Mouse Testicular Mkrn3 Expression Is Primarily Interstitial, Increases Peripubertally, and Is Responsive to LH/hCG.

Studies in humans and mice support a role for Makorin RING finger protein 3 (MKRN3) as an inhibitor of gonadotropin-releasing hormone (GnRH) secretion prepubertally, and its loss of function is the most common genetic cause of central precocious puberty in humans. Studies have shown that the gonads can synthesize neuropeptides and express MKRN3/Mkrn3 mRNA. Therefore, we aimed to investigate the spatiotemporal expression pattern of Mkrn3 in gonads during sexual development, and its potential regulation in the functional testicular compartments by gonadotropins. Mkrn3 mRNA was detected in testes and ovaries of wild-type mice at all ages evaluated, with a sexually dimorphic expression pattern between male and female gonads. Mkrn3 expression was highest peripubertally in the testes, whereas it was lower peripubertally than prepubertally in the ovaries. Mkrn3 is expressed primarily in the interstitial compartment of the testes but was also detected at low levels in the seminiferous tubules. In vitro studies demonstrated that Mkrn3 mRNA levels increased in human chorionic gonadotropin (hCG)-treated Leydig cell primary cultures. Acute administration of a GnRH agonist in adult mice increased Mkrn3 expression in testes, whereas inhibition of the hypothalamic-pituitary-gonadal axis by chronic administration of GnRH agonist had the opposite effect. Finally, we found that hCG increased Mkrn3 mRNA levels in a dose-dependent manner. Taken together, our developmental expression analyses, in vitro and in vivo studies show that Mkrn3 is expressed in the testes, predominantly in the interstitial compartment, and that Mkrn3 expression increases after puberty and is responsive to luteinizing hormone/hCG stimulation.

MKRN3 inhibits puberty onset via interaction with IGF2BP1 and regulation of hypothalamic plasticity.

Makorin ring finger protein 3 (MKRN3) was identified as an inhibitor of puberty initiation with the report of loss-of-function mutations in association with central precocious puberty. Consistent with this inhibitory role, a prepubertal decrease in Mkrn3 expression was observed in the mouse hypothalamus. Here, we investigated the mechanisms of action of MKRN3 in the central regulation of puberty onset. We showed that MKRN3 deletion in hypothalamic neurons derived from human induced pluripotent stem cells was associated with significant changes in expression of genes controlling hypothalamic development and plasticity. Mkrn3 deletion in a mouse model led to early puberty onset in female mice. We found that Mkrn3 deletion increased the number of dendritic spines in the arcuate nucleus but did not alter the morphology of GnRH neurons during postnatal development. In addition, we identified neurokinin B (NKB) as an Mkrn3 target. Using proteomics, we identified insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) as another target of MKRN3. Interactome analysis revealed that IGF2BP1 interacted with MKRN3, along with several members of the polyadenylate-binding protein family. Our data show that one of the mechanisms by which MKRN3 inhibits pubertal initiation is through regulation of prepubertal hypothalamic development and plasticity, as well as through effects on NKB and IGF2BP1.

Novel MKRN3 Missense Mutations Associated With Central Precocious Puberty Reveal Distinct Effects on Ubiquitination.

CONTEXT: Loss-of-function mutations in the maternally imprinted genes, MKRN3 and DLK1, are associated with central precocious puberty (CPP). Mutations in MKRN3 are the most common known genetic etiology of CPP. OBJECTIVE: This work aimed to screen patients with CPP for MKRN3 and DLK1 mutations and analyze the effects of identified mutations on protein function in vitro. METHODS: Participants included 84 unrelated children with CPP (79 girls, 5 boys) and, when available, their first-degree relatives. Five academic medical institutions participated. Sanger sequencing of MKRN3 and DLK1 5' upstream flanking and coding regions was performed on DNA extracted from peripheral blood leukocytes. Western blot analysis was performed to assess protein ubiquitination profiles. RESULTS: Eight heterozygous MKRN3 mutations were identified in 9 unrelated girls with CPP. Five are novel missense mutations, 2 were previously identified in patients with CPP, and 1 is a frameshift variant not previously associated with CPP. No pathogenic variants were identified in DLK1. Girls with MKRN3 mutations had an earlier age of initial pubertal signs and higher basal serum luteinizing hormone and follicle-stimulating hormone compared to girls with CPP without MRKN3 mutations. Western blot analysis revealed that compared to wild-type MKRN3, mutations within the RING finger domain reduced ubiquitination whereas the mutations outside this domain increased ubiquitination. CONCLUSION: MKRN3 mutations were present in 10.7% of our CPP cohort, consistent with previous studies. The novel identified mutations in different domains of MKRN3 revealed different patterns of ubiquitination, suggesting distinct molecular mechanisms by which the loss of MRKN3 results in early pubertal onset.

Liraglutide Activates Type 2 Deiodinase and Enhances ß3-Adrenergic-Induced Thermogenesis in Mouse Adipose Tissue.

