United States

Heart Rate Variability (HRV) results from the autonomic nervous system activity, it is a non-invasive marker of cardiac autonomic function. Loss of parasympathetic function (PNS) and sympathetic (SNS) override, reflected in decreased HRV, is one of the earliest subclinical manifestations of cardiac autonomic dysfunction. Lower HRV is associated with increased risk of cardiac events in adults.
We aimed to characterize HRV in normal weight and overweight children with and without dysglycemia and to investigate the determinants of HRV in these youth, including body composition, glycemia measures, beta-cell function and inflammatory markers.
We evaluated 94 adolescents (50 males/44 females), age 15 ± 2.1 years; 21 normal weight with normal glucose tolerance (NW-NGT), 23 overweight with NGT (OW-NGT) and 50 overweight with impaired glucose regulation (OW-IGR) including prediabetes (n= 27) and type 2 diabetes (n= 23). They underwent assessment of anthropometrics, body composition (DXA scan), inflammatory markers (hs-CRP and TNF-α), fasting labs and 2-hour oral glucose tolerance test (OGTT) with determination of glucose and insulin concentration. EndoPat was used to measure HRV: frequency-domain indices [high-frequency (HF) reflecting PNS, low-frequency (LF) reflecting both PNS and SNS, and LF/HF (higher is worse) estimating the ratio between SNS and PNS] and time-domain indices [the inter-beat interval of normal sinus beats (NN) and the standard deviation of NN (SDNN) measuring overall HRV, the square root of the mean squared difference of successive NN (RMSSD) and the NN intervals differing by more than 50 milliseconds (NN50) measuring PNS activity].
LF/HF was higher in the OW-IGR group compared with NW and OW-NGT (p=0.005). After controlling for sex, race and Tanner stage, fasting glucose (FBG) negatively correlated with NN (r=-0.22,p=0.04), SDNN (r=-0.21,p=0.05), RMSSD (r=-0.3,p=0.004), NN50 (r=-0.27,p=0.01) and HF (r=-0.26,p=0.02). LF/HF was positively related to FBG (r=0.39,p&lt;0.001), 2hr-glucose (r=0.31,p=0.004) and HbA1c (r=0.22,p=0.04), and negatively with the insulinogenic index (r=-0.27,p=0.02), but not fasting insulin or HOMA-IR; LF/HF also correlated with percent body fat (r=0.22,p=0.04), hs-CRP (r=0.33,p=0.002) and TNF-α (r=0.38,p=0.006).
In a linear regression model with LnLF/HF as the dependent variable and percent body fat, hs-CRP, FBG and HOMA-IR as the independent variables, FBG (beta=0.39,p=0.003) and hs-CRP (beta=0.21,p=0.09) were the significant determinants of LnLF/HF independent of age, sex, race and Tanner stage as covariates (R²= 0.23,p=0.013).
Youth with impaired glucose metabolism have evidence of early subclinical cardiac autonomic dysfunction with decreased HRV, loss of parasympathetic function and sympathetic overdrive as reflected by lower time-domain indices, lower HF and higher LF/HF, related to glycemia and systemic inflammation.

ENDO 2022

Hyperglycemia Is the Main Determinant of Cardiac Autonomic Dysfunction in Youth With Obesity Across the Spectrum of Glycemic Regulation.

**Program**
ENDO’s comprehensive program provides attendees the opportunity to learn about the latest developments in hormone science and medicine from renowned investigators, expert clinicians, and educators from all over the world. 

This year’s education program features:
more than 100 in-person and virtual sessions with another 70+ in-person only sessions;
six plenary sessions including plenaries on Envisioning Future Therapies for Type 1 Diabetes and Addressing Challenges in Fertility and Reproduction;
over 20 Meet the Professor sessions presented both in-person and live-streamed virtually and another 20+ presented live in Atlanta;
four Basic Science Pathways, including Diabetes and Metabolism; Neuroendocrinology; Nuclear Receptors and Signaling; and Reproductive Endocrinology; and
more than 30 oral sessions and a robust virtual poster hall for accepted scientific abstracts.

