Chellakkan S. Blesson

Elevated Testosterone Levels During Rat Pregnancy Cause Hypersensitivity to Angiotensin II and Attenuation of Endothelium-Dependent Vasodilation in Uterine Arteries

Elevated testosterone levels increase maternal blood pressure and decrease uterine blood flow in pregnancy, resulting in abnormal perinatal outcomes. We tested whether elevated testosterone alters uterine artery adaptations during pregnancy, and whether these alterations depend on endothelium-derived factors such as nitric oxide, endothelium-derived hyperpolarizing factor, and prostacyclin, or endothelium-independent mechanisms such as angiotensin II (Ang-II). Pregnant Sprague–Dawley rats were injected with vehicle (n=20) or testosterone propionate (0.5 mg/kg per day from gestation day 15 to 19; n=20). Plasma testosterone levels increased 2-fold in testosterone-injected rats compared with controls. Elevated testosterone significantly decreased placental and pup weights compared with controls. In endothelium-intact uterine arteries, contractile responses to thromboxane, phenylephrine, and Ang-II were greater in testosterone-treated rats compared with controls. In endothelium-denuded arteries, contractile responses to Ang-II (pD2=9.1±0.04 versus 8.7±0.04 in controls; P<0.05), but not thromboxane and phenylephrine, were greater in testosterone-treated rats. Ang-II type 1b receptor expression was increased, whereas Ang-II type 2 receptor was decreased in testosterone-exposed arteries. In endothelium-denuded arteries, relaxations to sodium nitroprusside were unaffected. Endothelium-dependent relaxation to acetylcholine was significantly lower in arteries from testosterone-treated dams (Emax=51.80±6.9% versus 91.98±1.4% in controls; P<0.05). The assessment of endothelial factors showed that nitric oxide–, endothelium-derived hyperpolarizing factor–, and prostacyclin-mediated relaxations were blunted in testosterone-treated dams. Endothelial nitric oxide synthase, small conductance calcium–activated potassium channel-3, and prostacyclin receptor expressions were significantly decreased in arteries from testosterone-treated dams. Hypoxia-inducible factor-1&agr;, Ankrd37, and Egln were significantly increased in testosterone-exposed placentas. These results suggest that elevated maternal testosterone impairs uterine vascular function, which may lead to an increased vascular resistance and a decrease in uterine blood flow.

Prenatal Testosterone Exposure Induces Hypertension in Adult Females via Androgen Receptor–Dependent Protein Kinase Cδ–Mediated Mechanism

Prenatal exposure to excess testosterone induces hyperandrogenism in adult females and predisposes them to hypertension. We tested whether androgens induce hypertension through transcriptional regulation and signaling of protein kinase C (PKC) in the mesenteric arteries. Pregnant Sprague–Dawley rats were injected with vehicle or testosterone propionate (0.5 mg/kg per day from gestation days 15 to 19, SC) and their 6-month-old adult female offspring were examined. Plasma testosterone levels (0.84±0.04 versus 0.42±0.09 ng/mL) and blood pressures (111.6±1.3 versus 104.5±2.4 mm Hg) were significantly higher in prenatal testosterone–exposed rats compared with controls. This was accompanied with enhanced expression of PKC&dgr; mRNA (1.5-fold) and protein (1.7-fold) in the mesenteric arteries of prenatal testosterone–exposed rats. In addition, mesenteric artery contractile responses to PKC activator, phorbol-12,13-dibutyrate, was significantly greater in prenatal testosterone–exposed rats. Treatment with androgen receptor antagonist flutamide (10 mg/kg, SC, BID for 10 days) significantly attenuated hypertension, PKC&dgr; expression, and the exaggerated vasoconstriction in prenatal testosterone–exposed rats. In vitro exposure of testosterone to cultured mesenteric artery smooth muscle cells dose dependently upregulated PKC&dgr; expression. Analysis of PKC&dgr; gene revealed a putative androgen responsive element in the promoter upstream to the transcription start site and an enhancer element in intron-1. Chromatin immunoprecipitation assays showed that androgen receptors bind to these elements in response to testosterone stimulation. Furthermore, luciferase reporter assays showed that the enhancer element is highly responsive to androgens and treatment with flutamide reverses reporter activity. Our studies identified a novel androgen-mediated mechanism for the control of PKC&dgr; expression via transcriptional regulation that controls vasoconstriction and blood pressure.

