Viscoelastic Properties of Zona Pellucida of Oocytes Characterized by Transient Electrical Impedance Spectroscopy.

The success rate in vitro fertilization is significantly linked to the quality of the oocytes. The oocyte's membrane is encapsulated by a shell of gelatinous extracellular matrix, called zona pellucida, which undergoes dynamic changes throughout the reproduction cycle. During the window of highest fertility, the zona pellucida exhibits a softening phase, while it remains rigid during oocyte maturation and again after fertilization. These variations in mechanical properties facilitate or inhibit sperm penetration. Since successful fertilization considerably depends on the state of the zona pellucida, monitoring of the hardening process of the zona pellucida is vital. In this study, we scrutinized two distinct genetic mouse models, namely, fetuin-B wild-type and fetuin-B/ovastacin double deficient with normal and super-soft zona pellucida, respectively. We evaluated the hardening with the help of a microfluidic aspiration-assisted electrical impedance spectroscopy system. An oocyte was trapped by a microhole connected to a microfluidic channel by applying suction pressure. Transient electrical impedance spectra were taken by microelectrodes surrounding the microhole. The time-depending recovery of zona pellucida deflections to equilibrium was used to calculate the Young's modulus and, for the first time, absolute viscosity values. The values were obtained by fitting the curves with an equivalent mechanical circuit consisting of a network of dashpots and springs. The observer-independent electrical readout in combination with a fitting algorithm for the calculation of the viscoelastic properties demonstrates a step toward a more user-friendly and easy-to-use tool for the characterizing and better understanding of the rheological properties of oocytes.

Population-based meta-analysis and gene-set enrichment identifies FXR/RXR pathway as common to fatty liver disease and serum lipids.

Nonalcoholic fatty liver disease (NAFLD) is prevalent worldwide. NAFLD is associated with elevated serum triglycerides (TG), low-density lipoprotein cholesterol (LDL), and reduced high-density lipoprotein cholesterol (HDL). Both NAFLD and blood lipid levels are genetically influenced and may share a common genetic etiology. We used genome-wide association studies (GWAS)-ranked genes and gene-set enrichment analysis to identify pathways that affect serum lipids and NAFLD. We identified credible genes in these pathways and characterized missense variants in these for effects on serum traits. We used MAGENTA to identify 58 enriched pathways from publicly available TG, LDL, and HDL GWAS (n = 99,000). Three of these pathways were also enriched for associations with European-ancestry NAFLD GWAS (n = 7176). One pathway, farnesoid X receptor (FXR)/retinoid X receptor (RXR) activation, was replicated for association in an African-ancestry NAFLD GWAS (n = 3214) and plays a role in serum lipids and NAFLD. Credible genes (proteins) in FXR/RXR activation include those associated with cholesterol/bile/bilirubin transport/absorption (ABCC2 (MRP2) [ATP binding cassette subfamily C member (multidrug resistance-associated protein 2)], ABCG5, ABCG8 [ATP-binding cassette (ABC) transporters G5 and G8], APOB (APOB) [apolipoprotein B], FABP6 (ILBP) [fatty acid binding protein 6 (ileal lipid-binding protein)], MTTP (MTP) [microsomal triglyceride transfer protein], SLC4A2 (AE2) [solute carrier family 4 member 2 (anion exchange protein 2)]), nuclear hormone-mediated control of metabolism (NR0B2 (SHP) [nuclear receptor subfamily 0 group B member 2 (small heterodimer partner)], NR1H4 (FXR) [nuclear receptor subfamily 1 group H member 4 (FXR)], PPARA (PPAR) [peroxisome proliferator activated receptor alpha], FOXO1 (FOXO1A) [forkhead box O1]), or other pathways (FETUB (FETUB) [fetuin B]). Missense variants in ABCC2 (MRP2), ABCG5 (ABCG5), ABCG8 (ABCG8), APOB (APOB), MTTP (MTP), NR0B2 (SHP), NR1H4 (FXR), and PPARA (PPAR) that associate with serum LDL levels also associate with serum liver function tests in UK Biobank. Conclusion: Genetic variants in NR1H4 (FXR) that protect against liver steatosis increase serum LDL cholesterol while variants in other members of the family have congruent effects on these traits. Human genetic pathway enrichment analysis can help guide therapeutic development by identifying effective targets for NAFLD/serum lipid manipulation while minimizing side effects. In addition, missense variants could be used in companion diagnostics to determine their influence on drug effectiveness.

