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The receptor tyrosine kinase, MERTK, plays an essential role in homeostasis of the retina via efferocytosis of shed outer nuclear segments of photoreceptors. The Royal College of Surgeons rat model of retinal degeneration has been linked to loss-of-function of MERTK, and together with the MERTK knock-out mouse, phenocopy retinitis pigmentosa in humans with MERTK mutations. Given recent efforts and interest in MERTK as a potential immuno-oncology target, development of a strategy to assess ocular safety at an early pre-clinical stage is critical. We have applied a state-of-the-art, multi-modal imaging platform to assess the in vivo effects of pharmacological inhibition of MERTK in mice. This involved the application of mass spectrometry imaging (MSI) to characterize the ocular spatial distribution of our highly selective MERTK inhibitor; AZ14145845, together with histopathology and transmission electron microscopy to characterize pathological and ultra-structural change in response to MERTK inhibition. In addition, we assessed the utility of a human retinal in vitro cell model to identify perturbation of phagocytosis post MERTK inhibition. We identified high localized total compound concentrations in the retinal pigment epithelium (RPE) and retinal lesions following 28 days of treatment with AZ14145845. These lesions were present in 4 of 8 treated animals, and were characterized by a thinning of the outer nuclear layer, loss of photoreceptors (PR) and accumulation of photoreceptor outer segments at the interface of the RPE and PRs. Furthermore, the lesions were very similar to that shown in the RCS rat and MERTK knock-out mouse, suggesting a MERTK-induced mechanism of PR cell death. This was further supported by the observation of reduced phagocytosis in the human retinal cell model following treatment with AZ14145845. Our study provides a viable, translational strategy to investigate the pre-clinical toxicity of MERTK inhibitors but is equally transferrable to novel chemotypes.

Objective Dominant loss‐of‐function mutations in the gene encoding the lysosomal protein, progranulin, cause 5‐10% of frontotemporal dementia cases. As progranulin undergoes secretion and endocytosis, a small number of progranulin‐expressing cells can potentially supply the protein to the entire central nervous system. Thus, gene therapy is a promising treatment approach. Methods We evaluated adeno‐associated viral vector administration into the cerebrospinal fluid as a minimally invasive approach to deliver the granulin gene to the central nervous system in a murine disease model and nonhuman primates. Results In progranulin‐deficient mice, vector delivery into the lateral cerebral ventricles increased progranulin levels in the cerebrospinal fluid and normalized histological and biochemical markers of progranulin deficiency. A single vector injection into the cisterna magna of nonhuman primates achieved CSF progranulin concentrations up to 40‐fold higher than those of normal human subjects and exceeded CSF progranulin levels of successfully treated mice. Animals treated with an adeno‐associated virus serotype 1 vector exhibited progranulin expression fivefold higher than those treated with an AAV5 vector or the AAV9 variant, AAVhu68, apparently due to remarkably efficient transduction of ependymal cells. Progranulin expression mediated by adeno‐associated viral vectors was well tolerated in nonhuman primates with no evidence of dose‐limiting toxicity, even at vector doses that induced supraphysiologic progranulin expression. Interpretation These findings support the development of AAV1‐based gene therapy for frontotemporal dementia caused by progranulin deficiency.

Cannabis Social Clubs (CSCs) are typically non-profit associations of adult cannabis users who collectively produce and distribute cannabis among themselves. Since the emergence of the model in Spain during the 1990s, other countries may have seen the appearance of CSCs (or CSC-like associations) but there is a dearth of knowledge about the phenomenon in Europe. The goals of this analysis are to: (1) map the presence of CSCs across the European Union; and (2) examine how CSCs are operating in such settings. The data included in our analysis derive from a 2018–19 survey. The 30-item questionnaire comprised questions about CSCs’ origins and relations with other stakeholders and organizations, the types of activities the CSCs developed and their views on cannabis regulation. The questionnaire was translated into all the official languages of the EU zone and sent via email to the participants. In total, 81 CSCs completed the questionnaire. Beyond Spain and Belgium, where the CSC presence has already been documented, we were able to identify CSCs in 11 other countries. The longest-running CSC in our sample was established in 1999, but most emerged in the past decade. The smallest CSC in our sample reported six registered members, whereas the largest counted a total of 5000 members. Most CSCs were cultivating or distributing cannabis to their members at the time of the survey, but engaged in other informative, entertainment and activist activities as well. The CSC model remains prohibited across the EU. CSC activists have thus by and large shaped the way CSCs operate, often adapting to domestic law particularities or law enforcement activities. In this article, we present and discuss the range of CSC practices from 13 different European countries, and what these represent for the consideration of the CSC model in current policy debates.