Gestational protein restriction impairs insulin-regulated glucose transport mechanisms in gastrocnemius muscles of adult male offspring.

Type II diabetes originates from various genetic and environmental factors. Recent studies showed that an adverse uterine environment such as that caused by a gestational low-protein (LP) diet can cause insulin resistance in adult offspring. The mechanism of insulin resistance induced by gestational protein restriction is not clearly understood. Our aim was to investigate the role of insulin signaling molecules in gastrocnemius muscles of gestational LP diet-exposed male offspring to understand their role in LP-induced insulin resistance. Pregnant Wistar rats were fed a control (20% protein) or isocaloric LP (6%) diet from gestational day 4 until delivery and a normal diet after weaning. Only male offspring were used in this study. Glucose and insulin responses were assessed after a glucose tolerance test. mRNA and protein levels of molecules involved in insulin signaling were assessed at 4 months in gastrocnemius muscles. Muscles were incubated ex vivo with insulin to evaluate insulin-induced phosphorylation of insulin receptor (IR), Insulin receptor substrate-1, Akt, and AS160. LP diet-fed rats gained less weight than controls during pregnancy. Male pups from LP diet-fed mothers were smaller but exhibited catch-up growth. Plasma glucose and insulin levels were elevated in LP offspring when subjected to a glucose tolerance test; however, fasting levels were comparable. LP offspring showed increased expression of IR and AS160 in gastrocnemius muscles. Ex vivo treatment of muscles with insulin showed increased phosphorylation of IR (Tyr972) in controls, but LP rats showed higher basal phosphorylation. Phosphorylation of Insulin receptor substrate-1 (Tyr608, Tyr895, Ser307, and Ser318) and AS160 (Thr642) were defective in LP offspring. Further, glucose transporter type 4 translocation in LP offspring was also impaired. A gestational LP diet leads to insulin resistance in adult offspring by a mechanism involving inefficient insulin-induced IR, Insulin receptor substrate-1, and AS160 phosphorylation and impaired glucose transporter type 4 translocation.

Prenatal Testosterone Exposure Leads to Gonadal Hormone-Dependent Hyperinsulinemia and Gonadal Hormone-Independent Glucose Intolerance in Adult Male Rat Offspring

ABSTRACT Elevated testosterone levels during prenatal life lead to hyperandrogenism and insulin resistance in adult females. This study evaluated whether prenatal testosterone exposure leads to the development of insulin resistance in adult male rats in order to assess the influence of gonadal hormones on glucose homeostasis in these animals. Male offspring of pregnant rats treated with testosterone propionate or its vehicle (control) were examined. A subset of male offspring was orchiectomized at 7 wk of age and reared to adulthood. At 24 wk of age, fat weights, plasma testosterone, glucose homeostasis, pancreas morphology, and gastrocnemius insulin receptor (IR) beta levels were examined. The pups born to testosterone-treated mothers were smaller at birth and remained smaller through adult life, with levels of fat deposition relatively similar to those in controls. Testosterone exposure during prenatal life induced hyperinsulinemia paralleled by an increased HOMA-IR index in a fasting state and glucose intolerance and exaggerated insulin responses following a glucose tolerance test. Prenatal androgen-exposed males had more circulating testosterone during adult life. Gonadectomy prevented hyperandrogenism, reversed hyperinsulinemia, and attenuated glucose-induced insulin responses but did not alter glucose intolerance in these rats. Prenatal androgen-exposed males had decreased pancreatic islet numbers, size, and beta-cell area along with decreased expression of IR in gastrocnemius muscles. Gonadectomy restored pancreatic islet numbers, size, and beta-cell area but did not normalize IRbeta expression. This study shows that prenatal testosterone exposure leads to a defective pancreas and skeletal muscle function in male offspring. Hyperinsulinemia during adult life is gonad-dependent, but glucose intolerance appears to be independent of postnatal testosterone levels.