Hepatokine Fetuin B expression is regulated by leptin-STAT3 signalling and associated with leptin in obesity.

Obesity is an expanding global public health problem and a leading cause of metabolic disorders. The hepatokine Fetuin B participates in regulating insulin resistance, glucose metabolism and liver steatosis. However, the mechanism underlying Fetuin B activation remains unclear. Our previous population-based study demonstrated a significant association between serum Fetuin B and body fat mass in an obese population, which indicates its potential in mediating obesity-related metabolic disorders. In the present study, we further revealed a significant correlation between Fetuin B and leptin, the classic adipokine released by expanding adipose tissue, in this obese population. Consistently, elevated Fetuin B and leptin levels were confirmed in diet-induced obese mice. Furthermore, an in vitro study demonstrated that the leptin signalling pathway directly activated the transcription and expression of Fetuin B in primary hepatocytes and AML12 cells in a STAT3-dependent manner. STAT3 binds to the response elements on FetuB promoter to directly activate FetuB transcription. Finally, the mediating effect of Fetuin B in insulin resistance induced by leptin was confirmed according to mediation analysis in this obese population. Therefore, our study identifies leptin-STAT3 as an upstream signalling pathway that activates Fetuin B and provides new insights into the pathogenic mechanisms of obesity-related metabolic disorders.

Fetuin B overexpression suppresses proliferation, migration, and invasion in prostate cancer by inhibiting the PI3K/AKT signaling pathway.

Fetuin B (FETUB) is a glycoprotein that is a member of the cysteine protease inhibitor family, and it is associated with cancer. However, the role of FETUB in prostate carcinogenesis is unknown. In this study, we overexpressed FETUB in prostate cancer cells by using lentivirus and then studied the impacts on cell apoptosis, migration and invasion. We found that apoptosis was increased and the migration and invasion of prostate cancer cells were significantly inhibited after overexpression. Then, we performed experiments in vivo and found that there were fewer tumors in the overexpression groups than in the control groups. In addition, we demonstrated that overexpression of FETUB inactivates the PI3K/AKT signaling pathway. Rescue assays revealed that intervention of 740Y-P reversed the anti-tumor effect of FETUB on prostate cancer cells. Taken together, our results revealed that FETUB may act as a novel regulator to promote apoptosis and inhibit the migration and invasion of prostate cancer cells and that FETUB is related to the inactivation of the PI3K/AKT signaling pathway.

Multiplex CRISPR/Cas screen in regenerating haploid limbs of chimeric Axolotls.

Axolotls and other salamanders can regenerate entire limbs after amputation as adults, and much recent effort has sought to identify the molecular programs controlling this process. While targeted mutagenesis approaches like CRISPR/Cas9 now permit gene-level investigation of these mechanisms, genetic screening in the axolotl requires an extensive commitment of time and space. Previously, we quantified CRISPR/Cas9-generated mutations in the limbs of mosaic mutant axolotls before and after regeneration and found that the regenerated limb is a highfidelity replicate of the original limb (Flowers et al. 2017). Here, we circumvent aforementioned genetic screening limitations and present methods for a multiplex CRISPR/Cas9 haploid screen in chimeric axolotls (MuCHaChA), which is a novel platform for haploid genetic screening in animals to identify genes essential for limb regeneration.

Association of Fetuin-B with Subclinical Atherosclerosis in Obese Chinese Adults.

AIM: We aimed to explore the independent associations of serum Fetuin-B and common genetic variants in FETUB locus with subclinical atherosclerosis. METHODS: A cross-sectional study of 1,140 obese adults, who underwent serum Fetuin-B testing, hepatic ultrasonography scanning, genotyping on four tagging single nucleotide polymorphisms (SNPs) in FETUB locus and atherosclerosis detection, was conducted in Xiamen, China. RESULTS: Increasing tertiles of brachial ankle pulse wave velocity (ba-PWV) were significantly associated with higher prevalence of nonalcoholic fatty liver disease (NAFLD) (48.8%, 61.5%, and 70.5% for tertiles of 1-3, respectively, p＜0.001) and serum Fetuin-B (3.85±1.39, 4.09±1.40, and 4.27±1.46 µg/ml, p=0.047). Multivariable linear regression analyses with adjustment for potential confounding factors, even NAFLD per se, showed that serum Fetuin-B were significantly and positively associated with ba-PWV, with standardized regression coefficients (ß) ranging from 0.055 to 0.075 (all p-values ＜0.05) in different models. However, the significant relationship between serum Fetuin-B and ba-PWV disappeared with further adjustment for insulin resistance. Serum Fetuin-B was not significantly associated with ankle-brachial index (ABI). All genotypes of the four tested FETUB tagging SNPs were not significantly associated with either ba-PWV or ABI with adjustment for potential confounding factors. CONCLUSION: Serum Fetuin-B was positively associated with ba-PWV and may link liver fat accumulation to subclinical atherosclerosis via insulin resistance.