**What Programming Is Included in My Virtual Meeting Registration?**
As you build your schedule during the conference, you can select the sessions that are tagged with 'Livestreamed', so you can view virtually. These sessions are being livestreamed, so they are available in real time to our virtual audience. In addition, our in-person and livestreamed sessions will be recorded, so you can access them on demand after 72 hours. We will have over 200 hours available on demand for Standard and Premium pass registrants.

Product theaters and the Communications and Career Center will also include hybrid content delivery.

**Plenaries**
Spanning the spectrum of science, clinical care, and social implications, an outstanding slate of world-renowned speakers will showcase the most cutting-edge advances on research and medicine.

**Symposia**
Our ENDO 2022 lineup of sessions offers a unique opportunity to dive into topics from nearly every major therapeutic area of endocrinology and hormone science. Listen, learn, and be inspired by world-class faculty who are at the forefront of scientific discovery and high-quality health care.

**Basic Science Pathways**

These special “meeting-within-a-meeting” tracks integrate focused discussions, poster sessions, and networking opportunities to provide you with the latest research in:
Diabetes and Metabolism
Neuroendocrinology
Nuclear Receptors and Signaling
Reproductive Endocrinology

**Meet the Professor & Meet the Scientist**
Discover insights from experts on treating a variety of endocrine conditions and emerging science in the field of endocrinology, and learn the latest in patient care and scientific research.

ENDO’s comprehensive program provides attendees the opportunity to learn about the latest developments in hormone science and medicine from renowned investigators, expert clinicians, and educators from all over the world. 

Heart Rate Variability (HRV) results from the autonomic nervous system activity, it is a non-invasive marker of cardiac autonomic function. Loss of parasympathetic function (PNS) and sympathetic (SNS) override, reflected in decreased HRV, is one of the earliest subclinical manifestations of cardiac autonomic dysfunction. Lower HRV is associated with increased risk of cardiac events in adults.
We aimed to characterize HRV in normal weight and overweight children with and without dysglycemia and to investigate the determinants of HRV in these youth, including body composition, glycemia measures, beta-cell function and inflammatory markers.
We evaluated 94 adolescents (50 males/44 females), age 15 ± 2.1 years; 21 normal weight with normal glucose tolerance (NW-NGT), 23 overweight with NGT (OW-NGT) and 50 overweight with impaired glucose regulation (OW-IGR) including prediabetes (n= 27) and type 2 diabetes (n= 23). They underwent assessment of anthropometrics, body composition (DXA scan), inflammatory markers (hs-CRP and TNF-α), fasting labs and 2-hour oral glucose tolerance test (OGTT) with determination of glucose and insulin concentration. EndoPat was used to measure HRV: frequency-domain indices [high-frequency (HF) reflecting PNS, low-frequency (LF) reflecting both PNS and SNS, and LF/HF (higher is worse) estimating the ratio between SNS and PNS] and time-domain indices [the inter-beat interval of normal sinus beats (NN) and the standard deviation of NN (SDNN) measuring overall HRV, the square root of the mean squared difference of successive NN (RMSSD) and the NN intervals differing by more than 50 milliseconds (NN50) measuring PNS activity].
LF/HF was higher in the OW-IGR group compared with NW and OW-NGT (p=0.005). After controlling for sex, race and Tanner stage, fasting glucose (FBG) negatively correlated with NN (r=-0.22,p=0.04), SDNN (r=-0.21,p=0.05), RMSSD (r=-0.3,p=0.004), NN50 (r=-0.27,p=0.01) and HF (r=-0.26,p=0.02). LF/HF was positively related to FBG (r=0.39,p<0.001), 2hr-glucose (r=0.31,p=0.004) and HbA1c (r=0.22,p=0.04), and negatively with the insulinogenic index (r=-0.27,p=0.02), but not fasting insulin or HOMA-IR; LF/HF also correlated with percent body fat (r=0.22,p=0.04), hs-CRP (r=0.33,p=0.002) and TNF-α (r=0.38,p=0.006).
In a linear regression model with LnLF/HF as the dependent variable and percent body fat, hs-CRP, FBG and HOMA-IR as the independent variables, FBG (beta=0.39,p=0.003) and hs-CRP (beta=0.21,p=0.09) were the significant determinants of LnLF/HF independent of age, sex, race and Tanner stage as covariates (R²= 0.23,p=0.013).
Youth with impaired glucose metabolism have evidence of early subclinical cardiac autonomic dysfunction with decreased HRV, loss of parasympathetic function and sympathetic overdrive as reflected by lower time-domain indices, lower HF and higher LF/HF, related to glycemia and systemic inflammation.

