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ENDO 2021

Do GLP-1 receptor agonists increase the risk of breast cancer? A systematic review and meta-analysis

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technical paper

Background: 17α-hydroxyprogesterone caproate (17-OHPC) is a synthetic progestogen introduced in the 1950s to treat habitual and threatened abortion in pregnant women. Although 17-OHPC is still available (tradename Makena), little is known about its effects on health of adult offspring, and questions concerning safety and effectiveness remain. For example, progestogens have been implicated in cancer, and trends in the use of 17-OHPC in early pregnancy during the 1950s and 60s parallel increasing incidence rates of certain cancers in young adults, such as early-onset colorectal cancer, born during that time. Methods: We examined the effect of 17-OHPC exposure in utero on risk of cancer in adult offspring in the Child Health and Development Studies, a cohort of women receiving prenatal care between June 1959 and September 1966, with deliveries through June 1967 (n=18,751 live births excluding neonatal deaths among 14,507 mothers). Diagnosed conditions and prescribed medications were abstracted from mothers’ medical records beginning 6 months prior to pregnancy through delivery. We identified mothers who received 17-OHPC (tradenames Delalutin and Proluton) in early pregnancy, defined as day 1 - 140 of gestation. Incident cancers diagnosed in offspring through 2018 were ascertained by linkage with the California Cancer Registry. Results: Among 18,751 live births, 954 cancers were diagnosed at ages 18 - 58 years. The most frequent cancers were breast (20.9%), cervical (10.9%), colorectal (7.1%), and prostate (5.9%) cancer and melanoma (9.2%). Although few mothers (n=181, 1.0%) received 17-OHPC in early pregnancy, in utero exposure was more common in offspring diagnosed with cancer (n=18, 1.9%) compared to those without cancer (n=163, 0.9%). Conditions indicating 17-OHPC included threatened abortion (54.0%), amnionitis (9.4%), and incompetent cervix (3.0%). 17-OHPC increased risk of any cancer in offspring (OR 2.08, 95% CI 1.27, 3.40), with particularly striking associations for colorectal (OR 4.78, 95% CI 1.49, 15.41) and prostate (OR 3.83, 95% CI 0.93, 15.83) cancer. There was no association between conditions indicating 17-OHPC and risk of any cancer in offspring (threatened abortion: n=1,891 mothers, OR 1.07, 95% CI 0.87, 1.32), or with use of other progestogens within 6 months prior to pregnancy (medroxyprogesterone acetate: n=50 mothers, OR 0.38, 95% CI 0.05, 2.76). Conclusions: Findings support susceptibility of multiple organ systems to endocrine disruption during early development and risk of cancer decades later - and may partly explain increasing rates of early-onset colorectal cancer. Even before mechanisms of carcinogenesis are elucidated, caution using 17-OHPC and other endocrine-active pharmaceuticals in early pregnancy is warranted, especially in the absence of a clear short-term benefit, and given the possible effect on risk of cancer in adult offspring.?? 

In Utero Exposure to 17α-Hydroxyprogesterone Caproate May Contribute to Increasing Incidence Rates of Early-Onset Cancer