The safety profile of a novel drug candidate is highly determinant of its chance of success to be launched for treatment of patients. The integration of predictive toxicology in the drug discovery process is essential to identify and mitigate potential safety issues in a timely manner and contributes to improved productivity in pharmaceutical R&D, especially for small molecules, which is the focus of this review. A range of predictive in vitro toxicology assays has been established, which we use as part of AstraZeneca's proactive discovery safety screening strategy, with the aim to influence chemical design and obtain candidate drugs with the right safety profile. Here, we discuss recent progress in some of the main areas of toxicity, such as cardiotoxicity, hepatotoxicity, CNS toxicity, genetic toxicity, and nephrotoxicity, and also considerations around assay validation, predictivity, and throughput, which are relevant to secure actual impact on drug design. Finally, we share our perspectives on future opportunities and innovation.

Background Disturbance in the oropharyngeal microbiota is common in hospitalized patients and contributes to the development of nosocomial pneumonia. Lactiplantibacillus plantarum 299 and 299v (Lp299 and Lp299v) are probiotic bacteria with beneficial effects on the human microbiome. Aim To investigate how Lp299 and Lp299v affect the growth of nosocomial oropharyngeal pathogens in vitro and to evaluate the efficacy in vivo when these probiotics are administered prophylactically in hospitalized patients. Methods The in vitro effect of Lp299 and Lp299v on nosocomial respiratory tract pathogens was evaluated using two methods, the co‐culture and agar overlay. In the clinical study, patients were randomized to orally receive either probiotics or placebo twice daily during their hospital stay. Oropharyngeal swabs were analyzed at inclusion and every fourth day throughout hospitalization. Findings All tested pathogens were completely inhibited by both Lp299 and Lp299v using the agar‐overlay method. In the co‐culture experiment, Lp299 and Lp299v significantly (p, This combined laboratory and clinical study showed that Lactiplantibacillus plantarum 299 and 299v significantly inhibited in vitro growth of nosocomial pathogens often found in the oropharynx of hospitalized patients. In the randomized controlled trial, hospitalized patients were randomized to receive either oral twice‐daily treatment with lactobacilli or placebo, and oropharyngeal swabs were performed regularly throughout hospitalization. No differences could be shown between the Lactobacilli and placebo groups regarding changes in oropharyngeal microbiota or the occurrence of nosocomial infections.

Att besitta en god läsförståelse är viktigt för att kunna fungera i samhället men också lyckas i skolan. Läsförståelse är en komplex process vilken innefattar många olika delar. För att eleverna ska utveckla sin läsförståelse är lärarens val i undervisningen avgörande. I forskning framkommer mycket information om hur läraren kan arbeta för att utveckla läsförståelse. Forskning är dock bristfällig rörande hur lärare beskriver att de arbetar i praktiken med läsförståelse och vad de uppfattar som gynnsamt för att alla elever ska utveckla läsförståelse. Särskilt lite forskning finns gällande elever i läsförståelsesvårigheter. Syftet med denna studie är därför att öka kunskapen om hur lärare i F-3 beskriver att de arbetar för att utveckla läsförståelse. Särskilt fokus läggs på elever i läsförståelsesvårigheter. Syftet avses att uppfyllas med hjälp av följande frågeställningar: · Hur förklarar och motiverar lärare i årskurs F-3 sitt arbete med att utveckla elevers läsförståelse? · Hur förklarar och motiverar lärare i årskurs F-3 sitt arbete med att utveckla läsförståelse för elever i läsförståelsesvårigheter? I studien användes en kvalitativ semistrukturerad fokusintervju för att samla in materialet. Det var sex lärare från tre skolor som deltog. Resultatet analyserades sedan med utgångspunkt i ett sociokulturellt perspektiv. Resultatet i studien visar att lärarna anser det är viktigt att arbeta med utveckling av förmågorna avkodning, ord- och begreppsförståelse samt förmågan att använda läsförståelsestrategier genom varierade arbetssätt. För de elever i läsförståelsesvårigheter är den komponent som eleven brister i gällande ovanstående det som hen behöver arbeta med både genom intensivträning och genom anpassade arbetsformer tillsammans i klassen, där arbete med bildstöd och förförståelse kan fungera som stödjande verktyg för eleven. Sammanfattningsvis är det svårt att beskriva exakt hur lärare arbetar för att bana väg för alla elevernas läsförståelseutveckling. Alla elever är olika och läraren behöver anpassa sin undervisning efter elevgruppen. Developing reading comprehension is an important competence for being able to function in society but also to succeed in school. Reading comprehension is a complex process which involves many different parts. For students to develop their reading comprehension, the teacher's choice in teaching is crucial. Research reveals a lot of information about how the teacher can work to develop reading comprehension. However, there is a lack in research regarding how teachers describe that they work in practice with reading comprehension and what they perceive as favorable for all students to develop reading comprehension. There is very little research regarding students with reading comprehension difficulties. The aim of this study is therefore to increase the knowledge of how teachers in grades F-3 describe that they work to develop reading comprehension. A special focus is placed on students with reading comprehension difficulties. The aim will be fulfilled with the help of the following questions: • How do teachers in grades F-3 explain and motivate their work to develop students' reading comprehension?• How do teachers in grades F-3 explain and motivate their work to develop reading comprehension for students with reading comprehension difficulties? In the study, qualitative semi-structured focus interviews are used to collect data. Six teachers from three schools participated. The results were then analyzed based on a socio-cultural perspective. The results of the study show that the teachers consider it important to work with the development of the abilities decoding, word and concept comprehension as well as the ability to use reading comprehension strategies through varied working methods. For students with reading comprehension difficulties, the component that the student lacks in the above is what he or she needs to work with both through intensive training and through adapted working methods together in class, where work with pictures and pre-understanding can serve as supportive tools for the student. In summary, it is difficult to describe exactly how teachers work to pave the way for all students' reading comprehension development. All students are different, and the teacher needs to adapt his or her teaching to the student group.

