Metabolic dysfunction in female mice with disruption of 5α-reductase 1

5α-Reductases irreversibly catalyse A-ring reduction of pregnene steroids, including glucocorticoids and androgens. Genetic disruption of 5α-reductase 1 in male mice impairs glucocorticoid clearance and predisposes to glucose intolerance and hepatic steatosis upon metabolic challenge. However, it is unclear whether this is driven by changes in androgen and/or glucocorticoid action. Female mice with transgenic disruption of 5α-reductase 1 (5αR1-KO) were studied, representing a ‘low androgen’ state. Glucocorticoid clearance and stress responses were studied in mice aged 6 months. Metabolism was assessed in mice on normal chow (aged 6 and 12 m) and also in a separate cohort following 1-month high-fat diet (aged 3 m). Female 5αR1-KO mice had adrenal suppression (44% lower AUC corticosterone after stress), and upon corticosterone infusion, accumulated hepatic glucocorticoids (~27% increased corticosterone). Female 5αR1-KO mice aged 6 m fed normal chow demonstrated insulin resistance (~35% increased area under curve (AUC) for insulin upon glucose tolerance testing) and hepatic steatosis (~33% increased hepatic triglycerides) compared with controls. This progressed to obesity (~12% increased body weight) and sustained insulin resistance (~38% increased AUC insulin) by age 12 m. Hepatic transcript profiles supported impaired lipid β-oxidation and increased triglyceride storage. Female 5αR1-KO mice were also predisposed to develop high-fat diet-induced insulin resistance. Exaggerated predisposition to metabolic disorders in female mice, compared with that seen in male mice, after disruption of 5αR1 suggests phenotypic changes may be underpinned by altered metabolism of glucocorticoids rather than androgens

A Stratified Transcriptomics Analysis of Polygenic Fat and Lean Mouse Adipose Tissues Identifies Novel Candidate Obesity Genes

Obesity and metabolic syndrome results from a complex interaction between genetic and environmetal factors. In addition to brain-regulated processes, recent genome wide association studies have indicated that genes highly expressed in adipose tissue affect the distribution and function of fat and thus contribute to obesity. Using a stratified transcriptome gene enrichment approach we attempted to identify adipose tissue-specific obesity genes in the unique polygenic fat (F) mouse strain generated by selective breeding over 60 generations for divergent adiposity from a comparator lean (L) strain. To enrich for adipose tissue obesity genes a ˝snap-shot˝ pooled-sample transcriptome comparison of key fat depots and non adipose tissue (muscle, liver, kidney) was performed. Known obesity quantitative trait loci (QTL) information for the model allowed us to further filter genes for increased likelihood of being causal or secondary for obesity. This successfully identified several genes previously linked to obesity (C1qr1, and Np3r) as positional QTL candidate genes elevated specifically in F line adipose tissue.A number of novel obesity candidate genes were also identified (Thbs1, Ppp1rd, Tmepai, Trp53inp2, Ttc7b, Tuba1a, Fgf13, Fmr) that have inferred rolesin fat cell function. Quantitative microarray analysis was then applied to the most phenotypically divergent adipose depot after exaggerating F and L strain differences with chronic high fat feeding which revealed a dictinct gene expression profile of line, fat depot and diet-responsive inflammatory, angiogenic and metabolic pathaways. Selected candidate genes Npr3 and Thbs1, as well as Gys2, a non-QTL gene that otherwise passed our enrichment criteria were characterised, revealing novel functional effects consistent with a contribution to obesity. A focussed candidate gene enrichment strategy in the unique F and L model has identified novel adipose tissue-enriched genes contributing to obesity

Relative adrenal insufficiency in mice deficient in 5α-reductase 1

Patients with critical illness or hepatic failure exhibit impaired cortisol responses to ACTH, a phenomenon known as ‘relative adrenal insufficiency’. A putative mechanism is that elevated bile acids inhibit inactivation of cortisol in liver by 5α-reductases type 1 and type 2 and 5β-reductase, resulting in compensatory downregulation of the hypothalamic–pituitary–adrenal axis and adrenocortical atrophy. To test the hypothesis that impaired glucocorticoid clearance can cause relative adrenal insufficiency, we investigated the consequences of 5α-reductase type 1 deficiency in mice. In adrenalectomised male mice with targeted disruption of 5α-reductase type 1, clearance of corticosterone was lower after acute or chronic (eightfold, P<0.05) administration, compared with WT control mice. In intact 5α-reductase-deficient male mice, although resting plasma corticosterone levels were maintained, corticosterone responses were impaired after ACTH administration (26% lower, P<0.05), handling stress (2.5-fold lower, P<0.05) and restraint stress (43% lower, P<0.05) compared with WT mice. mRNA levels of Nr3c1 (glucocorticoid receptor), Crh and Avp in pituitary or hypothalamus were altered, consistent with enhanced negative feedback. These findings confirm that impaired peripheral clearance of glucocorticoids can cause ‘relative adrenal insufficiency’ in mice, an observation with important implications for patients with critical illness or hepatic failure, and for patients receiving 5α-reductase inhibitors for prostatic disease

Interobserver reproducibility of cytologic p16INK4a/Ki-67 dual immunostaining in human papillomavirus-positive women

BACKGROUND: The accumulation of cyclin-dependent kinase inhibitor 2A (p16(ink4a)) protein in a cell is associated with neoplastic progression in precancerous cervical lesions. Dual staining for p16(ink4a) and Ki-67 has been proposed as a triage test in cervical cancer screening for women who test positive for human papillomavirus DNA. In this study, interobserver reproducibility of the interpretation of this test was assessed. METHODS: Forty-two immunostained, liquid-based cytology slides were divided into 2 sets and were interpreted by 17 to 21 readers from 9 different laboratories, yielding a total of 816 reports. Immunostaining results were classified as positive, negative, inconclusive, or inadequate. After evaluation of the first set of slides and before circulation of the second set, the results were discussed in a plenary meeting. The 10 slides with the most discordant results were evaluated again by selected expert cytopathologists. RESULTS: The overall kappa value was 0.612 (95% confidence interval [CI], 0.523-0.701), it was higher for the positive and negative categories (kappa=0.692 and kappa=0.641, respectively), and it was almost null for the inconclusive category (kappa=0.058). Considering only readers from laboratories with documented experience, the kappa value was higher (kappa=0.747; 95% CI, 0.643-0.839) compared with nonexperienced centers (kappa=0.498; 95% CI, 0.388-0.616). The results were similar in both sets of slides (kappa=0.505 [95% CI, 0.358-0.642] and kappa=0.521 [95% CI, 0.240-0.698] for the first and second sets, respectively). Reinterpretation of the slides with the most discordant results did not provide any improvement (first evaluation, kappa=0.616 [95% CI, 0.384-0.866]; second evaluation, kappa=0.403 [95% CI, 0.182-0.643]). CONCLUSIONS: Dual staining for p16(ink4a) and Ki-67 demonstrated good reproducibility, confirming its robustness, which is a necessary prerequisite for its adoption as a triage test in cervical cancer screening programs that use human papillomavirus DNA as a primary test. Cancer Cytopathol 2017; 125:212-20. (C) 2016 American Cancer Society