Hydroxysteroid (17β) dehydrogenases (HSD17Bs) form an enzyme family characterized by their ability to catalyze reactions in steroid and lipid metabolism. In the present study, we characterized the phenotype of HSD17B13-knockout (HSD17B13KO) mice deficient in Hsd17b13. In these studies, hepatic steatosis was detected in HSD17B13KO male mice, indicated by histologic analysis and by the increased triglyceride concentration in the liver, whereas reproductive performance and serum steroid concentrations were normal in HSD17B13KO mice. In line with these changes, the expression of key proteins in fatty acid synthesis, such as FAS, acetyl-CoA carboxylase 1, and SCD1, was increased in the HSD17B13KO liver. Furthermore, the knockout liver showed an increase in 2 acylcarnitines, suggesting impaired mitochondrial β-oxidation in the presence of unaltered malonyl CoA and AMPK expression. The glucose tolerance did not differ between wild-type and HSD17B13KO mice in the presence of lower levels of glucose 6-phosphatase in HSD17B13KO liver compared with wild-type liver. Furthermore, microgranulomas and increased portal inflammation together with up-regulation of immune response genes were observed in HSD17B13KO mice. Our data indicate that disruption of Hsd17b13 impairs hepatic-lipid metabolism in mice, resulting in liver steatosis and inflammation, but the enzyme does not play a major role in the regulation of reproductive functions.-Adam, M., Heikelä, H., Sobolewski, C., Portius, D., Mäki-Jouppila, J., Mehmood, A., Adhikari, P., Esposito, I., Elo, L. L., Zhang, F.-P., Ruohonen, S. T., Strauss, L., Foti, M., Poutanen, M. Hydroxysteroid (17β) dehydrogenase 13 deficiency triggers hepatic steatosis and inflammation in mice.

Hydroxysteroid (17b)-dehydrogenase type 1 (HSD17B1) catalyzes the conversion of low active 17-ketosteroids, androstenedione (A-dione) and estrone (E1) to highly active 17-hydroxysteroids, testosterone (T) and E2, respectively. In this study, the importance of HSD17B1 in ovarian estrogen production was determined using Hsd17b1 knockout (HSD17B1KO) mice. In these mice, the ovarian HSD17B enzyme activity was markedly reduced, indicating a central role of HSD17B1 in ovarian physiology. The lack of Hsd17b activity resulted in increased ovarian E1:E2 and A-dione:T ratios, but we also observed reduced progesterone concentration in HSD17B1KO ovaries. Accordingly with the altered steroid production, altered expression of Star, Cyp11a1, Lhcgr, Hsd17b7, and especially Cyp17a1 was observed. The ovaries of HSD17B1KO mice presented with all stages of folliculogenesis, while the corpus luteum structure was less defined and number reduced. Surprisingly, bundles of large granular cells of unknown origin appeared in the stroma of the KO ovaries. The HSD17B1KO mice presented with severe subfertility and failed to initiate pseudopregnancy. However, the HSD17B1KO females presented with normal estrous cycle defined by vaginal smears and normal puberty appearance. This study indicates that HSD17B1 is a key enzyme in ovarian steroidogenesis and has a novel function in initiation and stabilization of pregnancy.-Hakkarainen, J., Jokela, H., Pakarinen, P., Heikelä, H., Kätkänaho, L., Vandenput, L., Ohlsson, C., Zhang, F.-P., Poutanen, M. Hydroxysteroid (17b)-dehydrogenase 1-deficient female mice present with normal puberty onset but are severely subfertile due to a defect in luteinization and progesterone production. FASEB J. 29, 3806-3816 (2015). www.fasebj.org Key Words: ovary • estrogen • fertility • HSD17B enzymes HYDROXYSTEROID (17b)-DEHYDROGENASES (HSD17BS) ARE ENZYMES CATALYZING the conversion of low active 17-ketosteroids to the highly active 17b hydroxysteroids, and vice versa. Many of the enzymes catalyze both oxidative (17-hydroxy to 17-keto) and reductive (17-keto to 17-hydroxy) activity with a proper cofactor added in vitro. The conversion of 17-ketosteroid to the 17b-hydroxysteroid is an essential step in the formation of the classic highly active sex steroids E2 and testosterone (T). Most of the HSD17B enzymes belong to the family of short-chain dehydrogenases/ reductases, while some are members of the aldo-keto reductase family (1).All enzymes with HSD17B activity present with distinct cell-specific expression, different substrate specificities, and unique regulatory mechanisms (2, 3). Current evidence indicates differential physiologic roles of the various HSD17B enzymes, and it is expected that some of the HSD17B enzymes act also in metabolic pathways other than those involved in sex steroid metabolism. Among those, HSD17B4 has been shown to be involved in b--oxidation of fatty acids (1, 4), HSD17B7 in cholesterol biosynthesis (5), and HSD17B10 and -12 in fatty acid metabolism (6).The human HSD17B1 enzyme prefera...