Nitrate and perchlorate competitively inhibit iodide uptake in thyrocytes and disrupt thyroid function in rodents and humans. Our previous data indicated that the intrauterine exposure to perchlorate or nitrate induced thyroid dysfunction in the offspring rats during adult life. Therefore, this study aimed to investigate the effects of these endocrine disruptors during the embryonic period on the endoderm and thyroid development. Additionally, it was also investigated the role of epigenetic modifications in the programming of gene expression of the evaluated tissues. For this purpose, CD1 pregnant female mice received filtered water (control) or filtered water supplemented with sodium perchlorate (0.3 or 1 ppm) or sodium nitrate (20 or 50 ppm). At gestational day 16.5 (GD16.5), the embryonic thyroid lobes were collected and processed for molecular analysis. Besides the in vivo model, the effect of these thyroid disruptors was also evaluated during the differentiation of mouse embryonic stem cells (mESc) into endoderm and thyroid cells. Endoderm cells differentiation was achieved through the treatment of mESc with several growth factors. During the entire protocol the cells were exposed or not to sodium perchlorate or sodium nitrate (10-5 or 10-7 M). The effects of perchlorate and nitrate were also evaluated during the differentiation of mESC into thyroid cells. For this purpose, mESC-derived endoderm cells were transiently transfected with Pax8/Nkx2.1 expressing vectors. During the endoderm-to-thyrocytes differentiation protocol, the cells were also exposed or not to perchlorate or nitrate (10-5 or 10-7 M). The results demonstrated that both thyroid disruptors reduced the mRNA and protein expression of several endoderm markers (Foxa1, Gata4, Sox17) in the mESc-derived endoderm cells. Moreover, perchlorate or nitrate treatment also reduced the expression of thyroid transcription factors (Pax8, Nkx2.1, Foxe1) and thyroid differentiation markers (Slc5a5, Tpo, Tshr, Tg) both in the embryonic thyroid lobes and in the mESc-derived thyrocytes. Epigenetic mechanisms related to transcription repression seem to be involved in the gene expression downregulation both in vivo and in vitro, since perchlorate and nitrate increased the mRNA expression of Dnmt1, Dnmt3, Hdac and reduced the expression of Hat. Additionally, the methylation of histone H3 was increased, and the acetylation status of this histone was decreased in perchlorate- or nitrate-exposed thyroid lobes and mESc-derived endoderm/thyroid cells. In conclusion, our data strongly suggest that the programming of thyroid dysfunction induced by intrauterine exposure to perchlorate or nitrate involve the disruption of the endoderm and thyroid development during embryonic life through epigenetic mechanisms.?? 

Perchlorate and Nitrate Treatments Disrupt the Endoderm and Thyroid Development Through Epigenetic Mechanisms

The presence of progesterone receptor (PR)-interacting compounds in the environment may have serious health consequences for humans and wildlife, but the methods for their detection and monitoring are limited. Here we report the development and testing of a cell line expressing a chimeric construct containing ligand-binding domain of progesterone receptor and green fluorescent protein-tagged domain of the glucocorticoid receptor (GFP-GR-PR) under tetracycline regulation. Unlike the constitutively nuclear PR, this chimera is cytoplasmic in the absence of the ligand and translocates to the nucleus in response to the hormone or its analogues. The GFP-GR-PR chimera maintains specificity for binding to progesterone and does not cross-react with GR-activating hormones. A concentration- and time-dependent translocation in response to progesterone confirmed picomolar sensitivity for detecting PR ligands. Importantly, the assay can detect both agonist and antagonist activities and thus can be used for screening environmental samples for contamination with endocrine disruptors and for drug development. Using this approach, we screened water samples collected at 23 sites along 2 major rivers in Virginia: Mattaponi and Rappahannock Rivers. We detected a low, but reproducible PR-binding activity in 34.8 % of the sites tested. The calculated progesterone equivalent concentration (EQ) in some of these sites reached ~ 0.8 ng/L. The assay provides an effect-based approach for screening PR-interacting endocrine disrupting chemicals regardless of whether they exert agonist or antagonist activities. Either one could be seriously disruptive for the health of humans and wildlife.?? 

Novel Assay for Detection of Progesterone Receptor-Interacting Endocrine Disruptors

