Engineering of a hydroxysteroid dehydrogenase with simultaneous enhancement in activity and thermostability for efficient biosynthesis of ursodeoxycholic acid.

Hydroxysteroid dehydrogenases (HSDHs) catalyze the oxidation/reduction of hydroxyl/keto groups of steroids with high regio- or stereoselectivity, playing an essential role in producing optically pure chemicals. In this work, a novel approach was developed to simultaneously improve the stability and activity of 7ß-hydroxysteroid dehydrogenase (7ß-HSDH) by combining B-factor analysis and computer-aided prediction. Several advantageous mutants were identified, and the most promising variant, S51Y/P202Y, exhibited 2.3-fold improvements in catalytic activity, 3.3-fold in half-life at 40°C, and 4.7-fold in catalytic efficiency (kcat/Km), respectively. Structural modeling analysis showed that the shortened reversible oxidation reaction catalytic distance and the strengthened residue interactions compared to the wild type were attributed to the improved stability and activity of the obtained mutants. To synthesize ursodeoxycholic acid cost-effectively by mutant S51Y/P202Y, a NAD-kinase was employed to facilitate the substitution of nicotinamide adenine dinucleotide phosphate (NADP+) with nicotinamide adenine dinucleotide (NAD+) in the whole-cell catalysis system. The substrate 7-ketolithocholic acid (100 mM) was converted completely in 0.5 h, achieving a space-time yield of 1,887.3 g L-1 d-1. This work provided a general target-oriented strategy for obtaining stable and highly active dehydrogenase for efficient biosynthesis. IMPORTANCE: Hydroxysteroid dehydrogenases have emerged as indispensable tools in the synthesis of steroids, bile acids, and other steroid derivatives for the pharmaceutical and chemical industries. In this study, a novel approach was developed to simultaneously improve the stability and activity of a hydroxysteroid dehydrogenase by combining B-factor analysis and computer-aided prediction. This semi-rational method was demonstrated to be highly effective for enzyme engineering. In addition, NAD kinase was introduced to convert NAD+ to NADP+ for effective coenzyme regeneration in the whole-cell multienzyme-catalyzed system. This strategy reduces the significant economic costs associated with externally supplemented cofactors in NADP-dependent biosynthetic pathways.

Not exclusively the activity, but the sweet spot: a dehydrogenase point mutation synergistically boosts activity, substrate tolerance, thermal stability and yield.

Catalytic activity is undoubtedly a key focus in enzyme engineering. The complicated reaction conditions hinder some enzymes from industrialization even though they have relatively promising activity. This has occurred to some dehydrogenases. Hydroxysteroid dehydrogenases (HSDHs) specifically catalyze the conversion between hydroxyl and keto groups, and hold immense potential in the synthesis of steroid medicines. We underscored the importance of 7α-HSDH activity, and analyzed the overall robustness and underlying mechanisms. Employing a high-throughput screening approach, we comprehensively assessed a mutation library, and obtained a mutant with enhanced enzymatic activity and overall stability/tolerance. The superior mutant (I201M) was identified to harbor improved thermal stability, substrate susceptibility, cofactor affinity, as well as the yield. This mutant displayed a 1.88-fold increase in enzymatic activity, a 1.37-fold improvement in substrate tolerance, and a 1.45-fold increase in thermal stability when compared with the wild type (WT) enzyme. The I201M mutant showed a 2.25-fold increase in the kcat/KM ratio (indicative of a stronger binding affinity for the cofactor). This mutant did not exhibit the highest enzyme activity compared with all the tested mutants, but these improved characteristics contributed synergistically to the highest yield. When a substrate at 100 mM was present, the 24 h yield by I201M reached 89.7%, significantly higher than the 61.2% yield elicited by the WT enzyme. This is the first report revealing enhancement of the catalytic efficiency, cofactor affinity, substrate tolerance, and thermal stability of NAD(H)-dependent 7α-HSDH through a single-point mutation. The mutated enzyme reached the highest enzymatic activity of 7α-HSDH ever reported. High enzymatic activity is undoubtedly crucial for enabling the industrialization of an enzyme. Our findings demonstrated that, when compared with other mutants boasting even higher enzymatic activity, mutants with excellent overall robustness were superior for industrial applications. This principle was exemplified by highly active enzymes such as 7α-HSDH.

Therapeutic potential of targeting AKR1C2 in the treatment of prostate cancer.

