Sex-specific neuroprotection by inhibition of the Y-chromosome gene, SRY, in experimental Parkinson's disease.

Parkinson's disease (PD) is a debilitating neurodegenerative disorder caused by the loss of midbrain dopamine (DA) neurons. While the cause of DA cell loss in PD is unknown, male sex is a strong risk factor. Aside from the protective actions of sex hormones in females, emerging evidence suggests that sex-chromosome genes contribute to the male bias in PD. We previously showed that the Y-chromosome gene, SRY, directly regulates adult brain function in males independent of gonadal hormone influence. SRY protein colocalizes with DA neurons in the male substantia nigra, where it regulates DA biosynthesis and voluntary movement. Here we demonstrate that nigral SRY expression is highly and persistently up-regulated in animal and human cell culture models of PD. Remarkably, lowering nigral SRY expression with antisense oligonucleotides in male rats diminished motor deficits and nigral DA cell loss in 6-hydroxydopamine (6-OHDA)-induced and rotenone-induced rat models of PD. The protective effect of the SRY antisense oligonucleotides was associated with male-specific attenuation of DNA damage, mitochondrial degradation, and neuroinflammation in the toxin-induced rat models of PD. Moreover, reducing nigral SRY expression diminished or removed the male bias in nigrostriatal degeneration, mitochondrial degradation, DNA damage, and neuroinflammation in the 6-OHDA rat model of PD, suggesting that SRY directly contributes to the sex differences in PD. These findings demonstrate that SRY directs a previously unrecognized male-specific mechanism of DA cell death and suggests that suppressing nigral Sry synthesis represents a sex-specific strategy to slow or prevent DA cell loss in PD.

Potential treatment of keloid pathogenesis with follistatin 288 by blocking the activin molecular pathway.

Keloids are benign tumours caused by abnormal wound healing driven by increased expression of cytokines, including activin A. This study compared effects of activins on normal and keloid-derived human dermal fibroblasts and investigated a novel treatment for keloids using follistatin. Normal skin and keloid tissue samples from 11 patients were used to develop primary fibroblast cultures, which were compared in terms of their histology and relevant gene (qRT-PCR and RNAseq) and protein (ELISA) expression. Activin A (INHBA) and connective tissue growth factor (CTGF) gene expression were significantly upregulated in keloid fibroblasts, as was activin A protein expression in cell lysates and culture medium. Activator protein 1 inhibitor (SR11302) significantly decreased INHBA and CTGF expression in keloid fibroblasts and a single treatment of follistatin over 5 days significantly inhibited activin and various matrix-related genes in keloid fibroblasts when compared to controls. Follistatin, by binding activin A, suppressed CTGF expression suggesting a novel therapeutic role in managing keloids and perhaps other fibrotic diseases.

Immunolocalisation of aromatase regulators liver kinase B1, phosphorylated AMP-activated protein kinase and cAMP response element-binding protein-regulated transcription co-activators in the human testis.

Although oestrogens are essential for spermatogenesis and their biosynthesis is dependent on aromatase expression, the molecular mechanism of aromatase regulation is poorly understood. Our laboratory has demonstrated that liver kinase B1 (LKB1) is a negative regulator of aromatase in the breast by phosphorylating AMP-activated protein kinase (AMPK) and inhibiting the nuclear translocation of the cAMP response element-binding protein-regulated transcription co-activator (CRTC) 2. The aim of this study was to determine the location of testis-associated proteins in the LKB1-CRTC pathway. Aromatase, LKB1, phosphorylated AMPK (pAMPK) and CRTC1-3 were examined by selected immunofluorescent antibodies in testis samples from a prepubertal boy and three fertile men. Aromatase, pAMPK and LKB1 proteins were present in the seminiferous epithelium and interstitium of the testis and were expressed in a differential and developmental manner in particular cell types. The expression pattern of LKB1 was similar to that of pAMPK and inversely related to aromatase expression. CRTC1 and CRTC3 were localised in the seminiferous epithelium, whereas CRTC2 was barely detectable in testis. These results lead to the conclusion that LKB1 is involved in the molecular pathway that underpins aromatase regulation in the testis via CRTC1 and CRTC3 and may be important for the oestrogen-mediated development of germ cells.