Endocrine disruptors (EDs) interfere with hormonal signalling and, given that multiple developmental processes are hormone-driven, the prenatal period is a window of increased sensitivity. Maternal smoking is a real-life model of in utero exposure to a complex mixture of EDs. Cigarette smoke contains of >7,000 pollutants, including polycyclic aromatic hydrocarbons (PAHs), which are AhR ligands and cross-talk with the estrogen receptor (ER) system. Prenatal exposure to cigarette smoke is associated with adverse outcomes, including intrauterine growth restriction and increased risk of metabolic syndrome later in life. We aimed to evaluate ED effects associated with smoke exposure in human fetuses.Fetal tissues (plasma, n=48; placenta, n=30; liver, n=29) from elective terminations of normally progressing pregnancies, ranging from 10 to 20 gestation weeks, were collected (SAFeR and FEGO studies: REC 15/NS/0123, REC 04/S0802/21). PAHs and PAH-like compounds were extracted from placenta and fetal liver. Bioactivity levels in plasma, placenta and liver extracts were determined using ER and androgen receptor (AR) transactivation reporter gene assays. PAH burden was evaluated using the AhR-responsive DRhp-CALUX assay. Smoke exposure was associated with a 1.3-fold increase in plasma estrogenic activity. The developmental trajectory of androgenic activity was altered in plasma of smoke-exposed fetuses, with significant anti-androgenic activity in older fetuses (>16 weeks of gestation). In males, plasma androgenic activity was positively associated with testes weight and anogenital distance. In contrast, placentas from smoking mothers had significantly increased androgenic potential. Furthermore, AhR-like activity was 2.9-fold higher in smoke-exposed placentas compared to controls, and 2.3-fold higher in female compared to male fetal livers. Overall, all bioactivity levels were higher in placentas compared to fetal liver.Prenatal exposure to cigarette smoke is associated with higher placental AhR activation, indicative of increased xenotoxicants burden. We also report that smoke-exposed fetuses showed increased circulating estrogenic activity and disrupted androgenic potential, across 10-20 weeks of gestation, in both fetal plasma and placenta. This demonstrates that EDs present in cigarette smoke are able to interfere with hormonal signalling and alter dynamic endocrine activity profiles, which are critical to ensure appropriate, sex-specific, development. These ED effects are likely to disturb placental function and reprogramme fetal development and thus impacting on life-long health.?? 

Disruption of Estrogenic and Androgenic Bioactivities in Human Fetuses Exposed to Maternal Smoking

Background: A unique feature of muscle during aging, obesity and type 2 diabetes is the appearance of adipose tissue between skeletal muscle fibers, the intramuscular adipose tissue (IMAT). IMAT is generally associated with systemic insulin resistance, decreased muscle strength and, in older adults, impaired mobility. A large body of work over the last years have addressed the origin of IMAT. Skeletal muscle contains preadipocyte progenitors termed fibro/adipogenic progenitors (FAPs) that normally do not form adipocytes, but proliferate during muscle injury to support the commitment of myogenic progenitor cells during muscle repair. However, under the conditions outlined above, FAPs differentiate to adipocytes and give rise to IMAT. The molecular cues that trigger this pathogenesis are unknown and strategies that can modulate the fate of FAPs and their propensity to differentiate to adipocytes in disease states are urgently needed. Methods: We have used molecular cloning and genome editing techniques together with a variety of biochemical, molecular, genomic, transcriptomic and proteomic analyses in pre-adipocyte and muscle cell lines, primary cells isolated from human and mouse skeletal muscle, human skeletal muscle biopsies, and animal models. Results: Using multiple screening assays upstream and downstream of a master regulator of myogenesis, microRNA (miR)-29a, we located the secreted protein and adipogenic inhibitor SPARC to an FGF-2 signaling pathway that is conserved between skeletal muscle cells from mice and humans and that is activated in skeletal muscle of aged mice and humans. We identified FGF-2 as an upstream regulator that increases miR-29a promoter activity and gene expression via MEK1/2/MAPK signaling and consequently decreases the conserved miR-29a target SPARC. FGF-2 induces the miR-29a/SPARC axis through transcriptional activation of FRA-1, which binds and activates an evolutionary conserved AP-1 site element proximal in the miR-29a promoter. CRISPR/Cas9-mediated genomic deletions of miR-29a recognition elements located in the Sparc 3´UTR of muscle cells and AAV-mediated overexpression of FGF-2 or SPARC in mouse skeletal muscle revealed that this axis regulates differentiation of FAPs in vitro and IMAT formation in vivo. Skeletal muscle from human donors aged > 75 years versus < 55 years showed activation of FGF-2-dependent signaling and increased IMAT. Conclusion: FGF-2 not only stimulates muscle growth, but also promotes intramuscular adipogenesis via targeting the fate of FAPs. Thus, our data highlights a disparate role of FGF-2 in adult skeletal muscle and reveals a novel pathway, which has the potential to provide innovative diagnostic and therapeutic approaches to combat fat accumulation in aged human skeletal muscle.?? 