Contractile smooth muscle-like peritubular cells build the wall of seminiferous tubules in men. They are crucial for sperm transport and complement the functions of Sertoli cells by secreting factors, including glial cell line-derived neurotrophic factor. Previous studies revealed that they also secrete the chemokine C-X-C motif chemokine ligand 12 (CXCL12), which has known roles in spermatogenesis. Peritubular cells express the androgen receptor (AR), which is retained in isolated human testicular peritubular cells. We aimed to explore AR-regulated functions in human testicular peritubular cells. Bearing in mind that infertile men often have high aromatase activity, which may lower intratesticular androgen concentrations, an animal model for male infertility was studied. These mice display an age-dependent loss in spermatogenesis due to high aromatase activity. Human testicular peritubular cells were exposed to dihydrotestosterone or the antiandrogen flutamide. We studied AR, smooth muscle cell markers, glial cell line-derived neurotrophic factor and 15 secreted factors previously identified, including CXCL12. We used qPCR, Western blotting, ELISA or selected reaction monitoring (SRM). In the animal model for male infertility, we employed qPCR and immunohistochemistry. Dihydrotestosterone increased AR and flutamide prevented these actions. The smooth muscle cell markers calponin and smooth muscle actin were likewise increased, while cell size or cellular proliferation was not changed. Dihydrotestosterone did not increase glial cell line-derived neurotrophic factor or CXCL12 secretion but increased levels of serine proteinase inhibitor (SERPIN) E1. The animal model for male infertility with high aromatase activity showed reduced numbers of AR-immunoreactive testicular peritubular cells, suggesting that altered androgen and/or oestrogen levels could influence AR-mediated responses in peritubular cells. Androgens act on human testicular peritubular cells to enhance AR levels, their contractile phenotype and to modulate the secretion of some secreted factors. This study suggests that some aspects of human peritubular cell functions are regulated by androgens.

Hydroxysteroid 17-beta dehydrogenase 12 (HSD17B12) is suggested to be involved in the elongation of very long chain fatty acids. Previously, we have shown a pivotal role for the enzyme during mouse development. In the present study we generated a conditional Hsd17b12 knockout (HSD17B12cKO) mouse model by breeding mice homozygous for a floxed Hsd17b12 allele with mice expressing the tamoxifen-inducible Cre recombinase at the ROSA26 locus. Gene inactivation was induced by administering tamoxifen to adult mice. The gene inactivation led to a 20% loss of body weight within six days, associated with drastic reduction in both white (83% males, 75% females) and brown (65% males, 60% females) fat, likely due to markedly reduced food and water intake. Furthermore, the knockout mice showed sickness behavior and signs of liver toxicity, specifically microvesicular hepatic steatosis and increased serum alanine aminotransferase (4.6-fold in males, 7.7-fold in females). The hepatic changes were more pronounced in females than males. Pro-inflammatory cytokines, such as interleukin 6 (IL-6), IL-17 and granulocyte-colony stimulating factor were increased in the HSD17B12cKO mice indicating inflammatory response. Serum lipidomics study showed an increase in the amount of dihydroceramides, despite the dramatic overall loss of lipids. In line with the proposed role for HSD17B12 in the fatty acid elongation, we observed accumulation of ceramides, dihydroceramides, hexosylceramides and lactosylceramides with shorter than 18-carbon fatty acid side chains in the serum. The results indicate that HSD17B12 is essential for proper lipid homeostasis, and HSD17B12 deficiency rapidly results in fatal systemic inflammation and lipolysis in adult mice.