Cannabis Social Clubs are typically non-profit associations established by adult users of cannabis, which produce and distribute cannabis among their members. Such associations can be found in several European countries and beyond, but with exception of Uruguay, they are illegal or otherwise operate at the margins of domestic drug control legislation, at risk for law enforcement detection. We have conducted several individual and collaborative studies on Cannabis Social Clubs and their key actors (e.g. Cannabis Social Clubs leaders, users affiliated as members, cannabis growers, other stakeholders), primarily drawing on qualitative methods (e.g. ethnographic fieldwork, interviews, qualitative media analyses) but also employing a quantitative approach (e.g. online surveys). In this article, we reflect on the research experiences of the authors in studying Cannabis Social Clubs, providing insights for future research in this area and within criminology and socio-legal studies. In particular, we aim to examine our approaches with regard to the recruitment of participants and research design, identifying good practices, but also discussing what the less successful strategies were. We draw on research conducted since 2014 in different countries, with different legal frameworks, which provides an opportunity for a comparative and more in-depth critical consideration of what might be helpful ways of reaching and researching hard-to-reach populations.

Läsning är en förmåga som alla elever behöver utveckla för att kunna tillägna sig kunskaper och för att kunna orientera sig i ett komplext informationssamhälle. Många elever uppvisar dock svårigheter i utvecklingen av läsningens delkomponenter, vilket kan ses som problematiskt. På grund av detta är denna litteraturstudies syfte att belysa om musik kan användas som ett pedagogiskt redskap för att stödja elever i processen att utveckla läsning i förskoleklass och grundskolans årskurser 1-3. Följande frågeställningar avser att bidra till att uppnå ovanstående syfte: Hur kan elever utveckla läsförståelse och läsförståelsestrategier genom musik? Hur kan elever utveckla sitt ordförråd genom musik? Hur kan elever utveckla avkodningsfärdigheter genom musik? Vilka andra påverkande faktorer synliggörs i lärandeprocessen genom användning av musik som ett pedagogiskt redskap? Litteraturen samlades in genom sökningar i olika databaser och består av såväl nationell som internationell forskning. Det vetenskapliga materialet analyserades och inkluderades utifrån bestämda kriterier och sammanfattades i en översiktstabell (se bilaga 1, analysöversikt). Resultatet visar att musik som ett pedagogiskt redskap kan stödja elever i läsutvecklingen. Kombinerad musik- och läsundervisning kan utveckla avkodning, ordförråd och lässtrategier som i sin tur utvecklar elevernas läsförståelse. I resultatet framkom även andra påverkande faktorer på lärandet. Musik kan också påverka hjärnans funktioner som gör att kunskaper lagras i minnet. Musik kan även bidra till motivation för att lära. Då skolans elever innefattar en stor mångfald är variation i undervisningen inom läsutveckling betydande. Kombinerad musik- och läsundervisning kan därför bli ett stödjande redskap i läsutvecklingen för att tillgodose alla elevers olika lärstilar, förmågor och förutsättningar.