Nutrition affects Sertoli cell function but not Sertoli cell numbers in sexually mature male sheep.

We tested whether the reversible effects of nutrition on spermatogenesis in sexually mature sheep were mediated by Sertoli cells. Rams were fed with diets designed to achieve a 10% increase (High), no change (Maintenance) or a 10% decrease (Low) in body mass after 65 days. At the end of treatment, testes were lighter in the Low than the High group (PP<0.05) in the expression of seven Sertoli cell-specific genes. Under-nutrition appeared to reverse cellular differentiation leading to disruption of tight-junction morphology. In conclusion, in sexually mature sheep, reversible reductions in testis mass and spermatogenesis caused by under-nutrition were associated with impairment of basic aspects of Sertoli cell function but not with changes in the number of Sertoli cells.

Role of G protein-coupled receptor kinases (GRKs) in ß2 -adrenoceptor-mediated glucose uptake.

Truncation of the C-terminal tail of the ß2 -AR, transfection of ßARKct or over-expression of a kinase-dead GRK mutant reduces isoprenaline-stimulated glucose uptake, indicating that GRK is important for this response. We explored whether phosphorylation of the ß2 -AR by GRK2 has a role in glucose uptake or if this response is related to the role of GRK2 as a scaffolding protein. CHO-GLUT4myc cells expressing wild-type and mutant ß2 -ARs were generated and receptor affinity for [3 H]-CGP12177A and density of binding sites determined together with the affinity of isoprenaline and BRL37344. Following receptor activation by ß2 -AR agonists, cAMP accumulation, GLUT4 translocation, [3 H]-2-deoxyglucose uptake, and ß2 -AR internalization were measured. Bioluminescence resonance energy transfer was used to investigate interactions between ß2 -AR and ß-arrestin2 or between ß2 -AR and GRK2. Glucose uptake after siRNA knockdown or GRK inhibitors was measured in response to ß2 -AR agonists. BRL37344 was a poor partial agonist for cAMP generation but displayed similar potency and efficacy to isoprenaline for glucose uptake and GLUT4 translocation. These responses to ß2 -AR agonists occurred in CHO-GLUT4myc cells expressing ß2 -ARs lacking GRK or GRK/PKA phosphorylation sites as well as in cells expressing the wild-type ß2 -AR. However, ß2 -ARs lacking phosphorylation sites failed to recruit ß-arrestin2 and did not internalize. GRK2 knock-down or GRK2 inhibitors decreased isoprenaline-stimulated glucose uptake in rat L6 skeletal muscle cells. Thus, GRK phosphorylation of the ß2 -AR is not associated with isoprenaline- or BRL37344-stimulated glucose uptake. However, GRKs acting as scaffold proteins are important for glucose uptake as GRK2 knock-down or GRK2 inhibition reduces isoprenaline-stimulated glucose uptake.

The novel adrenergic agonist ATR-127 targets skeletal muscle and brown adipose tissue to tackle diabesity and steatohepatitis.