technical paper

Background: Pediatric obesity is a multifactorial disease characterized by a prolonged imbalance between energy intake and expenditure. In rare cases, it is caused by underlying medical disorders arising from disruptions in the leptin-melanocortin pathway which regulates satiety and energy expenditure.
Aim: To investigate and compare resting energy expenditure (REE) and body composition characteristics of children and adolescents with severe obesity with and without underlying medical causes.
Methods: This prospective observational study included pediatric patients who underwent an extensive diagnostic workup in our academic center in which endocrine, non-syndromic and syndromic genetic, hypothalamic, and medication-induced causes of obesity were evaluated. Patients in whom no underlying medical cause was identified were classified as multifactorial obesity. REE was assessed by indirect calorimetry; body composition by air displacement plethysmography. The ratio measured REE (mREE) vs predicted REE (Schofield equations) was expressed as REE%, with decreased mREE defined as REE% ≤90% and elevated mREE as ≥110%. Additionally, the ratio mREE vs fat-free-mass (FFM) was calculated.
Results: We included 292 patients, of which 218 (75%) patients had multifactorial obesity and 74 (25%) had an underlying medical cause: non-syndromic and syndromic genetic (n= 29 and 28, respectively), hypothalamic (n= 10), and medication-induced (n= 7) obesity. Mean age was 10.8 ± 4.3 years, 59% were female, mean BMI SDS was 3.8 ± 1.1, indicating severe obesity. Mean REE% was higher in children with non-syndromic genetic obesity (107.4% ± 12.7) and lower in children with hypothalamic obesity (87.6% ± 14.2) compared to multifactorial obesity (100.5% ± 12.6, both p<0.01). Measured REE was decreased in 60 (21%) patients (corresponding to an average overprediction of daily caloric needs of 341 kcal/day) and elevated in 69 (24%) patients. Only in hypothalamic obesity, a larger proportion of patients showed a decreased REE compared to multifactorial obesity (6/10 vs 41/218, p<0.01). FFM was higher in children with non-syndromic obesity compared to multifactorial obesity (+7.5kg, p<0.001), but lower in syndromic obesity (-5.2kg, p=0.03), hypothalamic obesity (-12.6kg, p<0.001), and similar in medication-induced obesity (+1.5kg FFM, p=0.80). Mean mREE/FFM was 46.5 ± 10.6 kcal/day/kg FFM and did not differ between patients with underlying medical causes compared to multifactorial obesity (all p>0.05).
Conclusion: In this cohort of children with severe obesity due to various etiologies, large inter-individual differences in mREE were found. Almost half of patients had decreased or elevated mREE. When relating mREE to FFM, no differences were found between children with underlying medical causes versus multifactorial obesity. Thus, our study underlines the importance of measuring REE and relating mREE to FFM in children with early-onset severe obesity with or without underlying medical causes. This knowledge is important for patient-tailored treatment, e.g. personalized dietary or physical activity interventions and consideration of pharmacotherapy affecting central energy expenditure regulation in children with decreased mREE.