Low-frequency electroacupuncture restores sex steroid synthesis and sympathetic activity in women with polycystic ovary syndrome, which may improve its metabolic disturbances likely by modulating sympathetic nerve activity or sex steroid synthesis. We investigated whether low-frequency electroacupuncture regulates the metabolic function to the same extent as treatment with estradiol or β-adrenergic blocking in a rat model of polycystic ovary syndrome induced by a P450 aromatase inhibitor (letrozole). Letrozole (200 μg per day) or placebo pellets were implanted in pre-pubertal Wistar rats. Six weeks thereafter, rats were treated for 5–6 weeks with: low-frequency electroacupuncture (5 days per week), a β-adrenergic blocker (propranolol hydrochloride, 0.1 mg kg−1) (5 days per week), or 17β-estradiol (2.0 μg) every fourth day. Body weight development, body composition, locomotor activity, insulin sensitivity, tissue specific glucose uptake, lipid profile, adipocyte size, adiponectin and insulin serum concentrations, and gene expression in inguinal fat were measured. All treatments increased circulating levels of LDL-cholesterol. Estradiol treatment restored locomotor activity and increased insulin sensitivity but did not modify the glucose uptake in muscle and fat. An upregulation of genes related to insulin sensitivity and downregulation of genes related to adipogenesis were observed in subcutaneous adipose tissue from rats exposed to letrozole. Only estradiol treatment normalized the expression of these genes. In conclusions, low-frequency electroacupuncture increased LDL-cholesterol without affecting the insulin sensitivity or adipose tissue function, which could suggest effects on hepatic lipid regulation probably mediated by estradiol action or β-adrenergic pathway. This article is protected by copyright. All rights reserved

Acupuncture with combined manual and low-frequency electrical stimulation, or electroacupuncture (EA), reduces endocrine and reproductive dysfunction in women with polycystic ovary syndrome (PCOS), likely by modulating sympathetic nerve activity or sex steroid synthesis. To test this hypothesis, we induced PCOS in rats by prepubertal implantation of continuous-release letrozole pellets (200 µg/day) or vehicle. Six weeks later, rats were treated for 5-6 weeks with low-frequency EA 5 days/week, subcutaneous injection of 17β-estradiol (2.0 µg) every fourth day, or a β-adrenergic blocker (propranolol hydrochloride, 0.1 mg/kg) 5 days/week. Letrozole controls were handled without needle insertion or injected with sesame oil every fourth day. Estrous cyclicity, ovarian morphology, sex steroids, gonadotropins, insulin-like growth factor I, bone mineral density, and gene and protein expression in ovarian tissue were measured. Low-frequency EA induced estrous-cycle changes, decreased high levels of circulating luteinizing hormone (LH) and the LH/follicle-stimulating hormone (FSH) ratio, decreased high ovarian gene expression of adiponectin receptor 2, and increased expression of adiponectin receptor 2 protein and phosphorylation of ERK1/2. EA also increased cortical bone mineral density. Propranolol decreased ovarian expression of Foxo3, Srd5a1, and Hif1a. Estradiol decreased circulating LH, induced estrous cycle changes, and decreased ovarian expression of Adipor1, Foxo3, and Pik3r1. Further, total bone mineral density was higher in the letrozole-estradiol group. Thus, EA modulates the circulating gonadotropin levels independently of sex steroids or β-adrenergic action and affects the expression of ovarian adiponectin system.

Acupuncture has been demonstrated to improve menstrual frequency and to decrease circulating testosterone in women with polycystic ovary syndrome (PCOS). Our aim was to investigate whether acupuncture affects ovulation frequency and to understand the underlying mechanisms of any such effect by analyzing LH and sex steroid secretion in women with PCOS. This prospective, randomized, controlled clinical trial was conducted between June 2009 and September 2010. Thirty-two women with PCOS were randomized to receive either acupuncture with manual and low-frequency electrical stimulation or to meetings with a physical therapist twice a week for 10–13 wk. Main outcome measures were changes in LH secretion patterns from baseline to after 10–13 wk of treatment and ovulation frequency during the treatment period. Secondary outcomes were changes in the secretion of sex steroids, anti-Müllerian hormone, inhibin B, and serum cortisol. Ovulation frequency during treatment was higher in the acupuncture group than in the control group. After 10–13 wk of intervention, circulating levels of estrone, estrone sulfate, estradiol, dehydroepiandrosterone, dehydroepiandrosterone sulfate, androstenedione, testosterone, free testosterone, dihydrotestosterone, androsterone glucuronide, androstane-3α,17β-diol-3-glucuronide, and androstane-3α,17β-diol-17-glucuronide decreased within the acupuncture group and were significantly lower than in the control group for all of these except androstenedione. We conclude that repeated acupuncture treatments resulted in higher ovulation frequency in lean/overweight women with PCOS and were more effective than just meeting with the therapist. Ovarian and adrenal sex steroid serum levels were reduced with no effect on LH secretion.