OBJECTIVE: Simultaneous activation of ß2- and ß3-adrenoceptors (ARs) improves whole-body metabolism via beneficial effects in skeletal muscle and brown adipose tissue (BAT). Nevertheless, high-efficacy agonists simultaneously targeting these receptors whilst limiting activation of ß1-ARs - and thus inducing cardiovascular complications - are currently non-existent. Therefore, we here developed and evaluated the therapeutic potential of a novel ß2-and ß3-AR, named ATR-127, for the treatment of obesity and its associated metabolic perturbations in preclinical models. METHODS: In the developmental phase, we assessed the impact of ATR-127's on cAMP accumulation in relation to the non-selective ß-AR agonist isoprenaline across various rodent ß-AR subtypes, including neonatal rat cardiomyocytes. Following these experiments, L6 muscle cells were stimulated with ATR-127 to assess the impact on GLUT4-mediated glucose uptake and intramyocellular cAMP accumulation. Additionally, in vitro, and in vivo assessments are conducted to measure ATR-127's effects on BAT glucose uptake and thermogenesis. Finally, diet-induced obese mice were treated with 5 mg/kg ATR-127 for 21 days to investigate the effects on glucose homeostasis, body weight, fat mass, skeletal muscle glucose uptake, BAT thermogenesis and hepatic steatosis. RESULTS: Exposure of L6 muscle cells to ATR-127 robustly enhanced GLUT4-mediated glucose uptake despite low intramyocellular cAMP accumulation. Similarly, ATR-127 markedly increased BAT glucose uptake and thermogenesis both in vitro and in vivo. Prolonged treatment of diet-induced obese mice with ATR-127 dramatically improved glucose homeostasis, an effect accompanied by decreases in body weight and fat mass. These effects were paralleled by an enhanced skeletal muscle glucose uptake, BAT thermogenesis, and improvements in hepatic steatosis. CONCLUSIONS: Our results demonstrate that ATR-127 is a highly effective, novel ß2- and ß3-ARs agonist holding great therapeutic promise for the treatment of obesity and its comorbidities, whilst potentially limiting cardiovascular complications. As such, the therapeutic effects of ATR-127 should be investigated in more detail in clinical studies.

Pharmacophore-guided Virtual Screening to Identify New ß3 -adrenergic Receptor Agonists.

The ß3 -adrenergic receptor (ß3 -AR) is found in several tissues such as adipose tissue and urinary bladder. It is a therapeutic target because it plays a role in thermogenesis, lipolysis, and bladder relaxation. Two ß3 -AR agonists are used clinically: mirabegron 1 and vibegron 2, which are indicated for overactive bladder syndrome. However, these drugs show adverse effects, including increased blood pressure in mirabegron patients. Hence, new ß3 -AR agonists are needed as starting points for drug development. Previous pharmacophore modeling studies of the ß3 -AR did not involve experimental in vitro validation. Therefore, this study aimed to conduct prospective virtual screening and confirm the biological activity of virtual hits. Ligand-based pharmacophore modeling was performed since no 3D structure of human ß3 -AR is yet available. A dataset consisting of ß3 -AR agonists was prepared to build and validate the pharmacophore models. The best model was employed for prospective virtual screening, followed by physicochemical property filtering and a docking evaluation. To confirm the activity of the virtual hits, an in vitro assay was conducted, measuring cAMP levels at the cloned ß3 -AR. Out of 35 tested compounds, 4 compounds were active in CHO-K1 cells expressing the human ß3 -AR, and 8 compounds were active in CHO-K1 cells expressing the mouse ß3 -AR.

The metabolic effects of mirabegron are mediated primarily by ß3 -adrenoceptors.

The ß3 -adrenoceptor agonist mirabegron is approved for use for overactive bladder and has been purported to be useful in the treatment of obesity-related metabolic diseases in humans, including those involving disturbances of glucose homeostasis. We investigated the effect of mirabegron on glucose homeostasis with in vitro and in vivo models, focusing on its selectivity at ß-adrenoceptors, ability to cause browning of white adipocytes, and the role of UCP1 in glucose homeostasis. In mouse brown, white, and brite adipocytes, mirabegron-mediated effects were examined on cyclic AMP, UCP1 mRNA, [3 H]-2-deoxyglucose uptake, cellular glycolysis, and O2 consumption. Mirabegron increased cyclic AMP levels, UCP1 mRNA content, glucose uptake, and cellular glycolysis in brown adipocytes, and these effects were either absent or reduced in white adipocytes. In brite adipocytes, mirabegron increased cyclic AMP levels and UCP1 mRNA content resulting in increased UCP1-mediated oxygen consumption, glucose uptake, and cellular glycolysis. The metabolic effects of mirabegron in both brown and brite adipocytes were primarily due to actions at ß3 -adrenoceptors as they were largely absent in adipocytes derived from ß3 -adrenoceptor knockout mice. In vivo, mirabegron increased whole body oxygen consumption, glucose uptake into brown and inguinal white adipose tissue, and improved glucose tolerance, all effects that required the presence of the ß3 -adrenoceptor. Furthermore, in UCP1 knockout mice, the effects of mirabegron on glucose tolerance were attenuated. Thus, mirabegron had effects on cellular metabolism in adipocytes that improved glucose handling in vivo, and were primarily due to actions at the ß3 -adrenoceptor.