Pregnancy Is a New Window of Susceptibility for Bisphenol A Exposure

Bisphenol A (BPA) is a well-known synthetic compound that is widely studied for its various toxic and endocrine disrupting properties. In our industrialized world, BPA is ubiquitous; it is present in our food, water, air, and clothes. At relevant levels, BPA is involved or associated with an array of diseases related to reproduction, development, metabolism, cancers, immunity, and inflammation (1, 2). Although several studies showed inconclusive or mixed results (3,–5), BPA showed toxic effects in many animal studies during certain windows of susceptibility (1). One such well-studied susceptibility window for BPA action is during early development. There is well-documented evidence in laboratory animals demonstrating that in utero exposure to BPA increases the risk of numerous adverse effects to the offspring (6). Consequently, the United States Food and Drug Administration (FDA) issued an assessment identifying BPA as a possible health risk to fetuses, infants, and children. The FDA, however, recently revised its assessment saying “FDAs current perspective, based on its most recent safety assessment, is that BPA is safe at the current levels occurring in foods” (7). Meanwhile, the FDA continues to review and monitor new scientific results on possible safety issues with the use of BPA. As the debate on the safety of BPA continues, an interesting new study published in this issue of Endocrinology identifies a possible new health concern for mothers (8). Alonso-Magdalena et al show that pregnancy is a critical window of susceptibility for BPA effects, potentially causing glucose intolerance and altered insulin sensitivity in mothers later in their lives (8). Their findings reveal that gestational BPA exposure has detrimental long-term effects in the glucose metabolism of the dams (8). In a series of well-designed experiments, they show that BPA exposure from days 9–16 of gestation caused hyperglycemia after 4 months and the condition deteriorated with time when compared with vehicle-treated controls and nonpregnant mice with a similar exposure. Further, these affected mice secreted less insulin due to impaired insulin production in the pancreatic β-cells, and Alonso-Magdalena et al (8) go on to explain the mechanism for decreased insulin production. They also show that these mice have significantly increased gonadal fat depot. This study is of immense importance because 1) it identifies a previously unknown effect of BPA on maternal health, and 2) it identifies pregnancy as a new window of susceptibility for the development of diabetes in mothers. Until recently, the prenatal and neonatal periods were thought to be most vulnerable to environmental exposures and investigations were focused on effects in offspring. It is well known that various adverse uterine environment causes hyperglycemia and insulin resistance in the offspring. Effects such as caloric restriction (9), low protein diet (10, 11), overnutrition (12), exposure to glucocorticoids (13), and BPA (14) are known to cause hyperglycemia and insulin resistance in offspring. However, this study has forced us to rethink and expand our attention to include the maternal wellbeing also. Effects of maternal BPA exposure in humans are not well understood. Several epidemiological studies have shown that parity is a risk for the development of type 2 diabetes in later life (15,–21); however, other studies have contradictory findings (22,–24). These conflicting findings have been suggested to be due to other confounding factors such as body weight, dietary habits, and socio-economic status (23). In the light of the new findings in mice, it is tempting to ask whether BPA is also a contributing factor in the development of type 2 diabetes in these women. Currently, we do not know whether BPA plays any role in the development of type 2 diabetes in women after pregnancy and further detailed investigations are needed. We need well-defined studies to correlate BPA levels during pregnancy in normal women and determine the odds of the development of type 2 diabetes in these women in subsequent years. We will need to gather this information in different ethnicities and geographical locations to arrive at a concrete conclusion. BPA is pervasive and its daily exposure is inevitable. Although the FDA considers that it is safe at current exposure levels in food, we still do not know many aspects of this compound. This study by Alonso-Magdalena and coworkers (8) shows that exposure levels alone can no longer be considered as the only criteria for toxicity studies, the timing of the exposure may be even more important. Even low levels of exposure that are currently considered “acceptable” may cause serious ill effects during certain vulnerable periods of life. Furthermore, the effects of exposure may not be immediate and may take months or years to develop. This study has identified pregnancy as a new window of susceptibility for BPA action. It is not known whether there are other such windows of susceptibility that could cause insulin resistance and hyperglycemia. We need to take into consideration not just the levels and duration of exposure but also when the exposure occurs. As with early development and pregnancy, are there other periods of vulnerability? Could exposure to BPA during puberty, menopause, or certain immunocompromised states have any health consequences? What levels of exposure, if any, are safe during these susceptibility periods? We do not know the answers to these questions. Hence, instead of downplaying the possible risks of BPA exposure, we must realize that we have a long way to go and many questions remain yet unanswered.