Studying the mechanisms for the complex pathogenesis of polycystic ovary syndrome (PCOS) requires animal models with endocrine, reproductive, and metabolic features of the syndrome. Hyperandrogenism seems to be a central factor in PCOS, leading to anovulation and insulin resistance. In female rats, continuous administration of letrozole, a nonsteroidal inhibitor of P450 aromatase, at 400 μg/d starting before puberty induces hyperandrogenemia and reproductive abnormalities similar to those in women with PCOS. However, despite high circulating testosterone levels, these rats do not develop metabolic abnormalities, perhaps because of their supraphysiological testosterone concentrations or because estrogen synthesis is completely blocked in insulin-sensitive tissues. To test the hypothesis that continuous administration of lower doses of letrozole starting before puberty would result in both metabolic and reproductive phenotypes of PCOS, we performed a 12-wk dose-response study. At 21 d of age, 46 female Wistar rats were divided into two letrozole groups (100 or 200 μg/d) and a control group (placebo). Both letrozole doses resulted in increased body weight, inguinal fat accumulation, anovulation, larger ovaries with follicular atresia and multiples cysts, endogenous hyperandrogemia, and lower estrogen levels. Moreover, rats that received 200 μg/d had insulin resistance and enlarged adipocytes in inguinal and mesenteric fat depots, increased circulating levels of LH, decreased levels of FSH, and increased ovarian expression of Cyp17a1 mRNA. Thus, continuous administration of letrozole, 200 μg/d, to female rats for 90 d starting before puberty results in a PCOS model with reproductive and metabolic features of the syndrome.

What is the central question of this study? The effectiveness of low-frequency electroacupuncture in the treatment of metabolic disorders associated with polycystic ovary syndrome (PCOS), an endocrine-metabolic disorder characterized by an imbalance in sex steroid production, is controversial. What is the main finding and its importance? In a rat model of PCOS induced by the inhibition of P450 aromatase, low-frequency electroacupuncture increased low-density lipoprotein-cholesterol but did not improve the insulin resistance or the adipose tissue dysfunction, suggesting that a balance of sex steroids is needed to restore the metabolic function in this rat model of PCOS. Low-frequency electroacupuncture restores sex steroid synthesis and sympathetic activity in women with polycystic ovary syndrome, which may ameliorate its metabolic disturbances, probably by modulating sympathetic nerve activity or sex steroid synthesis. We investigated whether low-frequency electroacupuncture regulates the metabolic function to the same extent as treatment with estradiol or β-adrenergic blocking in a rat model of polycystic ovary syndrome induced by a P450 aromatase inhibitor (letrozole). Letrozole (200 μg day

Both low-frequency electro-acupuncture (EA) and manual acupuncture improve menstrual frequency and decrease circulating androgens in women with polycystic ovary syndrome (PCOS). We sought to determine whether low-frequency EA is more effective than manual stimulation in regulating disturbed oestrous cyclicity in rats with PCOS induced by 5α-dihydrotestosterone. To identify the central mechanisms of the effects of stimulation, we assessed hypothalamic mRNA expression of molecules that regulate reproductive and neuroendocrine function. From age 70 days, rats received 2 Hz EA or manual stimulation with the needles five times per week for 4-5 weeks; untreated rats served as control animals. Specific hypothalamic nuclei were obtained by laser microdissection, and mRNA expression was measured with TaqMan low-density arrays. Untreated rats were acyclic. During the last 2 weeks of treatment, seven of eight (88%) rats in the EA group had epithelial keratinocytes, demonstrating oestrous cycle change (P = 0.034 versus control rats). In the manual group, five of eight (62%) rats had oestrous cycle changes (n.s. versus control animals). The mRNA expression of the opioid receptors Oprk1 and Oprm1 in the hypothalamic arcuate nucleus was lower in the EA group than in untreated control rats. The mRNA expression of the steroid hormone receptors Esr2, Pgr and Kiss1r was lower in the manual group than in the control animals. In rats with 5α-dihydrotestosterone-induced PCOS, low-frequency EA restored disturbed oestrous cyclicity but did not differ from the manual stimulation group, although electrical stimulation lowered serum testosterone in responders, those with restored oestrus cyclicity, and differed from both control animals and the manual stimulation group. Thus, EA cannot in all aspects be considered superior to manual stimulation. The effects of low-frequency EA may be mediated by central opioid receptors, while manual stimulation may involve regulation of steroid hormone/peptide receptors.