Aims: Liraglutide is a long-acting glucagon-like peptide 1 (GLP-1) receptor agonist used as an anti-hyperglycemic agent in type 2 diabetes treatment and recently approved for obesity management. Weight loss is attributed to appetite suppression, but therapy may also increase energy expenditure. To further investigate the effect of GLP-1 signaling in thermogenic fat, we assessed adipose tissue oxygen consumption and type 2 deiodinase (D2) activity in mice treated with liraglutide, both basally and after ß3-adrenergic treatment. Methods: Male C57BL/6J mice were randomly assigned to receive liraglutide (400 µg/kg, n=12) or vehicle (n=12). After 16 days, mice in each group were co-treated with the selective ß3-adrenergic agonist CL316,243 (1 mg/kg, n=6) or vehicle (n=6) for 5 days. Adipose tissue depots were assessed for gene and protein expression, oxygen consumption, and D2 activity. Results: Liraglutide increased interscapular brown adipose tissue (iBAT) oxygen consumption and enhanced ß3-adrenergic-induced oxygen consumption in iBAT and inguinal white adipose tissue (ingWAT). These effects were accompanied by upregulation of UCP-1 protein levels in iBAT and ingWAT. Notably, liraglutide increased D2 activity without significantly upregulating its mRNA levels in iBAT and exhibited additive effects to ß3-adrenergic stimulation in inducing D2 activity in ingWAT. Conclusions: Liraglutide exhibits additive effects to those of ß3-adrenergic stimulation in thermogenic fat and increases D2 activity in BAT, implying that it may activate this adipose tissue depot by increasing intracellular thyroid activation, adding to the currently known mechanisms of GLP-1A-induced weight loss.

Heat stress effects on sheep: Are hair sheep more heat resistant?

Climatic variables can trigger physiological, biochemical, haematological and hormonal alterations that influence the maintenance of homeothermy and can affect production and productivity in sheep. Different mechanisms are responsible for tolerance to heat stress (HS) including coat and skin colour, body size, fat distribution, physiological reactions and not just coat type (hair/wool). This review looks at physical, physiological, molecular and genetic aspects of heat tolerance in sheep and how they affect hair and wool sheep. We propose that it is the adaptation to hot environments and not the type of coat (wool/hair) itself that determines the capacity of the resistance of the animal to HS, due to modifications in essential pathways such as energy metabolism, physiological responses and body size. When studied in similar environments, commercial wool breeds tend to show higher heat stress, but hair breeds tend not to differ from wool breeds that are adapted to hot environments.

Effects of metformin on endometrial cancer: Systematic review and meta-analysis.

BACKGROUND: Endometrial cancer is one of the most common gynecological cancers, which is frequently preceded by atypical endometrial hyperplasia, a premalignant lesion. Metformin, an antidiabetic drug, has emerged as a new adjunctive strategy for different cancer types, including endometrial cancer. This systematic review and meta-analysis aimed to evaluate the effects of metformin in atypical endometrial hyperplasia and endometrial cancer patients. METHODS: The search was conducted on January 2017 and the articles were collected in Cochrane, LILACS, PubMed, Scopus and Web of Science. A grey literature search was undertaken using Google SCHOLAR, ProQuest and Open Grey. Nineteen studies were included, which contained information about the following outcomes: reversal of atypical endometrial hyperplasia, cellular proliferation biomarkers expression and overall survival in metformin-users compared to non-users. RESULTS: Metformin was associated with reversion of atypical endometrial hyperplasia to a normal endometrial, and with decreased cell proliferation biomarkers staining, from 51.94% (CI=36.23% to 67.46%) to 34.47% (CI=18.55% to 52.43%). However, there is a high heterogeneity among studies. Metformin-users endometrial cancer patients had a higher overall survival compared to non-metformin users and non-diabetic patients (HR=0.82; CI: 0.70-0.95; p=0.09, I2=40%). CONCLUSION: Regardless the high heterogeneity of the analyzed studies, the present review suggests that adjunct metformin treatment may assist in the reversal of atypical endometrial hyperplasia to normal endometrial histology, in the reduction of cell proliferation biomarkers implicated in tumor progression, and in the improvement of overall survival in endometrial cancer. Further work on prospective controlled trials designed to address the effects of adjunct metformin on clinical outcomes is necessary for definite conclusions.

GQ-16, a TZD-Derived Partial PPARγ Agonist, Induces the Expression of Thermogenesis-Related Genes in Brown Fat and Visceral White Fat and Decreases Visceral Adiposity in Obese and Hyperglycemic Mice.

BACKGROUND: Beige adipocytes comprise a unique thermogenic cell type in the white adipose tissue (WAT) of rodents and humans, and play a critical role in energy homeostasis. In this scenario, recruitment of beige cells has been an important focus of interest for the development of novel therapeutic strategies to treat obesity. PPARγ activation by full agonists (thiazolidinediones, TZDs) drives the appearance of beige cells, a process so-called browning of WAT. However, this does not translate into increased energy expenditure, and TZDs are associated with weight gain. Partial PPARγ agonists, on the other hand, do not induce weight gain, but have not been shown to drive WAT browning. The present study was designed to investigate the effects of GQ-16 on BAT and on browning of WAT in obese mice. METHODS: Male Swiss mice with obesity and hyperglycemia induced by high fat diet were treated with vehicle, rosiglitazone (4 mg/kg/d) or the TZD-derived partial PPARγ agonist GQ-16 (40 mg/kg/d) for 14 days. Fasting blood glucose, aspartate aminotransferase, alanine aminotransferase and lipid profile were measured. WAT and brown adipose tissue (BAT) depots were excised for determination of adiposity, relative expression of Ucp-1, Cidea, Prdm16, Cd40 and Tmem26 by RT-qPCR, histological analysis, and UCP-1 protein expression analysis by immunohistochemistry. Liver samples were also removed for histological analysis and determination of hepatic triglyceride content. RESULTS: GQ-16 treatment reduced high fat diet-induced weight gain in mice despite increasing energy intake. This was accompanied by reduced epididymal fat mass, reduced liver triglyceride content, morphological signs of increased BAT activity, increased expression of thermogenesis-related genes in interscapular BAT and epididymal WAT, and increased UCP-1 protein expression in interscapular BAT and in epididymal and inguinal WAT. CONCLUSION: This study suggests for the first time that a partial PPARγ agonist may increase BAT activity and induce the expression of thermogenesis-related genes in visceral WAT. GENERAL SIGNIFICANCE: These findings suggest that PPARγ activity might be modulated by partial agonists to induce WAT browning and treat obesity.