The C-terminal region of human plasma fetuin-B is dispensable for the raised-elephant-trunk mechanism of inhibition of astacin metallopeptidases.

Human fetuin-B plays a key physiological role in human fertility through its inhibitory action on ovastacin, a member of the astacin family of metallopeptidases. The inhibitor consists of tandem cystatin-like domains (CY1 and CY2), which are connected by a linker containing a "CPDCP-trunk" and followed by a C-terminal region (CTR) void of regular secondary structure. Here, we solved the crystal structure of the complex of the inhibitor with archetypal astacin from crayfish, which is a useful model of human ovastacin. Two hairpins from CY2, the linker, and the tip of the "legumain-binding loop" of CY1 inhibit crayfish astacin following the "raised-elephant-trunk mechanism" recently reported for mouse fetuin-B. This inhibition is exerted by blocking active-site cleft sub-sites upstream and downstream of the catalytic zinc ion, but not those flanking the scissile bond. However, contrary to the mouse complex, which was obtained with fetuin-B nicked at a single site but otherwise intact, most of the CTR was proteolytically removed during crystallization of the human complex. Moreover, the two complexes present in the crystallographic asymmetric unit diverged in the relative arrangement of CY1 and CY2, while the two complexes found for the mouse complex crystal structure were equivalent. Biochemical studies in vitro confirmed the differential cleavage susceptibility of human and mouse fetuin-B in front of crayfish astacin and revealed that the cleaved human inhibitor blocks crayfish astacin and human meprin α and ß only slightly less potently than the intact variant. Therefore, the CTR of animal fetuin-B orthologs may have a function in maintaining a particular relative orientation of CY1 and CY2 that nonetheless is dispensable for peptidase inhibition.

Serum and follicular fluid fetuin-B levels are correlated with fertilization rates in conventional IVF cycles.

PURPOSE: To investigate the relationship between serum/follicular fluid fetuin-B levels and fertilization outcomes in conventional IVF cycles. METHODS: A prospective cohort study of conventional IVF treatments including 78 cycles with low fertilization rates (two pronuclei [2PN] rate < 30%; LF group) and 104 cycles performed during the same period with 2PN rate > 70% (high fertilization group, HF). To calculate the required sample size, a two-sample t test power analysis was applied to data from our pilot study, using PASS 11.0 software. Fetuin-B was measured using a commercial sandwich enzyme-linked immunosorbent assay. RESULTS: Serum fetuin-B and follicular fluid fetuin-B were positively correlated (r = 0.703, P < 0.001). Compared to the HF group, the LF group had significantly lower levels of fetuin-B, both in serum (5.81 ± 1.53 vs. 7.19 ± 1.42, P < 0.001) and follicular fluid (5.06 ± 1.29 vs. 6.16 ± 1.52, P < 0.001). The serum fetuin-B level from cycles with polypronuclear (PPN) zygotes was significantly lower when compared to cycles without PPN zygotes (6.82 ± 1.65 vs. 6.10 ± 1.43, P = 0.006). However, serum fetuin-B level was not correlated with preimplantation embryo development or clinical pregnancy. CONCLUSION: Serum fetuin-B level is correlated with fertilization rate in conventional IVF and it may be used as a predictive marker of fertilization in IVF treatment.

Changes in plasma concentrations and mRNA expression of hepatokines fetuin A, fetuin B and FGF21 in physiological pregnancy and gestational diabetes mellitus.