Obesity-Associated Inflammatory Cytokines and Prostaglandin E2 Stimulate Glucose Transporter mRNA Expression and Glucose Uptake in Primary Human Adipose Stromal Cells.

Obesity is associated with chronic low-grade inflammation. This occurs largely as a result of the infiltration of immune cells within the obese adipose, which produce a number of inflammatory factors, including interleukin-6 (IL-6), IL-1ß, tumor necrosis factor-α (TNFα), and prostaglandin E(2) (PGE(2)). These factors have previously been shown to affect insulin-mediated glucose uptake in differentiated adipocytes. However, the insulin-independent effect of inflammation on adipocyte precursors, the adipose stromal cells, has not been explored. This study therefore aimed to examine the effect of obesity-associated inflammatory factors on the expression of insulin-independent glucose transporters (GLUT1 and GLUT3) and on the uptake of glucose within adipose stromal cells. Primary human subcutaneous adipose stromal cells were isolated from abdominoplasty, and the effect of inflammatory cytokines (IL-6, IL-1ß, and TNFα) and PGE(2) on GLUT mRNA expression and glucose transport was assessed using real-time polymerase chain reaction and radiolabeled deoxyglucose uptake assays, respectively. Results demonstrate that all four inflammatory mediators caused a dose-dependent increase in GLUT1 mRNA expression and glucose uptake. GLUT3 mRNA expression was also upregulated by IL-6 (0.5 ng/mL), TNFα (0.1 and 10 ng/mL), and PGE(2) (0.1 µM). Overall, these results demonstrate that obesity-associated inflammation increases insulin-independent glucose transporter expression and glucose uptake in undifferentiated adipose stromal cells.

Genome-wide analysis using exon arrays demonstrates an important role for expression of extra-cellular matrix, fibrotic control and tissue remodelling genes in Dupuytren's disease.

Dupuytren's disease (DD) is a classic example of pathological fibrosis which results in a debilitating disorder affecting a large sector of the human population. It is characterized by excessive local proliferation of fibroblasts and over-production of collagen and other components of extracellular matrix (ECM) in the palmar fascia. The fibrosis progressively results in contracture of elements between the palmar fascia and skin causing flexion deformity or clawing of the fingers and a severe reduction in hand function. While much is known about the pathogenesis and surgical treatment of DD, little is known about the factors that cause its onset and progression, despite many years of research. Gene expression patterns in DD patients now offers the potential to identify genes that direct the pathogenesis of DD. In this study we used primary cultures of fibroblasts derived from excisional biopsies of fibrotic tissue from DD patients to compare the gene expression profiles on a genome-wide basis with normal control fibroblasts. Our investigations have identified genes that may be involved with DD pathogenesis including some which are directly relevant to fibrosis. In particular, these include significantly reduced expression levels of three matrix metallopeptidases (MMP1, MMP3, MMP16), follistatin, and STAT1, and significantly increased expression levels of fibroblast growth factors (FGF9, FGF11), a number of collagen genes and other ECM genes in DD patient samples. Many of these gene products are known to be involved in fibrosis, tumour formation and in the normal processes of tissue remodelling. In addition, alternative splicing was identified in some DD associated genes. These highly sensitive genomic investigations provide new insight into the molecular mechanisms that may underpin the development and progression of DD.

Overexpression of aromatase associated with loss of heterozygosity of the STK11 gene accounts for prepubertal gynecomastia in boys with Peutz-Jeghers syndrome.