FGF-2-Dependent Signaling Activated in Aged Human Skeletal Muscle Promotes Intramuscular Adipogenesis

GPR17 is a G protein-coupled receptor (GPCR) implicated in the regulation of glucose metabolism and energy homeostasis. Our genetic knockout studies in rodents suggest that GPR17 is a potential therapeutic target for the treatment of metabolic diseases. However, the contributions of GPR17 to human metabolism and metabolic deficits are not well understood. Here, we analyzed the human GPR17 coding sequences of individuals from control and metabolic disease cohorts that were comprised of patients with clinical phenotypes including severe insulin resistance, hypercholesterolemia, and obesity. Across cohorts, 18 nonsynonymous GPR17 variants were identified, including eight variants that were exclusive to the disease cohort. We characterized the protein expression levels, cellular localization, and downstream functional signaling profiles of nine human GPR17 variants (F43L, V96M, V103M, D105N, A131T, G136S, R248Q, R301H, and G354V). We found that the protein expression levels and subcellular localization for each of the nine GPR17 variants were similar to that of the wild type GPR17. As the endogenous GPR17 ligand is still elusive, we used a synthetic GPR17 agonist to quantitatively measure the functional signaling profiles of GPR17 variants. We found some of the variants had distinctly altered signaling profiles. GPR17-G136S lost agonist-mediated cAMP, Ca2+, and beta-arrestin signaling. GPR17-V96M retained cAMP inhibition similar to GPR17-WT but had impaired Ca2+ and beta-arrestin signaling. GPR17-D105N displayed impaired cAMP and Ca2+ signaling but enhanced agonist-stimulated beta-arrestin recruitment. Also, GPR17-G354V retained cAMP and Ca2+ signaling function but had attenuated beta-arrestin recruitment. The identification and functional profiling of naturally occurring human GPR17 variants from individuals with metabolic diseases revealed receptor variants with distinct signaling profiles, including differential signaling perturbations that resulted in receptor signaling bias. These results are expected to contribute to our understanding of the molecular signaling mechanisms underlying GPR17 in metabolic regulation.?? 

Human GPR17 Nonsynonymous Variants Identified in Individuals with Metabolic Diseases Have Distinct Functional Signaling Profiles

The sodium-glucose co-transporter (SGLT)-2 inhibitor dapagliflozin was recently reported to reduce renal tyrosine hydroxylase (TH) and norepinephrine levels, lowering blood pressure and preventing endothelial dysfunction, in a murine model of neurogenic hypertension. This suggested that dapagliflozin may combat sympathetic nervous system (SNS) hyperactivity, which is known to accompany and aggravate chronic heart failure (CHF). Adrenal G protein-coupled receptor kinase (GRK)-2 upregulation is a major driver of circulating catecholamine (CA) elevation and SNS hyperactivity, especially in CHF. GRK2 severely dysregulates adrenal sympatho-inhibitory α2-adrenergic receptors (ARs), leading to unchecked, chronically elevated CA secretion. Therefore, we hypothesized herein that SGLT2 inhibition with dapagliflozin may lower SNS hyperactivity in the adrenal gland by antagonizing GRK2 actions on α2ARs in adrenal chromaffin cells. We used the rat pheochromocytoma PC12 cell line expressing human α2AAR, as well as freshly isolated adrenal glands from adult male Sprague-Dawley rats treated with dapagliflozin in vivo. We measured circulating norepinephrine (NE) in vivo via RIA, GRK2 & TH expression levels via real-time PCR and immunoblotting, adrenal α2AR density (Bmax) via saturation radioligand binding with [methyl-3H]-rauwolscine, and G protein activation via the GTPγS assay. Dapagliflozin treatment for 7 consecutive days (20 mg/kg/d in drinking water) led to a significant reduction in blood circulating NE levels (217+67 pg/ml, n=6), compared to control, vehicle-treated rats (363+77 pg/ml, n=6, p<.05), suggesting reduced SNS activity. This was accompanied by reduced GRK2 and TH mRNA and protein levels in dapagliflozin-treated rat adrenals vs. vehicle-treated animal-derived glands, indicating reduced adrenal CA synthesis and secretion. Finally, adrenal α2AR density was higher in dapagliflozin- vs. vehicle-treated rats (51.3+7.3 vs. 26.1+8.1 fmol/mg of protein, respectively; n=12 glands from 6 animals per group, p<.05). These results (i.e. GRK2 and TH downregulation) were completely recapitulated in PC12 α2AAR-expressing cells in culture, treated with 5 μM dapagliflozin (or vehicle) for 24 hours. Importantly, α2AR-dependent G protein-mediated signaling towards inhibition of CA secretion was markedly enhanced at 24 hrs post-dapagliflozin application in PC12 cells. This was the result of reduced GRK2-dependent receptor desensitization, since dapagliflozin lacked this effect in cells co-transfected with a GRK2-encoding adenovirus to acutely overexpress GRK2. In conclusion, dapagliflozin exerts a sympatholytic action in the adrenal medulla via downregulation of both TH, which reduces CA biosynthesis, and GRK2, which reduces α2AR desensitization in favor of enhanced inhibition of CA secretion.?? 