Prostate cancer development and progression are driven by androgens, and changes in androgen metabolic pathways can lead to prostate cancer progression or remission. AKR1C2 is a member of the aldo-keto reductase superfamily and plays an important role in the metabolism of steroids and prostaglandins. Alterations in the expression and activity of AKR1C2 affect the homeostasis of active androgens, which in turn affects the progression of prostate cancer. AKR1C2 reduces the highly active dihydrotestosterone to the less active 3α-diol in the prostate, resulting in lower androgen levels. Whereas the expression of AKR1C2 is significantly reduced in prostate cancer tissues relative to normal prostate tissues, this results in a weakening of the dihydrotestosterone metabolic inactivation pathway, leading to the retention of dihydrotestosterone in the prostate cancer cells, which promotes the progress of prostate cancer. Given the critical role of AKR1C2 in prostate cancer cells, targeting AKR1C2 for the treatment of prostate cancer may be an effective strategy. It has been demonstrated that curcumin and neem leaf extract effectively inhibit prostate cancer in vitro and in vivo by modulating AKR1C2.

Structure-based rational design of hydroxysteroid dehydrogenases for improving and diversifying steroid synthesis.

A group of steroidogenic enzymes, hydroxysteroid dehydrogenases are involved in steroid metabolism which is very important in the cell: signaling, growth, reproduction, and energy homeostasis. The enzymes show an inherent function in the interconversion of ketosteroids and hydroxysteroids in a position- and stereospecific manner on the steroid nucleus and side-chains. However, the biocatalysis of steroids reaction is a vital and demanding, yet challenging, task to produce the desired enantiopure products with non-natural substrates or non-natural cofactors, and/or in non-physiological conditions. This has driven the use of protein design strategies to improve their inherent biosynthetic efficiency or activate their silent catalytic ability. In this review, the innate features and catalytic characteristics of enzymes based on sequence-structure-function relationships of steroidogenic enzymes are reviewed. Combining structure information and catalytic mechanisms, progress in protein redesign to stimulate potential function, for example, substrate specificity, cofactor dependence, and catalytic stability are discussed.

Sequence and structure-guided discovery of a novel NADH-dependent 7ß-hydroxysteroid dehydrogenase for efficient biosynthesis of ursodeoxycholic acid.

7ß-Hydroxysteroid dehydrogenases (7ß-HSDHs) have attracted increasing attention due to their crucial roles in the biosynthesis of ursodeoxycholic acid (UDCA). However, most published 7ß-HSDHs are strictly NADPH-dependent oxidoreductases with poor activity and low productivity. Compared with NADPH, NADH is more stable and cheaper, making it the more popular cofactor for industrial applications of dehydrogenases. Herein, by using a sequence and structure-guided genome mining approach based on the structural information of conserved cofactor-binding motifs, we uncovered a novel NADH-dependent 7ß-HSDH (Cle7ß-HSDH). The Cle7ß-HSDH was overexpressed, purified, and characterized. It exhibited high specific activity (9.6 U/mg), good pH stability and thermostability, significant methanol tolerance, and showed excellent catalytic efficiencies (kcat/Km) towards 7-oxo-lithocholic acid (7-oxo-LCA) and NADH (70.8 mM-1s-1 and 31.8 mM-1s-1, respectively). Molecular docking and mutational analyses revealed that Asp42 could play a considerable role in NADH binding and recognition. Coupling with a glucose dehydrogenase for NADH regeneration, up to 20 mM 7-oxo-LCA could be completely transformed to UDCA within 90 min by Cle7ß-HSDH. This study provides an efficient approach for mining promising enzymes from genomic databases for cost-effective biotechnological applications.

Comparative study on the gene expression of corticosterone metabolic enzymes in embryonic tissues between Tibetan and broiler chickens.