We measured plasma concentrations, adipose tissue and placental mRNA expression of hepatokines fetuin A, fetuin B and fibroblast growth factor 21 (FGF21) in 12 healthy pregnant women (P group), 12 pregnant women with gestational diabetes (GDM) and 10 healthy non-pregnant women (N group) to explore their potential role in the etiopathogenesis of GDM. GDM and P group had comparable BMI, C-reactive protein (CRP) and glycated hemoglobin levels while IL-10 and TNF-alpha levels were higher in GDM group. Fetuin A and fetuin B levels were higher in pregnancy as compared to N group and decreased after delivery with no apparent influence of GDM. In contrast, the pattern of changes of circulating FGF21 levels differed between GDM and P group. Fetuin A concentrations positively correlated with CRP, TNF-alpha mRNA expression in adipose tissue and IL-6 mRNA expression in placenta. Fetuin B positively correlated with CRP. FGF21 levels correlated positively with IFN-gamma mRNA in adipose tissue and inversely with IL-8 mRNA in the placenta. Taken together, fetuin A and fetuin B levels were increased during pregnancy regardless of the presence of GDM. In contrast, FGF21 patterns differed between healthy pregnant women and GDM patients suggesting a possible role of this hepatokine in the etiopathogenesis of GDM.

Fetuin B links vitamin D deficiency and pediatric obesity: Direct negative regulation by vitamin D.

Vitamin D (VD) deficiency (VDD) correlates to obesity, with VD a recognized mediator of metabolic diseases. From a previous proteomic study identifying adiponectin as a link between VDD and pediatric obesity, herein we analysed another protein (SSP2301) increased with VDD. A focused 2D-electrophoretic analysis identified 4 corresponding plasma proteins, with one predicted to be fetuin B (FETUB). FETUB was studied due to its emerging role in metabolic diseases and cytogenetic location (3q27.3) with adiponectin. Results were confirmed in obese children, where plasma FETUB was higher with VDD. A direct effect by 1α,25-(OH)2D3 on hepatocellular FETUB synthesis was observed, with a time and dose dependent reduction. Further, we demonstrated the VD-receptor (VDR) is key, with FETUB "released" with VDR silencing. Finally, VD supplementation (6weeks) to juvenile mice fed a standard diet, reduced plasma FETUB. Only at 22weeks did liver FETUB correspond to plasma FETUB, highlighting the contribution of other VD-responsive tissues. Overall, FETUB is a key protein linking VDD to pediatric obesity. With an emerging role in metabolic diseases, we demonstrate that VD/VDR directly regulate FETUB.

The rs4686434 variant in the fetuin B (FETUB) locus is associated with intrahepatic triglyceride content in obese Chinese adults.

BACKGROUND: This study explored associations of genetic variants in the fetuin B (FETUB) locus with intrahepatic triglyceride (IHTG) content. METHODS: Four tagging single-nucleotide polymorphisms (SNPs) of the FETUB locus and patatin-like phospholipase domain containing 3 (PNPLA3) rs738409 and transmembrane 6 super family member 2 (TM6SF2) rs58542926 were genotyped in 418 obese Chinese adults in whom serum FETUB and IHTG were measured. RESULTS: Subjects carrying the minor G allele for FETUB rs4686434 (AG/GG) had lower serum FETUB levels (mean [±SD] 3.89 ± 1.36 vs 4.22 ± 1.46 µg/mL; P = 0.021) and IHTG content (12.7 ± 9.4% vs 14.6 ± 9.8%; P = 0.045) than their controls (AA), whereas IHTG content was higher in those carrying the minor G allele for PNPLA3 rs738409 (CG/GG) than in their controls (CC; 14.5 ± 10.1% vs 12.0 ± 8.6%; P = 0.012). After adjusting for potential confounders, IHTG content was lower in carriers of the minor G allele for FETUB rs4686434 (AG/GG vs AA, ß -2.27 ± 0.91, P = 0.012), but was higher in carriers of the minor G allele for PNPLA3 rs738409 (CG/GG vs CC, ß 2.65 ± 0.97, P = 0.006). There was a significant joint effect between FETUB rs4686434 and PNPLA3 rs738409 on IHTG content, with increasing genetic risk score (counting the risk allele of A in rs4686434 and G in rs738409) being associated with higher IHTG content (ß 1.85 ± 0.48, P <0.001). CONCLUSIONS: Carrying the minor G allele for FETUB rs4686434 was significantly associated with decreased IHTG content and may affect hepatic triglyceride accumulation in individuals at high risk of non-alcoholic fatty liver disease.