To compare the effect of a single session of acupuncture with either low-frequency electrical or manual stimulation on insulin sensitivity and molecular pathways in the insulin-resistant dihydrotestosterone-induced rat polycystic ovary syndrome (PCOS) model. Both stimulations cause activation of afferent nerve fibers. In addition, electrical stimulation causes muscle contractions, enabling us to differentiate changes induced by activation of sensory afferents from contraction-induced changes. Control and PCOS rats were divided into no-stimulation, manual-, and electrical stimulation groups and insulin sensitivity was measured by euglycemic hyperinsulinemic clamp. Manually stimulated needles were rotated 180° ten times every 5 min, or low-frequency electrical stimulation was applied to evoke muscle twitches for 45 min. Gene and protein expression were analyzed by real-time PCR and Western blot. The glucose infusion rate (GIR) was lower in PCOS rats than in controls. Electrical stimulation was superior to manual stimulation during treatment but both methods increased GIR to the same extent in the post-stimulation period. Electrical stimulation decreased mRNA expression of Adipor2, Adrb1, Fndc5, Erk2, and Tfam in soleus muscle and increased ovarian Adrb2 and Pdf. Manual stimulation decreased ovarian mRNA expression of Erk2 and Sdnd. Electrical stimulation increased phosphorylated ERK levels in soleus muscle. One acupuncture session with electrical stimulation improves insulin sensitivity and modulates skeletal muscle gene and protein expression more than manual stimulation. Although electrical stimulation is superior to manual in enhancing insulin sensitivity during stimulation, they are equally effective after stimulation indicating that it is activation of sensory afferents rather than muscle contraction per se leading to the observed changes.

Polycystic ovary syndrome (PCOS), the most common endocrine disorder among women of reproductive age, is characterized by the coexistence of hyperandrogenism, ovulatory dysfunction, and polycystic ovaries (PCO). PCOS also represents the largest part of female oligoovulatory infertility, and the management of ovulatory and menstrual dysfunction, comprises a third of the high costs of PCOS treatment. Current pharmacological and surgical treatments for reproductive symptoms are effective, however, associated with negative side effects, such as cardiovascular complications and multiple pregnancies. For menstrual irregularities and ovulation induction in women with PCOS, acupuncture has indicated beneficial effects. This review will focus on the results from randomized controlled acupuncture trials for regulation of menstrual dysfunction and for inducing ovulation in women with PCOS although there are uncontrolled trials with nonetheless interesting results. Animal experimental studies will be further discussed when they can provide a more mechanistic explanatory view.

In rats with dihydrotestosterone (DHT)-induced polycystic ovary syndrome (PCOS), repeated low-frequency electrical stimulation of acupuncture needles restores whole-body insulin sensitivity measured by euglycemic hyperinsulinemic clamp. We hypothesized that electrical stimulation causing muscle contractions and manual stimulation causing needle sensation have different effects on insulin sensitivity and related signaling pathways in skeletal muscle and adipose tissue, with electrical stimulation being more effective in DHT-induced PCOS rats. From age 70 days, rats received manual or low-frequency electrical stimulation of needles in abdominal and hind limb muscle five times/wk for 4-5 wks; controls were handled but untreated rats. Low-frequency electrical stimulation modified gene expression (decreased Tbc1d1 in soleus, increased Nr4a3 in mesenteric fat) and protein expression (increased pAS160/AS160, Nr4a3 and decreased GLUT4) by western blot and increased GLUT4 expression by immunohistochemistry in soleus muscle; glucose clearance during oral glucose tolerance tests was unaffected. Manual stimulation led to faster glucose clearance and modified mainly gene expression in mesenteric adipose tissue (increased Nr4a3, Mapk3/Erk, Adcy3, Gsk3b), but not protein expression to the same extent; however, Nr4a3 was reduced in soleus muscle. The novel finding is that electrical and manual muscle stimulation affect glucose homeostasis in DHT-induced PCOS rats through different mechanisms. Repeated electrical stimulation regulated key functional molecular pathways important for insulin sensitivity in soleus muscle and mesenteric adipose tissue to a larger extent than manual stimulation. Manual stimulation improved whole-body glucose tolerance, an effect not observed after electrical stimulation, but did not affect molecular signaling pathways to the same extent as electrical stimulation. Although more functional signaling pathways related to insulin sensitivity were affected by electrical stimulation, our findings suggest that manual stimulation of acupuncture needles has a greater effect on glucose tolerance. The underlying mechanism of the differential effects of the intermittent manual and the continuous electrical stimulation remains to be elucidated.