Glucocorticoids (GCs) are an essential mediator hormone that can regulate animal growth, behavior, the phenotype of offspring, and so on, while GCs in poultry are predominantly corticosterones. The biological activity of GCs is mainly regulated by the intracellular metabolic enzymes, including 11ß-hydroxysteroid dehydrogenases 1 (11ß-HSD1), 11ß-hydroxysteroid dehydrogenases 2 (11ß-HSD2), and 20-hydroxysteroid dehydrogenase (20-HSD). To investigate the embryonic mechanisms of phenotypic differences between breeds, we compared the expression of corticosterone metabolic enzyme genes in the yolk-sac membrane and chorioallantoic membrane (CAM). We described the tissue distribution and ontogenic patterns of corticosterone metabolic enzymes during embryonic incubation between Tibetan and broiler chickens. Forty fertilized eggs from Tibetan and broiler chickens were incubated under hypoxic and normoxic conditions, respectively. Real-time fluorescence quantitative PCR was used to examine the expression of 11ß-HSD1/2, and 20-HSD mRNA in embryonic tissues. The results showed that the expression levels of yolk-sac membrane mRNA of 11ß-HSD2 and 20-HSD in Tibetan chickens on E14 (embryonic day of 14) were significantly lower than those of broiler chickens (P < 0.05), and these genes expression of CAM in Tibetan chickens were higher than those of broiler chickens (P < 0.05). In addition, the three genes in the yolk-sac membrane and CAM were followed by a down-regulation on E18 (embryonic day of 18). The 11ß-HSD1 and 11ß-HSD2 genes followed a similar tissue-specific pattern: the expression level was more abundantly in the liver, kidney, and intestine, with relatively lower abundance in the hypothalamus and muscle, and the expression level of 20-HSD genes in all tissues tested was higher. In the liver, 20-HSD of both Tibetan and broiler chickens showed different ontogeny development patterns, and hepatic mRNA expression of 20-HSD in broiler chickens was significantly higher than that of Tibetan chickens of the same age from E14 to E18 (P < 0.05). This study preliminarily revealed the expression levels of cortisol metabolic genes in different tissues during the development process of Tibetan and broiler chicken embryos. It provided essential information for in-depth research of the internal mechanism of maternal GCs programming on offspring.

Resistance to Resveratrol Treatment in Experimental PTSD Is Associated with Abnormalities in Hepatic Metabolism of Glucocorticoids.

Glucocorticoids are metabolized by the CYP3A isoform of cytochrome P450 and by 11-ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD-1). Experimental data suggest that post-traumatic stress disorder (PTSD) is associated with an increase in hepatic 11ß-HSD-1 activity and a concomitant decrease in hepatic CYP3A activity. Trans-resveratrol, a natural polyphenol, has been extensively studied for its antipsychiatric properties. Recently, protective effects of trans-resveratrol were found in relation to PTSD. Treatment of PTSD rats with trans-resveratrol allowed the rats to be divided into two phenotypes. The first phenotype is treatment-sensitive rats (TSR), and the second phenotype is treatment-resistant rats (TRRs). In TSR rats, trans-resveratrol ameliorated anxiety-like behavior and reversed plasma corticosterone concentration abnormalities. In contrast, in TRR rats, trans-resveratrol aggravated anxiety-like behavior and decreased plasma corticosterone concentration. In TSR rats, hepatic 11ß-HSD-1 activity was suppressed, with a concomitant increase in CYP3A activity. In TRR rats, the activities of both enzymes were suppressed. Thus, the resistance of PTSD rats to trans-resveratrol treatment is associated with abnormalities in hepatic metabolism of glucocorticoids. The free energy of binding of resveratrol, cortisol, and corticosterone to the human CYP3A protein was determined using the molecular mechanics Poisson-Boltzmann surface area approach, indicating that resveratrol could affect CYP3A activity.

SRSF3-Mediated Ki67 Exon 7-Inclusion Promotes Head and Neck Squamous Cell Carcinoma Progression via Repressing AKR1C2.

Ki67 is a well-known proliferation marker with a large size of around 350 kDa, but its biological function remains largely unknown. The roles of Ki67 in tumor prognosis are still controversial. Ki67 has two isoforms generated by alternative splicing of exon 7. The roles and regulatory mechanisms of Ki67 isoforms in tumor progression are not clear. In the present study, we surprisingly find that the increased inclusion of Ki67 exon 7, not total Ki67 expression level, was significantly associated with poor prognosis in multiple cancer types, including head and neck squamous cell carcinoma (HNSCC). Importantly, the Ki67 exon 7-included isoform is required for HNSCC cell proliferation, cell cycle progression, cell migration, and tumorigenesis. Unexpectedly, Ki67 exon 7-included isoform is positively associated with intracellular reactive oxygen species (ROS) level. Mechanically, splicing factor SRSF3 could promote exon 7 inclusion via its two exonic splicing enhancers. RNA-seq revealed that aldo-keto reductase AKR1C2 is a novel tumor-suppressive gene targeted by Ki67 exon 7-included isoform in HNSCC cells. Our study illuminates that the inclusion of Ki67 exon 7 has important prognostic value in cancers and is essential for tumorigenesis. Our study also suggested a new SRSF3/Ki67/AKR1C2 regulatory axis during HNSCC tumor progression.