The hepatokines fetuin-A and fetuin-B are upregulated in the state of hepatic steatosis and may differently impact on glucose homeostasis in humans.

The liver is a central regulator of whole body glucose, and lipid homeostasis and hepatokines, like fetuin-A, have been identified as markers and mediators of fatty liver-induced cardiometabolic risk. The closely related protein fetuin-B was shown to be upregulated in the fatty liver and to impact on glucose homeostasis in mice. In the present study we aimed to test the relevance of these findings in humans. In 55 subjects, hepatic mRNA expression of both hepatokines, fetuin-A and fetuin-B, associated positively with liver triglyceride content, whereas only fetuin-A expression associated with the homeostatic model assessment of insulin resistance. In 220 subjects who underwent precise metabolic phenotyping, circulating fetuin-A, but not fetuin-B, associated positively with liver fat content, and negatively with insulin sensitivity, measured during the oral glucose tolerance test (OGTT) and during the euglycemic, hyperinsulinemic clamp. Both circulating fetuin-A and fetuin-B correlated positively with the glucose area under the curve during the OGTT, but after additional adjustment for insulin sensitivity this relationship remained significant only for fetuin-B. In conclusion, despite the fact that the two hepatokines, fetuin-A and fetuin-B, are upregulated in the state of hepatic steatosis in humans, it appears that they differently impact on glucose homeostasis. Our data are in agreement with observations that fetuin-A can alter insulin signaling and that fetuin-B may regulate glucose homeostasis via so far unknown effects, possibly on glucose effectiveness.

Intracellular activation of ovastacin mediates pre-fertilization hardening of the zona pellucida.

STUDY QUESTION: How and where is pro-ovastacin activated and how does active ovastacin regulate zona pellucida hardening (ZPH) and successful fertilization? STUDY FINDING: Ovastacin is partially active before exocytosis and pre-hardens the zona pellucida (ZP) before fertilization. WHAT IS KNOWN ALREADY: The metalloproteinase ovastacin is stored in cortical granules, it cleaves zona pellucida protein 2 (ZP2) upon fertilization and thereby destroys the ZP sperm ligand and triggers ZPH. Female mice deficient in the extracellular circulating ovastacin-inhibitor fetuin-B are infertile due to pre-mature ZPH. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: We isolated oocytes from wild-type and ovastacin-deficient (Astlnull) FVB mice before and after fertilization (in vitro and in vivo) and quantified ovastacin activity and cleavage of ZP2 by immunoblot. We assessed ZPH by measuring ZP digestion time using α-chymotrypsin and by determining ZP2 cleavage. We determined cellular distribution of ovastacin by immunofluorescence using domain-specific ovastacin antibodies. Experiments were performed at least in triplicate with a minimum of 20 oocytes. Data were pre-analyzed using Shapiro-Wilk test. In case of normal distribution, significance was determined via two-sided Student's t-test, whereas in case of non-normal distribution via Mann-Whitney U-test. MAIN RESULTS AND THE ROLE OF CHANCE: Metaphase II (MII) oocytes contained both inactive pro-ovastacin and activated ovastacin. Immunoblot and ZP digestion assays revealed a partial cleavage of ZP2 even before fertilization in wild-type mice. Partial cleavage coincided with germinal-vesicle breakdown and MII, despite the presence of fetuin-B protein, an endogenous ovastacin inhibitor, in the follicular and oviductal fluid. Upon exocytosis, part of the C-terminal domain of ovastacin remained attached to the plasmalemma, while the N-terminal active ovastacin domain was secreted. This finding may resolve previously conflicting data showing that ovastacin acts both as an oolemmal receptor termed SAS1B (sperm acrosomal SLLP1 binding protein; SLLP, sperm lysozyme like protein) and a secreted protease mediating ZP2 cleavage. LIMITATIONS, REASONS FOR CAUTION: For this study, only oocytes isolated from wild-type and ovastacin-deficient FVB mice were investigated. Some experiments involved oocyte activation by the Ca2+ ionophore A23187 to trigger ZPH. WIDER IMPLICATIONS OF THE FINDINGS: This study provides a detailed spatial and temporal view of pre-mature cleavage of ZP2 by ovastacin, which is known to adversely affect IVF rate in mice and humans. LARGE SCALE DATA: None. STUDY FUNDING AND COMPETING INTEREST(S): This work was supported by the Center of Natural Sciences and Medicine and by a start-up grant of the Johannes Gutenberg University Mainz to W.S., and by a grant from Deutsche Forschungsgemeinschaft and by the START program of the Medical Faculty of RWTH Aachen University to J.F. and W.J.D. There are no competing interests to declare.