Resting Energy Expenditure and Body Composition in Children and Adolescents with Genetic, Hypothalamic, Medication-Induced or Multifactorial Severe Obesity

Background: Turner syndrome (TS) is caused by the absence of whole/part of the second X-chromosome. Primary ovarian insufficiency (POI) is common in TS and serum anti-Mullerian hormone (AMH) of <4 pmol/L (0.56 ng/mL) is suggestive of POI and lack of spontaneous puberty.
Objective: To describe the distribution of AMH and spontaneous puberty in a cross-sectional cohort of patients in a newly-established pediatric TS specialty clinic.
Methods: Data on all TS patients seen in the multidisciplinary clinic between 1/1/2019 and 10/27/2021 were abstracted from retrospective chart review into a REDCap database. Descriptive analysis, one way ANOVA and two sample t-tests for pair-wise comparisons was completed (SPSSv28.0 and Excelv2102), with statistical significance set at p<0.05.
Results: Ninety-six patients (9% Asian, 13.5% African American and 37.5% Caucasian, and rest unknown/other self-reported race, and 29.2% Hispanic ethnicity) were enrolled in our clinic. While the majority were diagnosed postnatally (74%, median age 5 years), 6% were prenatal and 20% had positive prenatal screen with postnatal confirmation. Karyotype distribution: 41% non-mosaic monosomy X, 17% Xq (including 14% isochromosome Xq) and 14% Xp structural abnormalities, 10% ringX, 7% 45,X/46,XX mosaicism and 9% with Y-chromosome. Median age at first TS clinic visit was 10 years (IQR 9.7) with median height at 2%ile, weight 31%ile and BMI 73%ile. AMH was measured in 88 patients and only 33 (37.5%) had detectable (>0.015 ng/mL) AMH; 8 (9%) had AMH >0.56ng/mL. Mean AMH was significantly lower in 45, X monosomy (0.03+/- 0.14) vs. other karyotypes (0.44+/-1.13)(p=0.01). A one-way ANOVA revealed a statistically significant difference in AMH between 45,X, structural abnormalities in Xp/Xq and, mosaic 45,X/46,XX karyotypes (p<0.001). Pair-wise comparisons confirmed mean AMH in 45,X/46,XX was significantly higher (3.1ng/mL) (p<0.001) compared with 45, X (0.03ng/mL), Xq (0.02ng/mL) and Xp (0.08ng/mL) abnormalities but no other comparisons were statistically significant.
Spontaneous thelarche was reported in 23/58 (39.6%) pubertal patients with mean AMH (0.65ng/mL +/-1.3) compared to absent spontaneous thelarche (N=35, 0.02 +/- 0.04) (p=0.03). Spontaneous menarche was reported in 9 patients, with mean AMH 1.8+/- 1.7ng/mL; 4/9 had 45,X/46, XX karyotype and 1 each had 45,X, Xp, Xq, isoXq, and ringX karyotypes. The mean age at spontaneous thelarche was lower than age at thelarche with hormonal induction (11 + 1 years vs. 14 + 2 years, p < 0.001). Median age at estrogen initiation was 12 years (IQR 3); 86% received transdermal estradiol with menarche at a median age of 15 years. While 78/96 (81%) received fertility preservation counseling at a mean age of 11 + 6 years, only 4 utilized fertility preservation options (1 oocyte cryopreservation and 3 ovarian tissue cryopreservation).
Although cross-sectional data limit inferences on AMH as a marker of puberty, our data represents a large and diverse cohort of TS patients in the US.

AMH and Spontaneous Puberty in a Diverse US Turner Syndrome Clinic Cohort: A Cross-sectional study