Gestational Protein Restriction impairs Glucose Disposal in the Gastrocnemius muscles of Female Rats.

Gestational low-protein (LP) diet causes hyperglycemia and insulin resistance in adult offspring, but the mechanism is not clearly understood. In this study, we explored the role of insulin signaling in gastrocnemius muscles of gestational LP-exposed female offspring. Pregnant rats were fed a control (20% protein) or an isocaloric LP (6%) diet from gestational day 4 until delivery. Normal diet was given to mothers after delivery and to pups after weaning until necropsy. Offspring were euthanized at 4 months, and gastrocnemius muscles were treated with insulin ex vivo for 30 minutes. Messenger RNA and protein levels of molecules involved in insulin signaling were assessed at 4 months. LP females were smaller at birth but showed rapid catchup growth by 4 weeks. Glucose tolerance test in LP offspring at 3 months showed elevated serum glucose levels (P < 0.01; glycemia Δ area under the curve 342 ± 28 in LP vs 155 ± 23 in controls, mmol/L * 120 minutes) without any change in insulin levels. In gastrocnemius muscles, LP rats showed reduced tyrosine phosphorylation of insulin receptor substrate 1 upon insulin stimulation due to the overexpression of tyrosine phosphatase SHP-2, but serine phosphorylation was unaffected. Furthermore, insulin-induced phosphorylation of Akt, glycogen synthase kinase (GSK)-3α, and GSK-3β was diminished in LP rats, and they displayed an increased basal phosphorylation (inactive form) of glycogen synthase. Our study shows that gestational protein restriction causes peripheral insulin resistance by a series of phosphorylation defects in skeletal muscle in a mechanism involving insulin receptor substrate 1, SHP-2, Akt, GSK-3, and glycogen synthase causing dysfunctional GSK-3 signaling and increased stored glycogen, leading to distorted glucose homeostasis.

Novel lean type 2 diabetic rat model using gestational low-protein programming.

BACKGROUND Type 2 diabetes (T2D) in lean individuals is not well studied and up to 26% of diabetes occurs in these individuals. Although the cause is not well understood, it has been primarily attributed to nutritional issues during early development. OBJECTIVE Our objective was to develop a lean T2D model using gestational low-protein (LP) programming. STUDY DESIGN Pregnant rats were fed control (20% protein) or isocaloric LP (6%) diet from gestational day 4 until delivery. Standard diet was given to dams after delivery and to pups after weaning. Glucose tolerance test was done at 2, 4, and 6 months of age. Magnetic resonance imaging of body fat for females was done at 4 months. Rats were sacrificed at 4 and 8 months of age and their perigonadal, perirenal, inguinal, and brown fat were weighed and expressed relative to their body weight. Euglycemic-hyperinsulinemic clamp was done around 6 months of age. RESULTS Male and female offspring exposed to a LP diet during gestation developed glucose intolerance and insulin resistance (IR). Further, glucose intolerance progressed with increasing age and occurred earlier and was more severe in females when compared to males. Euglycemic-hyperinsulinemic clamp showed whole body IR in both sexes, with females demonstrating increased IR compared to males. LP females showed a 4.5-fold increase in IR while males showed a 2.5-fold increase when compared to their respective controls. Data from magnetic resonance imaging on female offspring showed no difference in the subcutaneous, inguinal, and visceral fat content. We were able to validate this observation by sacrificing the rats at 4 and 8 months and measuring total body fat content. This showed no differences in body fat content between control and LP offspring in either males or females. Additionally, diabetic rats had a similar body mass index to that of the controls. CONCLUSION LP gestational programming produces a progressively worsening T2D model in rats with a lean phenotype without obesity.