HB-EGF upregulates StAR expression and stimulates progesterone production through ERK1/2 signaling in human granulosa-lutein cells.

BACKGROUND: Heparin-binding epidermal growth factor-like growth factor (HB-EGF) belongs to the epidermal growth factor (EGF) family of growth factors. HB-EGF and its receptors, epidermal growth factor receptor (EGFR) and HER4, are expressed in the human corpus luteum. HB-EGF has been shown to regulate luteal function by preventing cell apoptosis. Steroidogenesis is the primary function of the human corpus luteum. Steroidogenic acute regulatory protein (StAR) plays a critical role in steroidogenesis. StAR expression and progesterone (P4) production in human granulosa-lutein (hGL) cells have been shown to be upregulated by a ligand of EGFR, amphiregulin. However, whether HB-EGF can achieve the same effects remains unknown. METHODS: A steroidogenic human ovarian granulosa-like tumor cell line, KGN, and primary culture of hGL cells obtained from patients undergoing in vitro fertilization treatment were used as experimental models. The underlying molecular mechanisms mediating the effects of HB-EGF on StAR expression and P4 production were explored by a series of in vitro experiments. RESULTS: Western blot showed that EGFR, HER2, and HER4 were expressed in both KGN and hGL cells. Treatment with HB-EGF for 24 h induced StAR expression but did not affect the expression of steroidogenesis-related enzymes, P450 side chain cleavage enzyme, 3ß-hydroxysteroid dehydrogenase, and aromatase. Using pharmacological inhibitors and a siRNA-mediated knockdown approach, we showed that EGFR, HER4, but not HER2, were required for HB-EGF-stimulated StAR expression and P4 production. In addition, HB-EGF-induced upregulations of StAR expression and P4 production were mediated by the activation of the ERK1/2 signaling pathway. CONCLUSION: This study increases the understanding of the physiological role of HB-EGF in human luteal functions. Video Abstract.

Reconstruction of Acinetobacter johnsonii ICE_NC genome using hybrid de novo genome assemblies and identification of the 12α-hydroxysteroid dehydrogenase gene.

AIMS: The role of a Acinetobacter johnsonii strain, isolated from a soil sample, in the biotransformation of bile acids (BAs) was already described but the enzymes responsible for these transformations were only partially purified and molecularly characterized. METHODS AND RESULTS: This study describes the use of hybrid de novo assemblies, that combine long-read Oxford Nanopore and short-read Illumina sequencing strategies, to reconstruct the entire genome of A. johnsonii ICE_NC strain and to identify the coding region for a 12α-hydroxysteroid dehydrogenase (12α-HSDH), involved in BAs metabolism. The de novo assembly of the A. johnsonii ICE_NC genome was generated using Canu and Unicycler, both strategies yielded a circular chromosome of about 3.6 Mb and one 117 kb long plasmid. Gene annotation was performed on the final assemblies and the gene for 12α-HSDH was detected on the plasmid. CONCLUSIONS: Our findings illustrate the added value of long read sequencing in addressing the challenges of whole genome characterization and plasmid reconstruction in bacteria. These approaches also allowed the identification of the A. johnsonii ICE_NC gene for the 12α-HSDH enzyme, whose activity was confirmed at the biochemical level. SIGNIFICANCE AND IMPACT OR THE STUDY: At present, this is the first report on the characterization of a 12α-HSDH gene in an A. johnsonii strain able to biotransform cholic acid into ursodeoxycholic acid, a promising therapeutic agent for several diseases.

Lipid metabolism regulator human hydroxysteroid dehydrogenase-like 2 (HSDL2) modulates cervical cancer cell proliferation and metastasis.

