mimic the carbohydrate moieties of cell matrix glycoprotein. The chitosan-amino acid derivatives were characterized by using Fourier transform infrared and ultraviolet spectra. The amino acid content on chitosan-amino acid derivatives was determined by using a ninhydrin-ultraviolet method. A new strategy, entrapment, was therefore used to modify the PDL-LA membrane with chitosan and chitosan-amino acid derivatives. The results of X-ray photoelectron spectroscopy, attenuated total reflectance-Fourier transform infrared, and contact angle confirmed that a stable thin film of chitosan and its derivatives can be entrapped on the surface of the PDL-LA membrane. From the results of chondrocyte cytocompatibility, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assays, and cell morphology, the chitosan-amino acid derivative modified PDL-LA membranes were shown to promote chondrogenesis. The novel surface treatment method combines the good mechanical property of PDL-LA with the good cytocompatibility of chitosan derivatives, which may have potential for tissue engineering.

Selective adsorption of serum albumin on biomedical polyurethanes modified by a poly(ethylene oxide) coupling-polymer with cibacron blue (F3G-A) endgroups.

A tri-block-coupling polymer, "PEO-MDI-PEO" ["poly(ethylene oxide)-4,4'-methylene diphenyl diisocyanate-poly(ethylene oxide)", abbreviated "MPEO"], was used to react with a triazine dye, Cibacron Blue F3G-A (ciba), in an alkaline environment. The product of this nucleophilic reaction was a penta-block-coupling polymer, "ciba-PEO-MDI-PEO-ciba" (abbreviated "cibaMPEO"). The cibaMPEO-modified poly(ether urethane) (PEU) surfaces were prepared by dip-coating and detected by XPS. The surface enrichment of both ciba endgroups and poly(ethylene oxide) spacer-arms was revealed. On the modified surfaces, bovine serum albumin (BSA)-adsorbing experiments were carried out, respectively, in the low and high BSA bulk-concentration solutions, and accordingly, the methods of radioactive (125)I-probe and ATR-FTIR were, respectively, employed for the characterization. The competitive adsorption of BSA and bovine serum fibrinogen (Fg) in the BSA-Fg binary solutions was also studied using a (125)I-probe, and through which the reversibly BSA-selective adsorption on cibaMPEO-modified PEU surfaces was confirmed. Finally, the improvement of blood-compatibility on the modified surfaces was verified by the plasma recalcification time (PRT) test.

Surface engineering of poly(DL-lactic acid) by entrapment of alginate-amino acid derivatives for promotion of chondrogenesis.

Alginate-amino acid derivatives were explored to engineer poly(DL-lactic acid)(PDL-LA) as glycocalyx-like surface to promote cell adhesion and growth. Four different kinds of alginate-amino acid derivatives were synthesized to mimic the glycocalyx of cell membrane to promote chondrogenesis. The alginate-amino acid derivatives were characterized by FT-IR, 1H NMR and UV spectra and the amino acid content on alginate-amino acid derivatives was given by ninhydrin-UV method. A new strategy, entrapment, was then employed to modify PDL-LA membranes with alginate and its amino acid derivatives. The results of XPS, ATR-FTIR and contact angle confirmed that a stable thin film of alginate and its amino acid derivatives can be entrapped on the surface of PDL-LA membrane. The chondrocyte cytocompatibility test and MTT assays indicated that the alginate-amino acid derivatives modified PDL-LA membranes could promote chondrogenesis. The novel surface treatment method may have potentials for tissue engineering and other biomedical applications.

Preparation of albumin preferential surfaces on poly(vinyl chloride) membranes via surface self-segregation.

Poly(vinyl chlorides)-graft-[omega-stearyl-poly(ethylene oxide)] (PVC-g-SPEO), which has a poly(vinyl chloride) (PVC) backbone, poly(ethylene oxide) (PEO) side chain, and stearyl end groups, has been synthesized. Self-organizing blends of the amphiphilic comb polymer in poly(vinyl chlorides) have been examined as a means to create albumin preferential surfaces on polymer films. X-ray photoelectron spectroscopy (XPS) analysis indicates substantial surface segregation of the PVC-g-SPEO. A surface concentration of 59.9 EO wt % is achieved by the solution casting and heat treatment of a film with a bulk concentration of only 3.78 EO wt %. In the aqueous environment, the surface rearrangement of PVC-g-SPEO/PVC blend film is limited and presents a high interfacial energy and high depolar component of interfacial energy due to the "tail-like" SPEO side chain. Protein adsorption tests confirm that PVC-g-SPEO/PVC blend films absorb high levels of albumin and dramatically resist fibrinogen adsorption. Surfaces to attract and reversibly bind albumin, which might diminish the occurrence of thrombosis, inflammation, and infection, are developed by self-organizing blends of the amphiphilic comb polymer in poly(vinyl chlorides).

[Quantitative characterization of adsorbed bovine serum albumin on modified surfaces of poly(ether urethane) materials using ATR-FT-IR spectroscopy].

Attenuated total reflection (ATR) FT-IR spectroscopy was used to quantitatively characterize the extent of bovine serum albumin (BSA) adsorbed on the surface-coating-modified poly(ether urethane) (PEU) matrix. The two surface modifying additives (SMA) were respectively a tri-block-coupling-polymer of stearyl poly (ethylene oxide)-4,4'-methylene diphenyl diisocyanate-stearyl poly(ethylene oxide), for short MSPEO, and another similar block-coupling polymer with the Cibacron Blue F3G-A endgroups, for short cibaMPEO. The experiments of static BSA adsorption were composed by two parts. One was static isothermal adsorption, and the other was static adsorption kinetics. The quantitative characterization was based on the optical principles of FT-IR, method of experiment and index of the apparatus, by which the enhancement of BSA adsorption on the SMA-modified surfaces was confirmed.

A novel urethane containing copolymer as a surface modification additive for blood contact materials.

Surface modification to develop a biomolecules-presenting surface is of interest, both from a scientific and an industrial point of view. In this research, a penta-block-coupling polymer of warfarin-PEO-MDI-PEO-warfarin was specially designed as the surface modifying additive (SMA). The warfarin-modified polyurethane surfaces were then prepared by dip-coating method. Attenuated total reflection fourier transform infra-red (ATR-FTIR) spectra revealed that the urethane segments in the SMA could penetrate into the hard block of segmented polyurethane (SPU) via intermolecular hydrogen bonds. The X-ray photoelectron spectroscopy (XPS) results indicated that the intermolecular hydrogen bonds were strong enough to form stable warfarin-PEO composite surfaces in an aqueous environment. Fibrinogen and albumin adsorption onto unmodified and SMA-modified SPU was investigated by the (125)I-labeled method. The surface for attracting and reversibly binding albumin, which was proved to suppress the platelet adhesion and prolong the clotting time, has been developed by the simple coating of the novel SMA in SPU.