Adrenomedullin2 (ADM2)/Intermedin (IMD) in Rat Ovary: Changes in Estrous Cycle and Pregnancy and Its Role in Ovulation and Steroidogenesis

ABSTRACT Adrenomedullin2 (ADM2) is reported to facilitate embryo implantation and placental development. Therefore, the current study was undertaken to identify if ADM2 has a functional role in ovary to facilitate its reproductive actions. This study shows that the expression of ADM2 is differentially regulated in rat estrous cycle and that ADM2 increases the synthesis and secretion of 17beta-estradiol accompanied with an increase in the expression of steroidogenic factor 1 (Sf1), estrogen receptor Esr1, and enzymes involved in steroidogenesis in equine chorionic gonadotropin (eCG)-treated rat ovaries. In addition, inhibition of endogenous ADM2 function in eCG-treated immature rats caused impaired ovulation. Furthermore, the mRNA expression of Adm2 and receptor activity modifying protein 3 is higher in the ovary on Day 18 compared to nonpregnant and pregnant rats on Day 22. ADM2-like immunoreactivity is localized in granulosa cells, blood vessels, oocytes, cumulous oophorus, and corpus luteum of pregnant ovaries, suggesting a potential role for ADM2 in the ovary. This is supported by the presence of ADM2-like immunoreactivity in the corpus luteum during pregnancy and a decline in aromatase immunoreactivity in corpus luteum on Day 9 of gestation in rats infused with ADM2 antagonist during implantation and decidualization phase. Taken together, this study suggests a potential involvement of ADM2 in the rat ovary in regulating synthesis of estradiol to support ovulation and facilitate efficient implantation and placental development for a successful pregnancy.

Polycystic Ovary Syndrome: A Multifaceted Enigma

Polycystic Ovary Syndrome: A Multifaceted Enigma Polycystic ovary syndrome (PCOS) is the most common form of ovulatory dysfunction, affecting up to 8% of the population worldwide, or over 100 million women [1-3]. While subject to some controversy, it is diagnosed most frequently by the Rotterdam criteria, which includes irregular menses, laboratory or clinical evidence of hyperandrogenemia, and polycystic ovarian morphology. The diagnosis may be made if a patient has at least two of these criteria, and other possible etiologies are excluded, such as prolactin or thyroid disorders [3]. One needs only to consider the history of the diagnosis of PCOS for a glimpse of the complex nature of this disease. Up to present day, medical professionals and organizations have engaged in contentious debate as to the best way to characterize and define this heterogenous cohort of patients [4,5]. As an example, the finding of polycystic ovarian morphologyon ultrasound exists in a large proportion of women without PCOS, and may in these scenarios be normal [6,7].Thus, some have argued for more weight placed on the criteria of menstrual regularity or hyperandrogenemia [1]. Hyperandrogenemia may be the most essential component of the Rotterdam criteria; not only due to the poor sensitivity and specificity of polycystic ovarian morphologyand broad differential diagnosis of oligo-ovulation, but also due to the inherent pathology due to increased androgen production in an affected female. Hyperandrogenemia from PCOS stems from increased ovarian and adrenal androgen synthesis as well as decreased production of sex hormone binding globulin, resulting in a surplus bioavailability [8]. Downstream effects include alterations in enzymes in the steroid pathway [9-12], and the hypothalamic-pituitary axis [13-15], just to name a few. Further, clinical manifestations of hyperandrogenemia include hirsutism and virilization which cause significant morbidity for patients and can prove to be quite difficult to treat [16]. Despite any controversy in the diagnosis, it remains an important disease to recognize as it carries with it an array of associated risks and comorbidities [17]. PCOS patients have increased risk of chronic metabolic derangements such as hypertension, hyperlipidemia, obesity, and diabetes mellitus which ultimately lead to long term complications such as cardiovascular and renal disease. Patients also have increased risk of depression and anxiety, further affecting their quality of life [18]. Additionally, there is an increased risk of certain cancers, such as endometrial cancer, likely due to anovulation and unopposed estrogen effects. Reproductive physiology is affected as well, with research showing increased incidence of infertility, higher rates of miscarriage [19,20], and significant obstetric risks including preeclampsia, gestational diabetes, preterm delivery, higher neonatal intensive care unit admission, and overall infant mortality [17]. From a research standpoint, PCOS is a fascinating subject, though fraught with difficulty, as it represents a wide array of endocrinopathy leading to its phenotype. PCOS may be described as the common endpoint of several metabolic disturbances, including but not limited to aberrant hypothalamic-pituitary-ovarian signaling, insulin resistance and glucose intolerance, obesity and the metabolic syndrome, hyperandrogenism, environmental and genetic factors, among several others [1,21,22]. This diverse array of pathology provides a helpful illustration of the difficulty inherent in looking at this population. While this makes PCOS a topic of great interest, it also requires a high level of scrutiny and scientific rigor. There are many contradictory and conflicting studies in the field of PCOS, and this may be in large part due to its heterogeneity. When diagnostic criteria are debated, and patients do not share all the same baseline pathology, confounders are inherently introduced that will skew any results [23]. The quintessential example in the case of PCOS is obesity [24]. Approximately 60-80% of PCOS patients are obese, and obesity has well documented adverse outcomes independent of PCOS, though similar to those listed *Corresponding author:

Folate treatment partially reverses gestational low protein diet induced glucose intolerance and the magnitude of reversal is age and sex dependent

OBJECTIVES Gestational low-protein (LP) programming causes glucose intolerance (GI) and insulin resistance (IR) in adult offspring. Folate supplementation has been shown to rescue the offspring from various programming effects. The aim of this study was to investigate whether folate supplementation during pregnancy reverses LP-induced GI and IR. METHODS Pregnant rats were fed control (20% protein), isocaloric low-protein (LP, 6%) or LP with 5 mg/kg folate (LPF) diets from gestational day 4 to delivery. The control diet was given during lactation and to pups after weaning. Glucose tolerance test was done at 1, 2, and 3 mo of age followed by euglycemic-hyperinsulinemic clamp at 4 mo. Rats were sacrificed at 4 mo and their gonadal, renal, inguinal, brown fat, and pancreas were weighed and expressed relative to their body weight. RESULTS LP- and LPF-fed dams showed similar weight loss during late pregnancy after decreased feed intake. Both LP and LPF pups were smaller at birth but their weights caught up like that of controls by 3 mo. In males, folate supplementation reduced LP-induced GI at 2 mo (glucose area under the curve [AUC]: 1940 mmol/L × 180 min in LP, 1629 mmol/L × 180 min in LPF, and 1653 mmol/L × 180 min in controls; P <0.05, LP versus control and P <0.01, LP versus LPF) but the effect diminished at 3 mo. In females, folate reduced GI at 1 mo (glucose AUC: 1406 mmol/L × 180 min in LP, 1264 mmol/L × 180 min in LPF, and 1281 mmol/L × 180 min in controls; P <0.05, LP versus control and LP versus LPF) but had no effect at 2 and 3 mo. Interestingly, the LPF group had higher pancreatic weights than other groups, suggesting that folate helps in pancreatic development enabling the LPF rats to produce/secrete more insulin to maintain euglycemia. Euglycemic-hyperinsulinemic clamp shows both LP and LPF are insulin resistant compared with controls by 4 mo with LPF more severe than LP in males. Interestingly, females were more insulin resistant than males. CONCLUSIONS Folate treatment partially reverses LP-induced GI and the magnitude of reversal is age and sex dependent. Furthermore, folate treatment does not reverse IR in either sex but makes it worse in males at 4 mo. The present study demonstrated that folate treatment is not sufficient to rescue the LP programming effects.