Identification and expression analysis of fetuin B (FETUB) in turbot (Scophthalmus maximus L.) mucosal barriers following bacterial challenge.

Fetuin B (FETUB), a recently described cysteine proteinase inhibitor, has numerous conserved N-glycosylation sites, species-specific O-glycosylation sites, and two cystatin (CY) domains. FETUB is likely to play regulatory roles in acute inflammation, female infertility, fish organogenesis and tumor suppression. In the present study, transcript of turbot FETUB gene was captured, its protein structure and expression patterns in different tissues with emphasis on mucosal barriers following different bacterial infection were characterized. Turbot FETUB gene showed the closest relationship with Takifugu rubripes in phylogenetic analysis. In addition, FETUB was ubiquitously expressed in all examined tissues with the highest expression level in skin. Finally, FETUB gene showed different expression patterns following both bacterial challenge. The rapidly and significantly differential expression patterns of FETUB in mucosal surfaces against bacterial infections might indicate its key roles to prevent pathogen attachment and entry in turbot mucosal immunity. Functional studies should be carried out to further characterize the FETUB and avail utilization of its function to increase the disease resistance of turbot in maintaining the integrity of the mucosal barriers against infections and to facilitate selection of the fine family/varieties of disease resistance in turbot.

Identification and characterization of secreted factors that are upregulated during somatic cell reprogramming.

The process of cell reprogramming has been characterized considerably since the successful generation of induced pluripotent stem cells. However, the importance of cell-cell communications for cellular reprogramming remains largely unknown. Secreted factors, which are expressed and secreted during reprogramming, may influence the reprogramming efficiency. Here, we have identified Sostdc1, Glb1l2, Fetub, Dpp4, Gdf3, Trh, and Tdgf1 as prominently upregulated secreted factors during reprogramming. Our detailed analysis reveals that these seven factors may be categorized into four groups based on their expression patterns in relation to the reprogramming stages. Remarkably, knockdown of Sostdc1, which is the most prominently upregulated factor and which is expressed earlier than the other six factors, results in reduced reprogramming efficiency, suggesting its involvement in the reprogramming process.

Down-regulation of the liver-derived plasma protein fetuin-B mediates reversible female infertility.