Human hydroxysteroid dehydrogenase-like 2 (HSDL2) is a potent regulator in cancers and is also involved in lipid metabolism, but the role of HSDL2 in cervical cancer and whether it regulates the progress of cervical cancer through lipid metabolism remains unclear. In this study, we found that the overexpression of HSDL2 was in relation with cervical cancer progression including lymph nodes metastasis and recurrence. HSDL2 could serve as a novel marker of early diagnosis in cervical cancer. HSDL2 also gave impetus to tumorigenesis by initiating and promoting proliferation, invasion and migration of cervical cancer cells (Hela, C33A and SiHa) through EMT. Interestingly, we also searched that HSDL2 participated in oncogenesis by regulating lipid metabolism. In sum, our results gave the novel insight of HSDL2 functions which could be the potential for being the biomarker of prognosis and new target of therapy.

Alterations in bile acid metabolizing gut microbiota and specific bile acid genes as a precision medicine to subclassify NAFLD.

Multiple mechanisms for the gut microbiome contributing to the pathogenesis of nonalcoholic fatty liver disease (NAFLD) have been implicated. Here, we aim to investigate the contribution and potential application for altered bile acids (BA) metabolizing microbes in NAFLD by post hoc analysis of whole metagenome sequencing (WMS) data. The discovery cohort consisted of 86 well-characterized patients with biopsy-proven NAFLD and 38 healthy controls. Assembly-based analysis was performed to identify BA-metabolizing microbes. Statistical tests, feature selection, and microbial coabundance analysis were integrated to identify microbial alterations and markers in NAFLD. An independent validation cohort was subjected to similar analyses. NAFLD microbiota exhibited decreased diversity and microbial associations. We established a classifier model with 53 differential species exhibiting a robust diagnostic accuracy [area under the receiver-operator curve (AUC) = 0.97] for detecting NAFLD. Next, eight important differential pathway markers including secondary BA biosynthesis were identified. Specifically, increased abundance of 7α-hydroxysteroid dehydrogenase (7α-HSDH), 3α-hydroxysteroid dehydrogenase (baiA), and bile acid-coenzyme A ligase (baiB) was detected in NAFLD. Furthermore, 10 of 50 BA-metabolizing metagenome-assembled genomes (MAGs) from Bacteroides ovatus and Eubacterium biforme were dominant in NAFLD and interplayed as a synergetic ecological guild. Importantly, two subtypes of patients with NAFLD were observed according to secondary BA metabolism potentials. Elevated capability for secondary BA biosynthesis was also observed in the validation cohort. These bacterial BA-metabolizing genes and microbes identified in this study may serve as disease markers. Microbial differences in BA-metabolism and strain-specific differences among patients highlight the potential for precision medicine in NAFLD treatment.

Ile258Met mutation of Brucella melitensis 7α-hydroxysteroid dehydrogenase significantly enhances catalytic efficiency, cofactor affinity, and thermostability.

NAD(H)-dependent 7α-hydroxysteroid dehydrogenase catalyzes the oxidation of chenodeoxycholic acid to 7-oxolithocholic acid. Here, we designed mutations of Ile258 adjacent to the catalytic pocket of Brucella melitensis 7α-hydroxysteroid dehydrogenase. The I258M variant gave a 4.7-fold higher kcat, but 4.5-fold lower KM, compared with the wild type, resulting in a 21.8-fold higher kcat/KM value for chenodeoxycholic acid oxidation. It presented a 2.0-fold lower KM value with NAD+, suggesting stronger binding to the cofactor. I258M produced 7-oxolithocholic acid in the highest yield of 92.3% in 2 h, whereas the wild-type gave 88.4% in 12 h. The I258M mutation increased the half-life from 20.8 to 31.1 h at 30 °C. Molecular dynamics simulations indicated increased interactions and a modified tunnel improved the catalytic efficiency, and enhanced rigidity at three regions around the ligand-binding pocket increased the enzyme thermostability. This is the first report about significantly improved catalytic efficiency, cofactor affinity, and enzyme thermostability through single site-mutation of Brucella melitensis 7α-hydroxysteroid dehydrogenase. KEY POINTS: • Sequence and structure analysis guided the site mutation design. • Thermostability, catalytic efficiency and 7-oxo-LCA production were determined. • MD simulation was performed to indicate the improvement by I258M mutation.

Differences in expression of genes related to steroidgenesis in abdominal subcutaneous adipose tissue of pregnant women with and without PCOS; a case control study.