CONTEXT: Peutz-Jeghers syndrome (PJS) is an autosomal-dominant disorder that arises as a consequence of mutations in the STK11 gene that encodes LKB1. PJS males often have estrogen excess manifesting as gynecomastia and advanced bone age. We and others have previously described an increase in testicular aromatase expression in PJS patients. However, the underlying mechanism has not yet been explored. OBJECTIVE: The aim of this study was to characterize the role of LKB1 in regulating the expression of aromatase in boys with PJS via signaling pathways involving AMP-activated protein kinase (AMPK) and cyclic AMP-responsive element binding protein-regulated transcription coactivators (CRTCs). PATIENTS: We studied testicular biopsies from two boys with STK11 mutations: a 13-year-old boy and an unrelated 4-year-old boy with prepubertal gynecomastia and advanced bone age, as well as breast tissue from the 13-year-old boy. RESULTS: Loss of heterozygosity of STK11, measured by the absence of LKB1 immunofluorescence, was observed in Sertoli cells of abnormal cords of testis samples from affected individuals. This was associated with loss of p21 expression and decreased phosphorylation of AMPK, known downstream targets of LKB1, as well as the increased expression of aromatase. Similar results of low LKB1 expression in cells expressing aromatase were observed in the mammary epithelium from one of these individuals. Nuclear expression of the CRTC proteins, potent stimulators of aromatase and known to be inhibited by AMPK, was significantly correlated with aromatase. CONCLUSIONS: Loss of heterozygosity of the STK11 gene leads to an increase in aromatase expression associated with an increase in CRTC nuclear localization, thereby providing a mechanism whereby PJS results in increased endogenous estrogens in affected males.

Promoter-specific effects of metformin on aromatase transcript expression.

Phase III aromatase inhibitors (AIs) are proving successful in the treatment of hormone-dependent postmenopausal breast cancer. Side-effects associated with total body aromatase inhibition have prompted new research into the development of breast-specific AIs. The identification of tissue- and disease-specific usage of aromatase promoters has made the inhibition of aromatase at the transcriptional level an interesting approach. We have previously demonstrated that AMPK-activating drugs, including metformin, were potent inhibitors of aromatase expression in primary human breast adipose stromal cells (hASCs). This study examines the promoter-specific effects of metformin on inhibiting aromatase expression in hASCs. Tumour-associated promoters PII/PI.3 were activated using forskolin (FSK)/phorbol ester (PMA), whereas normal adipose associated promoter PI.4 was activated using dexamethasone (DEX)/tumour necrosis factor-α (TNFα). Results demonstrate that metformin significantly decreased the FSK/PMA-, but not the DEX/TNFα-mediated expression of total aromatase at concentrations of 10, 20, and 50 µM (P ≤ 0.05). Using PCR to amplify promoter-specific transcripts of aromatase, it appears that the inhibition of the FSK/PMA-mediated expression of aromatase is due to decreases in PII/PI.3-specific transcripts, whereas no effect of metformin is observed on any promoter-specific transcript, including PI.4, in DEX/TNFα-treated hASCs. This report therefore supports the hypothesis that metformin would act as a breast-specific inhibitor of aromatase expression in the context of postmenopausal breast cancer.

Teasing out the role of aromatase in the healthy and diseased testis.

Scientific discoveries over the past decade have shifted the stereotypical view of androgens as male hormones and estrogens as female hormones. It is now recognized that a delicate balance of both androgens and estrogens, a process controlled by aromatase, is fundamental for normal testicular development and fertility. While the site-specific actions of these two classes of steroids within the testis are becoming better documented, the role and regulation of estrogen biosynthesis by aromatase within the testis remains unclear. The majority of data comes from a wide range of animal species, particularly genetically modified mouse models; aromatase knockout (ArKO) and overexpressing (AROM(+)), with limited information on humans, however the existence of congenital aromatase mutations has provided some insight into its effects on individual parameters of the testis. This review dissects out the localization and activity of aromatase in the healthy and diseased testis, addresses the cellular insult to the testis that occurs in its absence and over abundance and proposes potential molecular mechanisms of aromatase regulation in the testis.