Dapagliflozin Exerts Adrenal Sympatholysis via G protein-Coupled Receptor Kinase-2 & Tyrosine Hydroxylase Downregulation

Background: Cardiometabolic conditions in adulthood are more common in children born SGA1. The relationship of the transcriptome (gene expression) and epigenome (DNA methylation) to birth size and the future development of cardiometabolic disease has not been characterized. Aims: To identify I) the relationship between epigenome at age 0, 7 and 17 years, transcriptome at age 9 years and birth size in a normal population; II) links between the transcriptome and epigenome in childhood and adult cardiometabolic risk. Study Design: Normal children (n=6487) from the Avon Longitudinal Study of Parents and Children were assigned to groups based on birth size using bodyweight (BW) and gestation and divided into groups using the population 10th centile. Adverse cardiometabolic risk at age 17 years was defined by the National Heart Lung and Blood Institute criteria of prehypertension using systolic and diastolic blood pressure as well as HDL and LDL2. Blood transcriptome at age 9 and blood epigenome at age 0, 7, and 17 years and were available from 980 and 947 children, respectively. Hypernetworks were used to integrate differentially expressed genes in the transcriptome (DEGs) and differentially methylated points (DMPs) in the epigenome, identifying functional links. Random Forest, a machine learning approach, was used to determine the predictive value of ‘omic data presented as the area under the curve (AUC) of the receiver operating characteristic. Results: Pre-hypertensive participants at age 17 years were distinguished from normotensive participants and this group was enriched for children born small who caught up by age 7 years (155/611 unhealthy/healthy SGA compared to 1979/12746 in all other BW groups; 1.6-fold, p<1x10-5). This group had a greater height velocity during their catch-up period than the normotensive participants (1.2-fold, p=0.027). Hypernetwork integration of ‘omic data identified a functional relationship between 55 DEGs at age 9 years and DMPs at age 7 years. Random forest analysis was able to accurately predict the presence of pre-hypertensive young adults from the age 9 transcriptome (AUC: 0.973). Using a gene-level contraction of DMPs which map to the 55 DEGs (i.e. cis-DMPs), we demonstrated accurate classification of pre-hypertensive young adults from their blood methylome at age 0 (AUC [95% CI]: 0.92 [0.89-0.95]), 7 (0.90 [0.87-0.93]), and 17 (0.91 [0.88-0.94]) years. Conclusions: Through the integration of transcriptome and epigenome, we have identified a set of genes with an epigenomic and transcriptomic signature which predict pre-hypertension in children born SGA who catch up. Specifically, we have shown that the associated epigenomic signature tracks from birth to early adulthood, indicating the possibility of early detection of risk and primordial prevention. 1 Barker etal. (1988) BMJ 297(6641):134-135. 2 Chobanian etal. (2003) JAMA 289(19):2560-2571.?? 