BACKGROUND: It was reported that steroid-related gene expressions in the adipose tissue (AT) of women differ between women affected with polycystic ovary syndrome (PCOS) and non-PCOS. Although association between PCOS in mother and offspring's health is a crucial issue, there are few studies focusing on AT of pregnant women suffering from PCOS. Our objectives were to determine the differences between mRNA expression levels of key steroid-converting enzymes in abdominal subcutaneous AT of pregnant women afflicted with PCOS and non-PCOS. METHODS: Twelve pregnant women with PCOS (case) and thirty six non-PCOS pregnant women (control) (1:3 ratio; age- and BMI-matched) undergoing cesarean section were enrolled for the present study. Expressions of fifteen genes related to steriodogenesis in abdominal subcutaneous AT were investigated using quantitative real-time PCR. RESULTS: No significant differences were detected with respect to age, BMI (prior pregnancy and at delivery day), gestational period and parity among pregnant women with PCOS and non-PCOS. Most of the sex steroid-converting genes except 17ß-Hydroxysteroid dehydrogenases2 (17BHSD2), were highly expressed on the day of delivery in subcutaneous AT. Women with PCOS showed significantly higher mRNA levels of steroidgenic acute regulator (STAR; P < 0.001), cytochrome P450 monooxygenase (CYP11A1; P < 0.05), 17α-hydroxylase (CYP17A1; P < 0.05), and 11ß-Hydroxysteroid dehydrogenase (11BHSD1 and 11BHSD2; P < 0.05). The expression of steroid 21-hydroxylase (CYP21) in non-PCOS was fourfold higher than those of women with PCOS (P < 0.001). There were no significant differences between relative expression of aromatase cytochrome P450 (CYP19A1), 3ß-hydroxysteroid dehydrogenase (3BHSD1 and 3BHSD2), and 17BHSD family (1, 3, 5, 7, and 12) between the two groups. CONCLUSION: The expression levels of genes related to sex steroids metabolism were similar to age-matched and BMI- matched pregnant non-PCOS and pregnant women with PCOS at delivery day. However, the alterations in gene expressions involved in glucocorticoids and mineralocorticoids metabolism were shown. It is necessary to point out that further studies regarding functional activity are required. More attention should be given to AT of pregnant women with PCOS that was previously ignored.

Structural and biochemical characterization of 20ß-hydroxysteroid dehydrogenase from Bifidobacterium adolescentis strain L2-32.

Anaerobic bacteria inhabiting the human gastrointestinal tract have evolved various enzymes that modify host-derived steroids. The bacterial steroid-17,20-desmolase pathway cleaves the cortisol side chain, forming pro-androgens predicted to impact host physiology. Bacterial 20ß-hydroxysteroid dehydrogenase (20ß-HSDH) regulates cortisol side-chain cleavage by reducing the C-20 carboxyl group on cortisol, yielding 20ß-dihydrocortisol. Recently, the gene encoding 20ß-HSDH in Butyricicoccus desmolans ATCC 43058 was reported, and a nonredundant protein search yielded a candidate 20ß-HSDH gene in Bifidobacterium adolescentis strain L2-32. B. adolescentis 20ß-HSDH could regulate cortisol side-chain cleavage by limiting pro-androgen formation in bacteria such as Clostridium scindens and 21-dehydroxylation by Eggerthella lenta Here, the putative B. adolescentis 20ß-HSDH was cloned, overexpressed, and purified. 20ß-HSDH activity was confirmed through whole-cell and pure enzymatic assays, and it is specific for cortisol. Next, we solved the structures of recombinant 20ß-HSDH in both the apo- and holo-forms at 2.0-2.2 Å resolutions, revealing close overlap except for rearrangements near the active site. Interestingly, the structures contain a large, flexible N-terminal region that was investigated by gel-filtration chromatography and CD spectroscopy. This extended N terminus is important for protein stability because deletions of varying lengths caused structural changes and reduced enzymatic activity. A nonconserved extended N terminus was also observed in several short-chain dehydrogenase/reductase family members. B. adolescentis strains capable of 20ß-HSDH activity could alter glucocorticoid metabolism in the gut and thereby serve as potential probiotics for the management of androgen-dependent diseases.

Hypoxia-Preconditioned Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Mitigate Hypoglycemic Testicular Injury Induced by Insulin in Rats.