Here, we tested the hypothesis that excess maternal androgen in late pregnancy reduces placental and fetal growth, increases placental steroidogenesis, and adversely affects glucose and lipid metabolism in adult female offspring. Pregnant Wistar rats were randomly assigned to treatment with testosterone (daily injections of 5 mg of free testosterone from gestational days 16 to 19) or vehicle alone. In experiment 1, fetal and placental weights, circulating maternal testosterone, estradiol, and corticosterone levels, and placental protein expression and distribution of estrogen receptor-α and -β, androgen receptor, and 17β-hydroxysteroid dehydrogenase 2 were determined. In experiment 2, birth weights, postnatal growth rates, circulating testosterone, estradiol, and corticosterone levels, insulin sensitivity, adipocyte size, lipid profiles, and the presence of nonalcoholic fatty liver were assessed in female adult offspring. Treatment with testosterone reduced placental and fetal weights and increased placental expression of all four proteins. The offspring of testosterone-treated dams were born with intrauterine growth restriction; however, at 6 wk of age there was no difference in body weight between the offspring of testosterone- and control-treated rats. At 10–11 wk of age, the offspring of the testosterone-treated dams had less fat mass and smaller adipocyte size than those born to control rats and had no difference in insulin sensitivity. Circulating triglyceride levels were higher in the offspring of testosterone-treated dams, and they developed nonalcoholic fatty liver as adults. We demonstrate for the first time that prenatal testosterone exposure alters placental steroidogenesis and leads to dysregulation of lipid metabolism in their adult female offspring.

Both low-frequency electro-acupuncture (EA) and manual acupuncture improve menstrual frequency and decrease circulating androgens in women with polycystic ovary syndrome (PCOS). We sought to determine whether low-frequency EA is more effective than manual stimulation in regulating disturbed oestrous cyclicity in rats with PCOS induced by 5α-dihydrotestosterone. To identify the central mechanisms of the effects of stimulation, we assessed hypothalamic mRNA expression of molecules that regulate reproductive and neuroendocrine function. From age 70 days, rats received 2 Hz EA or manual stimulation with the needles five times per week for 4-5 weeks; untreated rats served as control animals. Specific hypothalamic nuclei were obtained by laser microdissection, and mRNA expression was measured with TaqMan low-density arrays. Untreated rats were acyclic. During the last 2 weeks of treatment, seven of eight (88%) rats in the EA group had epithelial keratinocytes, demonstrating oestrous cycle change (P = 0.034 versus control rats). In the manual group, five of eight (62%) rats had oestrous cycle changes (n.s. versus control animals). The mRNA expression of the opioid receptors Oprk1 and Oprm1 in the hypothalamic arcuate nucleus was lower in the EA group than in untreated control rats. The mRNA expression of the steroid hormone receptors Esr2, Pgr and Kiss1r was lower in the manual group than in the control animals. In rats with 5α-dihydrotestosterone-induced PCOS, low-frequency EA restored disturbed oestrous cyclicity but did not differ from the manual stimulation group, although electrical stimulation lowered serum testosterone in responders, those with restored oestrus cyclicity, and differed from both control animals and the manual stimulation group. Thus, EA cannot in all aspects be considered superior to manual stimulation. The effects of low-frequency EA may be mediated by central opioid receptors, while manual stimulation may involve regulation of steroid hormone/peptide receptors.