STUDY QUESTION: Does antisense oligonucleotide (ASO)-mediated down-regulation of serum fetuin-B cause infertility like fetuin-B gene deficiency in female mice? SUMMARY ANSWER: Pharmacological fetuin-B down-regulation by ASO therapy results in reversible infertility in female mice. WHAT IS KNOWN ALREADY: Female fetuin-B deficient (Fetub-/-) mice are infertile owing to premature zona pellucida (ZP) hardening. Enzyme activity studies demonstrated that fetuin-B is a potent and highly specific inhibitor of the zona proteinase ovastacin, which cleaves ZP protein 2 (ZP2) and thus mediates definitive ZP hardening. STUDY DESIGN, SIZE, DURATION: Ten fetuin-B ASO boli (100 mg/kg) were injected s.c. over 20 days in 12 female mice, and 10 phosphate-buffered saline (PBS)-treated mice were used as control. At day 20 females were mated to evaluate fetuin-B as a potential molecular target for contraception. ASO and PBS treatment was continued for ten injections. After treatment cessation at day 50, mating was continued to investigate if infertility was reversible. PARTICIPANTS/MATERIALS, SETTING, METHODS: We generated fetuin-B/ovastacin double deficient (Fetub-/-, Astl-/-) mice by conventional breeding to test if fertility of Fetub-/- female mice was restored when the target proteinase would likewise be deleted. At least five matings with each female genotype (Fetub-/- single deficient, Astl-/- single deficient, Fetub-/-, Astl-/- double deficient) were performed. To test the contraceptive effect of fetuin-B down-regulation, 22 female mice (6-13 weeks old) were treated with repetitive boli of 100 mg/kg fetuin-B ASO (n = 12) or PBS (n = 10) and mated continuously. Serum fetuin-B was determined by immunoblot before, during and after the ASO treatment. After 3 weeks of ASO treatment, in 6 females Fetub mRNA in liver was analyzed by PCR, and six PBS-treated females were used as control. Aspartate (AST) and alanine aminotransferase (ALT) were also measured in serum of six mice in each group. To determine the minimum permissive serum fetuin-B concentration required for successful fertilization IVF was performed in five fetuin-B ASO-treated mice. As a control, six females were injected with control oligonucleotides and six females were left untreated. MAIN RESULTS AND THE ROLE OF CHANCE: Fertility of Fetub-/- female mice was restored by additional ovastacin deficiency (Astl-/-). Unlike Fetub-/- mice, female Fetub-/-, Astl-/- mice were fertile, confirming ovastacin as a primary molecular target of fetuin-B. At day 20, after receiving 10 fetuin-B ASO boli, serum fetuin-B was down-regulated to 8 ± 6% (mean ± SD) of baseline level. Fetuin-B down-regulation was confirmed at the mRNA level. Fetuin-B ASO-treated females had 12.1 ± 3.1% of the liver Fetub mRNA level seen in PBS-treated females. In the following mating study, 11 out of 12 mated females failed to become pregnant during 50 days of ASO treatment and continuous mating from day 20 onwards. IVF of oocytes derived from ASO-treated females suggested that a serum fetuin-B level of less than 10 µg/ml was required to prevent pregnancy. Withdrawal of ASO treatment normalized serum fetuin-B and restored fertility; all female mice became pregnant and had litters within 60.3 ± 35.9 days after cessation of ASO treatment. The first litter was significantly smaller than that of control mice (4.6 ± 2.3 versus 6.7 ± 1.8 pups, n = 20, P = 0.04) but the smaller litter size was only temporary. The size of the second litter was similar to the first litter of control mice (7.6 ± 1.3 versus 6.7 ± 1.8 pups, n = 18, P = 0.25). LIMITATIONS, REASONS FOR CAUTION: The repeated dose of 100 mg/kg fetuin-B ASO boli caused an increased serum ALT and AST activity, suggesting hepatotoxicity. Daily vaginal plug checks indicated successful mating, but mating plugs in ASO-treated mice were less stable (vaginal tract not closed) than in control mice. WIDER IMPLICATIONS OF THE FINDINGS: Pharmacological fetuin-B down-regulation in mice caused reversible infertility. Control of ovastacin proteinase activity by fetuin-B is a necessary determinant of female fertility that can serve as a target for female contraception. Although promising in terms of human contraception, further studies analyzing the balance between sufficient fetuin-B down-regulation and tolerable side effects are required to improve safety before transfer into human reproductive biology can be considered. LARGE SCALE DATA: None. STUDY FUNDING AND COMPETING INTERESTS: The research was supported by a grant from Deutsche Forschungsgemeinschaft and by the START program of the Medical Faculty of RWTH Aachen University. The authors E.D., J.F. and W.J.-D. are named inventors on a patent application of RWTH Aachen University covering the use of fetuin-B in ovary and oocyte culture. No conflict of interest is declared by C.S. and A.C.

Common genetic variants in the FETUB locus, genetically predicted fetuin-B levels, and risk of insulin resistance in obese Chinese adults.

Elevated serum fetuin-B is suggested to be associated with insulin resistance, but it is unknown if this association is causal. The aim of this study was to explore the potential causal relationship between fetuin-B and insulin resistance.We used Mendelian randomization analysis by incorporating information of genetic variants in FETUB and serum fetuin-B concentrations with insulin resistance in 1148 obese Chinese adults.Common genetic variants (FETUB rs4686434, rs6785067, and rs3733159) were significantly associated with serum fetuin-B concentrations but not with insulin resistance. Higher serum fetuin-B levels were significantly associated with increased homeostasis model assessment of insulin resistance (HOMA-IR) (0.17 [95%CI: 0.01 to 0.32, P = .037] 10 mol IU L higher per SD). However, Mendelian randomization analysis using 3 single-nucleotide polymorphisms as instrumental variables did not support a significant association between genetically predicted fetuin-B levels and HOMA-IR (-0.09 [95%CI: -0.62 to 0.44, P = .738] 10 mol IU L lower per SD). The regression coefficients for measured and genetically predicted fetuin-B concentrations on HOMA-IR were significantly different (P <.001).This study suggests the association between fetuin-B and insulin resistance may not be causal. Future studies on the nongenetic determinants of serum fetuin-B concentration to assess if such unmeasured factors may confound the association between fetuin-B and insulin resistance as well as more pathway analysis for this association are warranted.