Hypoglycemia is a neglected metabolic disorder. Thus, we evaluated the protective effect of hypoxia-preconditioned human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) on hypoglycemic testicular injury. We examined 56 testes from 28 animals: 7 rats with insulin-induced hypoglycemia (HG group), 7 hypoglycemic rats which received an intratesticular injection of hUCB-MSCs (HG-MSC group), and 14 untreated control rats. Testosterone level, testicular catalase (CAT) activity, and malondialdehyde (MDA) level were analyzed. Immunostaining for specific testicular germ and somatic cell markers was performed. Proliferating and apoptotic cells were detected by anti-PCNA and anti-caspase-3, respectively. Morphometrical data were statistically analyzed. The hypoglycemic rats showed a significant decrease in testosterone level and CAT activity and a significant increase in MDA production. Examination of histological structure and protein expression of diverse germ cell markers revealed collapsed tubules that were lined by degenerated germ cells, decreased lactate dehydrogenase type C immune expression, as well as decreased proliferating and increased apoptotic cells number in hypoglycemic testes. Injection of MSCs improved testicular biochemical parameters, preserved germ cells and somatic cells, and decreased apoptosis. In conclusion, hypoxia-preconditioned hUCB-MSCs attenuate rat testicular injury caused by insulin-induced hypoglycemia. Avoidance and rapid management of hypoglycemia are necessary to avoid significant testicular injury.

RDH1 suppresses adiposity by promoting brown adipose adaptation to fasting and re-feeding.

RDH1 is one of the several enzymes that catalyze the first of the two reactions to convert retinol into all-trans-retinoic acid (atRA). Here, we show that Rdh1-null mice fed a low-fat diet gain more weight as adiposity (17% males, 13% females) than wild-type mice by 20 weeks old, despite neither consuming more calories nor decreasing activity. Glucose intolerance and insulin resistance develop following increased adiposity. Despite the increase in white fat pads, epididymal white adipose does not express Rdh1, nor does muscle. Brown adipose tissue (BAT) and liver express Rdh1 at relatively high levels compared to other tissues. Rdh1 ablation lowered body temperatures during ambient conditions. Given the decreased body temperature, we focused on BAT. A lack of differences in BAT adipogenic gene expression between Rdh1-null mice and wild-type mice, including Pparg, Prdm16, Zfp516 and Zfp521, indicated that the phenotype was not driven by brown adipose hyperplasia. Rather, Rdh1 ablation eliminated the increase in BAT atRA that occurs after re-feeding. This disruption of atRA homeostasis increased fatty acid uptake, but attenuated lipolysis in primary brown adipocytes, resulting in increased lipid content and larger lipid droplets. Rdh1 ablation also decreased mitochondrial proteins, including CYCS and UCP1, the mitochondria oxygen consumption rate, and disrupted the mitochondria membrane potential, further reflecting impaired BAT function, resulting in both BAT and white adipose hypertrophy. RNAseq revealed dysregulation of 424 BAT genes in null mice, which segregated predominantly into differences after fasting vs after re-feeding. Exceptions were Rbp4 and Gbp2b, which increased during both dietary conditions. Rbp4 encodes the serum retinol-binding protein-an insulin desensitizer. Gbp2b encodes a GTPase. Because Gbp2b increased several hundred-fold, we overexpressed it in brown adipocytes. This caused a shift to larger lipid droplets, suggesting that GBP2b affects signaling downstream of the ß-adrenergic receptor during basal thermogenesis. Thus, Rdh1-generated atRA in BAT regulates multiple genes that promote BAT adaptation to whole-body energy status, such as fasting and re-feeding. These gene expression changes promote optimum mitochondria function and thermogenesis, limiting adiposity. Attenuation of adiposity and insulin resistance suggests that RDH1 mitigates metabolic syndrome.

[Xiongcan Yishen Prescription up-regulates the expressions of cholesterol transport proteins, steroidogenic enzymes and SF-1 in the Leydig cells of rats with late-onset hypogonadism].