The causes of anxiety and depression in women with polycystic ovary syndrome (PCOS) remain elusive. To identify steps linking androgen signaling to the regulation of affective symptoms in vivo, we compared behavioral responses in female rats continuously exposed to DHT from puberty (a model of DHT-induced PCOS) and in rats exposed to DHT for 1 week. Continuous and 1 week of DHT exposure resulted in a general decrease in locomotor activity and time spent on the open arms in the elevated plus maze, indicating anxiety-like behavior. Rats with DHT-induced PCOS have increases in adiposity and circulating leptin levels accompanied by leptin resistance. One week of DHT exposure decreased androgen receptor (AR) expression in the hypothalamus and leptin synthesis and function in adipocytes; it also inhibited signal transducer and activator of transcription 3 (STAT3) and attenuated leptin activity by increasing levels of soluble leptin receptor, a leptin-binding protein, in the hypothalamus. This may affect the androgen-induced anxiety-related behavior in female rats. In conclusion, our results highlight the central role of androgens in behavioral function in female rats and suggest that androgens directly regulate the AR by decreasing its hypothalamic expression. Androgens also increase leptin synthesis in adipocytes, which drives central leptin signaling, and may regulate anxiety-related behaviors. Elucidating mechanisms by which androgens modulate female anxiety-like behavior may uncover useful approaches for treating women with PCOS who have symptoms of anxiety.

Polycystic ovary syndrome (PCOS) is associated with hyperandrogenism and insulin resistance, possibly reflecting defects in skeletal muscle and adipocyte insulin signaling. Low-frequency (2 Hz) electroacupuncture (EA) increases insulin sensitivity in female rats with dihydrotestosterone (DHT)-induced PCOS, but the mechanism is unclear. We hypothesized that low-frequency EA regulates mediators involved in skeletal muscle glucose uptake and metabolism and alters the lipid profile in rats with DHT-induced PCOS. To test this hypothesis, we implanted in prepubescent female rats 90-day continuous-release pellets containing DHT (PCOS). At 70 days of age, the rats were randomly subdivided into two groups: one received low-frequency EA (evoking muscle twitches) for 20–25 min five times/wk for 4–5 wk; the other did not. Controls were implanted with pellets containing vehicle only. All three groups were otherwise handled similarly. Lipid profile was measured in fasting blood samples. Insulin sensitivity was determined by euglycemic hyperinsulinemic clamp, soleus muscle protein expression of glucose transporter 4 (GLUT4), and phosphorylated and nonphosphorylated Akt, and Akt substrate of 160 kDa was determined by Western blot analysis and GLUT4 location by immunofluorescence staining. PCOS EA rats had normalized insulin sensitivity, lower levels of total high-density lipoprotein and low-density lipoprotein cholesterol, and increased expression of GLUT4 in different compartments of skeletal muscle compared with PCOS rats. Total weight and body composition did not differ in the groups. Thus, in rats with DHT-induced PCOS, low-frequency EA has systemic and local effects involving intracellular signaling pathways in muscle that may, at least in part, account for the marked improved insulin sensitivity.

Adult female rats continuously exposed to androgens from prepuberty have reproductive and metabolic features of polycystic ovary syndrome (PCOS). We investigated whether such exposure adversely affects estrous cyclicity and the expression and distribution of gonadotropin-releasing hormone (GnRH), GnRH receptors, and corticotrophin-releasing hormone (CRH) in the hypothalamus and whether the effects are mediated by the androgen receptor (AR). We also assessed the effect of low-frequency electro-acupuncture (EA) on those variables. At 21 days of age, rats were randomly divided into three groups (control, PCOS, and PCOS EA; n = 12/group) and implanted subcutaneously with 90-day continuous-release pellets containing vehicle or 5alpha-dihydrostestosterone (DHT). From age 70 days, PCOS EA rats received 2-Hz EA (evoking muscle twitches) five times/week for 4-5 weeks. Hypothalamic protein expression was measured by immunohistochemistry and western blot. DHT-treated rats were acyclic, but controls had regular estrous cycles. In PCOS rats, hypothalamic medial preoptic AR protein expression and the number of AR- and GnRH-immunoreactive cells were increased, but CRH was not affected; however, GnRH receptor expression was decreased in both the pituitary and hypothalamus. Low-frequency EA restored estrous cyclicity within 1 week and reduced the elevated hypothalamic GnRH and AR expression levels. EA did not affect GnRH receptor or CRH expression. Interestingly, nuclear AR co-localized with GnRH in the hypothalamus. Thus, rats with DHT-induced PCOS have disrupted estrous cyclicity and an increased number of hypothalamic cells expressing GnRH, most likely mediated by AR activation. Repeated low-frequency EA normalized estrous cyclicity and restored GnRH and AR protein expression. These results may help explain the beneficial neuroendocrine effects of low-frequency EA in women with PCOS.