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.

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.

Downregulation of fetuin-B and zinc-α2-glycoprotein is linked to impaired fatty acid metabolism in liver cells.

BACKGROUND: Our recent proteomic study has shown that plasma protein levels of fetuin-B (Ft-B) and zinc-α2-glycoprotein (ZAG) are significantly elevated in obesity-resistant (OR) rats exposed to a high fat diet. Time profiling of the plasma concentrations of Ft-B and ZAG in OR rats has shown stable regulation of these proteins throughout the entire period of rat breeding. METHODS: To firmly establish roles for these proteins in lipogenesis, we efficiently knocked down (KD) the genes FETUB and AZGP1 encoding Ft-B and ZAG, respectively, using siRNA in Chang liver cells. RESULTS: Reduced expression of FETUB and AZGP1 led to a significant increase in the expression of lipogenic genes, thereby resulting in higher lipid levels in both KD cells. Collectively with our previous findings, we confirmed that Ft-B was similarly regulated with Ft-A, in that their plasma protein levels were commonly reduced in diet-induced obese rats. CONCLUSION: Our results provide a possible relationship between reduced plasma protein levels of Ft-B and ZAG and higher risk of diet-induced obesity through impaired fatty acid metabolism in hepatocytes.