OBJECTIVE: To investigate the effects of Xiongcan Yishen Prescription (XYP) on the expressions of cholesterol transport proteins, steroidogenic enzymes and steroidogenic factor-1 (SF-1) in the Leydig cells of the rats with late-onset hypogonadism (LOH). METHODS: Twenty-five 18-month-old male SD rats were randomly divided into five groups of equal number, LOH model control, testosterone propionate (TP) and low-, medium- and high-dose XYP, and another 5 two-month-old male SD rats included as normal controls. After modeling, the animals in the TP group were treated by intramuscular injection of TP at 5.21 mg/kg qd alt, those in the low-, medium- and high-dose XYP groups intragastrically with XYP at 10.4, 20.8 and 41.6 g/kg qd alt respectively, and those in the LOH model and normal control groups with saline, all for 28 successive days. Then, all the rats were sacrificed for determination of the expressions of the cholesterol transport proteins StAR and TSPO, steroidogenic enzymes CYP11A1, HSD3B7 and HSD17B4, and SF-1 in the Leydig cells by Western blot. RESULTS: The expressions of StAR, TSPO, CYP11A1, HSD3B7, HSD17B4 and SF-1 in the Leydig cells were significantly decreased in the LOH model controls compared with those in the normal controls (P< 0.05), but remarkably increased in the low-, medium- and high-dose XYP groups in comparison with those in the LOH model control group (P< 0.05). CONCLUSIONS: Xiongcan Yishen Prescription can up-regulate the expressions of the cholesterol transport proteins StAR and TSPO, steroidogenic enzymes CYP11A1, HSD3B7 and HSD17B4, and SF-1 in the rat Leydig cells, which might be one of the possible mechanisms of the prescription in the treatment of LOH.

The 7α-hydroxysteroid dehydratase Hsh2 is essential for anaerobic degradation of the steroid skeleton of 7α-hydroxyl bile salts in the novel denitrifying bacterium Azoarcus sp. strain Aa7.

Bile salts are steroid compounds from the digestive tract of vertebrates and enter the environment via defecation. Many aerobic bile-salt degrading bacteria are known but no bacteria that completely degrade bile salts under anoxic conditions have been isolated so far. In this study, the facultatively anaerobic Betaproteobacterium Azoarcus sp. strain Aa7 was isolated that grew with bile salts as sole carbon source under anoxic conditions with nitrate as electron acceptor. Phenotypic and genomic characterization revealed that strain Aa7 used the 2,3-seco pathway for the degradation of bile salts as found in other denitrifying steroid-degrading bacteria such as Sterolibacterium denitrificans. Under oxic conditions strain Aa7 used the 9,10-seco pathway as found in, for example, Pseudomonas stutzeri Chol1. Metabolite analysis during anaerobic growth indicated a reductive dehydroxylation of 7α-hydroxyl bile salts. Deletion of the gene hsh2 Aa7 encoding a 7-hydroxysteroid dehydratase led to strongly impaired growth with cholate and chenodeoxycholate but not with deoxycholate lacking a hydroxyl group at C7. The hsh2 Aa7 deletion mutant degraded cholate and chenodeoxycholate to the corresponding C19 -androstadienediones only while no phenotype change was observed during aerobic degradation of cholate. These results showed that removal of the 7α-hydroxyl group was essential for cleavage of the steroid skeleton under anoxic conditions.

Enhanced activity and substrate tolerance of 7α-hydroxysteroid dehydrogenase by directed evolution for 7-ketolithocholic acid production.

7-Ketolithocholic acid (7-KLCA) is an important intermediate for the synthesis of ursodeoxycholic acid (UDCA). UDCA is the main effective component of bear bile powder that is used in traditional Chinese medicine for the treatment of human cholesterol gallstones. 7α-Hydroxysteroid dehydrogenase (7α-HSDH) is the key enzyme used in the industrial production of 7-KLCA. Unfortunately, the natural 7α-HSDHs reported have difficulty meeting the requirements of industrial application, due to their poor activities and strong substrate inhibition. In this study, a directed evolution strategy combined with high-throughput screening was applied to improve the catalytic efficiency and tolerance of high substrate concentrations of NADP+-dependent 7α-HSDH from Clostridium absonum. Compared with the wild type, the best mutant (7α-3) showed 5.5-fold higher specific activity and exhibited 10-fold higher and 14-fold higher catalytic efficiency toward chenodeoxycholic acid (CDCA) and NADP+, respectively. Moreover, 7α-3 also displayed significantly enhanced tolerance in the presence of high concentrations of substrate compared to the wild type. Owing to its improved catalytic efficiency and enhanced substrate tolerance, 7α-3 could efficiently biosynthesize 7-KLCA with a substrate loading of 100 mM, resulting in 99% yield of 7-KLCA at 2 h, in contrast to only 85% yield of 7-KLCA achieved for the wild type at 16 h.