The Central Role of KNG1 Gene as a Genetic Determinant of Coagulation Pathway-Related Traits: Exploring Metaphenotypes.

Traditional genetic studies of single traits may be unable to detect the pleiotropic effects involved in complex diseases. To detect the correlation that exists between several phenotypes involved in the same biological process, we introduce an original methodology to analyze sets of correlated phenotypes involved in the coagulation cascade in genome-wide association studies. The methodology consists of a two-stage process. First, we define new phenotypic meta-variables (linear combinations of the original phenotypes), named metaphenotypes, by applying Independent Component Analysis for the multivariate analysis of correlated phenotypes (i.e. the levels of coagulation pathway-related proteins). The resulting metaphenotypes integrate the information regarding the underlying biological process (i.e. thrombus/clot formation). Secondly, we take advantage of a family based Genome Wide Association Study to identify genetic elements influencing these metaphenotypes and consequently thrombosis risk. Our study utilized data from the GAIT Project (Genetic Analysis of Idiopathic Thrombophilia). We obtained 15 metaphenotypes, which showed significant heritabilities, ranging from 0.2 to 0.7. These results indicate the importance of genetic factors in the variability of these traits. We found 4 metaphenotypes that showed significant associations with SNPs. The most relevant were those mapped in a region near the HRG, FETUB and KNG1 genes. Our results are provocative since they show that the KNG1 locus plays a central role as a genetic determinant of the entire coagulation pathway and thrombus/clot formation. Integrating data from multiple correlated measurements through metaphenotypes is a promising approach to elucidate the hidden genetic mechanisms underlying complex diseases.

Fetuin B Is a Secreted Hepatocyte Factor Linking Steatosis to Impaired Glucose Metabolism.

Liver steatosis is associated with the development of insulin resistance and the pathogenesis of type 2 diabetes. We tested the hypothesis that protein signals originating from steatotic hepatocytes communicate with other cells to modulate metabolic phenotypes. We show that the secreted factors from steatotic hepatocytes induce pro-inflammatory signaling and insulin resistance in cultured cells. Next, we identified 168 hepatokines, of which 32 were differentially secreted in steatotic versus non-steatotic hepatocytes. Targeted analysis showed that fetuin B was increased in humans with liver steatosis and patients with type 2 diabetes. Fetuin B impaired insulin action in myotubes and hepatocytes and caused glucose intolerance in mice. Silencing of fetuin B in obese mice improved glucose tolerance. We conclude that the protein secretory profile of hepatocytes is altered with steatosis and is linked to inflammation and insulin resistance. Therefore, preventing steatosis may limit the development of dysregulated glucose metabolism in settings of overnutrition.

The serum protein fetuin-B is involved in the development of acute myocardial infarction.

The rupture of an atherosclerotic plaque is one of the main causes of coronary artery thrombotic occlusion, leading to myocardial infarction. However, the exact mechanism and causal risk factors for plaque rupture remain unclear. To identify a potential molecule that can influence atherosclerotic plaque rupture, we investigated protein expression in serum from patients with acute myocardial infarction (AMI) and stable angina (SA), using proteomic analysis. The expression of six proteins, including fibrinogen, fetuin-B, keratin 9, proapolipoprotein and fibrinogen, were altered in serum from patients with AMI compared with serum from those with SA. Of these, fetuin-B, proapolipoprotein, fibrinogen γ-B-chain precursors and fibrinogen expression were greater in serum from patients with AMI than from patients with SA. Increased fetuin-B expression in serum from AMI patients was also confirmed by Western blot analysis. Treatment with recombinant human fetuin-B increased the migration in monocytes and macrophages in a concentration-dependent manner. Fetuin-B also affected vascular plaque-stabilizing factors, including lipid deposition and cytokine production in macrophages, the activation of matrix metalloproteinase (MMP)-2 in monocytes, and the activation of apoptosis and MMP-2 in vascular smooth muscle cells. Moreover, in vivo administration of fetuin-B decreased the collagen accumulation and smooth muscle cell content and showed an increased number of macrophages in the vascular plaque. From these results, we suggest that fetuin-B may act as a modulator in the development of AMI. This study may provide a therapeutic advantage for patients at high risk of AMI.