An Epigenomic Signature in Children Born Small for Gestational Age (SGA) With “Catch-up” Growth Is Present From Birth and Predicts Early Adulthood Pre-Hypertension

The pituitary gland regulates key physiological functions, including growth, sexual maturation, reproduction, and lactation. Here, we present a paired single-nuclei (sn) transcriptome and chromatin accessibility characterization of six post-mortem human pituitaries. These samples were from juvenile, adult, and elderly male and female subjects. Well-correlated snRNAseq and snATACseq datasets facilitated robust identification of the major pituitary cell types in each sample. Using latent variable pathway analysis, we uncovered previously unreported coordinated gene expression modules and chromatin accessibility programs for each major cell type as well as an age-specific program across all the endocrine cell types. These largely appear to be congruent between human and mouse datasets. Given the importance of murine models in the study of human pituitary disorders and pituitary physiology, we next sought to compare expression profiles of pituitary cell types in mouse vs. human. Murine and human cell types were well correlated, exemplified by coordinated gene expression programs, especially for undifferentiated stem cells (SCs). In both species, we identified clusters corresponding to naive and committing SCs. All human SC clusters expressed the established SC markers SOX2 and SOX9, as well as genes involved in SC regulatory pathways (WWTR1, YAP1 andPITX2). Additional markers previously reported in murine pituitary SCs were also found in human SC, including WIF1, LGR5, FOS, CDH1, EGFR, LGR4, and WLS. Remarkably, in human, the main naive SC cluster was roughly divided into a high-JUN and a low-JUN expressing subgroup, whereas Jun expression was less pronounced in the murine SC cluster. In both species, committing SC clusters expressed the endocrine markers for POU1F1, TSHB, or POMC, while SCs committing to an intermediate lobe/melanotrope cell identity were distinguishable based on PAX7 expression. In addition, in the human datasets we identify a population of cells as originating from the pars tuberalis. We offer a range of markers that can be utilized for in vivo validation of these cells.Overall, the characterization of the murine and human pituitary SCs strongly suggests the co-existence of subpopulations with different lineage commitments in addition to a single uncommitted SC population. This sn atlas of the human pituitary is a valuable resource that will be made web-accessible.?? 

Single Nucleus Transcriptome and Chromatin Accessibility Landscapes of Human Pituitaries

The pituitary gland is the major endocrine organ that produces and secretes hormones in response to hypothalamic signals to regulate important processes like growth, reproduction, and stress. The anterior pituitary adapts to metabolic and reproductive needs by exhibiting cellular plasticity, resulting in altered hormone production and secretion. The adipokine, leptin, serves a critical role to couple energy status to pituitary function. We have recently reported that the cell fate determinant, Musashi, functions as a post-transcriptional regulator of target mRNA translation in the mouse pituitary and have speculated that Musashi may modulate pituitary cell plasticity. However, the underlying mechanisms governing such pituitary plasticity are not fully understood. Musashi is an mRNA binding protein that is required for self-renewal, proliferation, and to control the differentiation of stem and progenitor cells. We have recently shown that Musashi is expressed in Sox2+ pituitary stem cells and surprisingly, we also found Musashi expression in all differentiated hormone expressing cell lineages in the adult anterior pituitary. The role of Musashi in these mature differentiated cells is unknown. We have observed that a range of critical pituitary mRNAs, including the lineage specification transcription factors Prop1 and Pou1f1, as well as hormone mRNAs including Tshb, Prl, and Gnrhr, all contain consensus Musashi binding elements (MBEs) in their 3’ untranslated regions (3’ UTRs). Using RNA electrophoretic mobility shift assays (EMSAs) and luciferase mRNA translation reporter assays we show that Musashi binds to these mRNAs and exerts inhibitory control of mRNA translation. Moreover, we determined that leptin stimulation opposes the ability of Musashi to exert translational repression of the Pou1f1 and Gnrhr 3’ UTRs. This de-repression does not require regulatory phosphorylation of Musashi on two conserved C-terminal serine residues. Interestingly in the same cell assay system, Musashi exerts translational activation of the Prop1 3’ UTR. We observed that this translational activation requires Musashi phosphorylation on the two regulatory C-terminal serine residues, consistent with the requirement for regulatory phosphorylation to drive translational activation of Musashi target mRNAs during Xenopus oocyte cell maturation. The distinction between MBEs in 3’ UTRs that exert repression (Pou1f1, Prl, Tshb, and Gnrhr) and the Prop1 3’ UTR that directs translational activation is under investigation. We propose that Musashi acts as a bifunctional regulator of pituitary hormone production and lineage specification and may function to maintain pituitary hormone plasticity in response to changing organismal needs.?? 

Do GLP-1 receptor agonists increase the risk of breast cancer? A systematic review and meta-analysis

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