Extracellular ATP has been described to be involved in inflammatory cytokine production by human testicular peritubular cells (HTPCs). The ectonucleotidases ENTPD1 and NT5E degrade ATP and have been reported in rodent testicular peritubular cells. We hypothesised that if a similar situation exists in human testis, ATP metabolites may contribute to cytokine production. Indeed, ENTPD1 and NT5E were found in situ and in vitro in HTPCs. Malachite green assays confirmed the enzyme activities in HTPCs. Pharmacological inhibition of ENTPD1 (by POM-1) significantly reduced pro-inflammatory cytokines evoked by ATP treatment, suggesting that metabolites of ATP, including adenosine, are likely involved. We focused on adenosine and detected three of the four known adenosine receptors in HTPCs. One, A2B, was also found in situ in peritubular cells of human testicular sections. The A2B agonist BAY60-6583 significantly elevated levels of IL6 and CXCL8, a result also obtained with adenosine and its analogue NECA. Results of siRNA-mediated A2B down-regulation support a role of this receptor. In mouse peritubular cells, in contrast to HTPCs, all four of the known adenosine receptors were detected; when challenged with adenosine, cytokine expression levels significantly increased. Organotypic short-term testis cultures yielded comparable results and indicate an overall pro-inflammatory action of adenosine in the mouse testis. If transferable to the in-vivo situation, our results may implicate that interference with the generation of ATP metabolites or interference with adenosine receptors could reduce inflammatory events in the testis. These novel insights may provide new avenues for treatment of sterile inflammation in male subfertility and infertility.

Hydroxysteroid 17‐beta dehydrogenase (HSD17B) enzymes have an important role in the regulation of sex steroid metabolism by catalyzing the conversion between 17‐keto and 17‐hydroxysteroids. In addition, some HSD17B enzymes are involved in lipid metabolism. Our data together with other previous studies suggest that HSD17B12 is involved in the elongation of very long chain fatty acids (VLCFAs), presumably in producing arachidonic acid. The enzyme presents with universal expression pattern both in human and mouse. Our previous studies have shown that the global knockout of Hsd17b12 gene in mice is embryonic lethal, which is likely due to its pivotal role in the elongation of VLCFAs during development. The aim of the present study was to determine the role of HSD17B12 at adulthood. We, thus, generated conditional Hsd17b12 knockout mice with exon 2 flanked by loxP sites. The gene was globally deleted by crossing the floxed mice with those expressing the tamoxifen‐inducible Cre recombinase in the Rosa26 locus (HSD17B12cKOrosa26 mice) and by providing tamoxifen treatment at the age of eight weeks. Interestingly, inducing the global gene deletion led to 20% reduction in body weight, associated with drastically reduced amount of both white and brown adipose tissue. Our results with indirect calorimetry showed that food intake and water consumption were dramatically reduced (44% and 65%, respectively) in HSD17B12cKOrosa26 mice, although the hypothalamic regulation of feeding behavior was intact. The loss of appetite might be a consequence of general indisposition of the animals, however, no differences in motor activity was observed between KO and control mice. To study whether the strong metabolic phenotype is caused by an altered adipose tissue function, we then generated an inducible, adipocyte‐specific Hsd17b12 KO model (HSD17B12cKOadipoq) by crossbreeding the floxed mice with those expressing the tamoxifen‐inducible Cre recombinase under adiponectin promoter (Adipoq‐cre/ERT2 mice). Surprisingly, the HSD17B12cKOadipoq mice were viable, presented with normal body weight, and were free of any obvious abnormalities. Because of the high hepatic expression of HSD17B12 in both human and mouse, we generated a liver‐specific knockout mouse by crossbreeding the floxed mouse strain with Albumin‐Cre mouse strain. These mice were viable but presented with lipid accumulation in the hepatocytes at a young age (2 months). The results indicate that HSD17B12 is essential for the metabolic homeostasis in adult mice, while the vital role of the enzyme in metabolic regulation is not due to the high HSD17B12 activity in the adipocytes or hepatocytes. Moreover, the hepatocyte‐specific deletion of Hsd17b12 leads to liver steatosis in mice.Support or Funding InformationThis research was funded by Sigrid Jusélius foundation, Academy of Finland and Drug Research Doctoral Program at the University of Turku.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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