Background
Pituitary stalk interruption syndrome (PSIS) may associate with brain midline defects and also with polydactyly (GLI2 mutations) but rarely with brain cortex anomalies.
Clinical Case
We report a boy referred at age 19 months because of growth failure. Bilateral postaxial polydactyly, syndactyly of the toes, and a nodule on the tongue were noted at term birth. The neonatal course was complicated by marked but transient hypoglycemia. During infancy, the acquisition of gross motor skills was slightly delayed. Upon referral, body length was 4.1 SD below the level of mid-parental height. Extremely low concentrations of circulating IGF-I (8 mcg/L) and IGFBP-3 (861 mcg/L) were suggestive of growth hormone (GH) deficiency which was corroborated by glucagon test (peak GH 3.3 mcg/L, peak cortisol 14.6 mcg/dL) in an euthyroid and normoprolactinemic state, and in the absence of polydipsia or polyuria. Brain MRI disclosed not only a PSIS triad (with a virtual absence of the pituitary stalk and the anterior pituitary, and with an ectopic position of the posterior pituitary) but also abnormal sulcation and polymicrogyria (pointing to abnormal lamination in the cortex) on both sides in the posterior cingulum. Familial history is positive for holoprosencephaly and polydactyly in two male relatives, who died neonatally.
Whole exome sequencing showed a rare maternally inherited variant in ZRSR2 on the X chromosome (c. 1207_1208delAG (p.Arg403Glyfs*24)) (OMIM 300028). ZRSR2 isdepleted from loss-of-function variants in the reference population, and has not been associated with congenital anomalies or with pituitary dysfunction. This ZRSR2 variant escapes nonsense mediated decay and segregates in this family according to an X-linked recessive pattern. X-inactivation studies and gene expression studies are ongoing to correlate the ZRSR2 variant to the patient's phenotype.
Conclusion
This case describes a toddler with polydactyly and short stature, based on GH deficiency due to PSIS, in combination with polymicrogyria. Studies are ongoing to link the patient's phenotype to a rare variant in ZRSR2.

Pituitary stalk interruption syndrome, polydactyly, polymicrogyria and a ZRSR2 variant

Weakening of the lower abdominal wall muscle via fibrosis causes visceral contents to bulge out, forming inguinal hernias. Humanized aromatase (Aromhum) transgenic male mice develop this weakness in the lower abdominal muscle (LAM) tissue, characterized by extensive fibrosis, muscle atrophy, and scrotal hernias. This mouse model incorporates the human version of the aromatase gene allowing for excessive estradiol (E2) production in skeletal muscle tissue. We have previously shown that the LAM fibroblasts are sensitive to local E2 production due to high levels of estrogen receptor alpha (ERα, Esr1)¹. Here, we explore the origins of estrogen-driven muscle fibrosis in the LAM tissue of Aromhum mice at a single-cell level.
Wild-type (WT) and Aromhum LAM tissues were processed via single-cell RNA sequencing (n = 3) and analyzed using Seurat. We observed a total of 22 UMAP clusters, including six fibroblast-like clusters. Five of these clusters express the fibro-adipogenic progenitor (FAP) marker Pdgfrα, and two of them are exclusively found in Aromhum LAM tissues. One Aromhum-enriched cluster highly expresses Esr1 and its downstream estrogen-responsive genes such as Pgr and Greb1. Pseudo-time analysis suggested that this cluster differentiates into a pathogenic fibroblast cluster characterized by high expression pro-inflammatory (Il33, Il6, Ccl8, C4b) and fibrosis-associated (Mmp3, Cthrc1, Saa3, Ptx3) genes. We further validated our findings via flow cytometry, immunocytochemistry, and western blots in LAM, upper abdominal muscle tissues, and quadriceps of both WT and Aromhum mice. We show that protein levels of Pdgfrα, ERα, Mmp3, C4b are greater in the fibroblasts freshly isolated from LAM tissues of Aromhum mice compared to WT mice. Moreover, Aromhum LAM tissue cell cycle analysis reveals that ~15% of all ERα+ cells are in the G2-S phase compared with <1% of ERα- cells. Overall, we provide an insight into the cell types, genes, and proteins that are dysregulated in the LAM tissues from Aromhum mice with hernias.
Inguinal hernias are found in ~25% of the elderly male population². Using an inguinal hernia mouse model that mimics the hernia pathophysiology of elderly men with estrogen excess and testosterone deficiency, we defined a specific population of estrogen-sensitive fibroblasts responsible for fibrosis and weakening of lower abdominal skeletal muscle tissue associated with hernias. We provide an insight into the specific disease-causing fibroblast populations and molecular targets that may be the basis for future therapeutics to prevent or treat inguinal hernias.
References:
1. Zhao H, Zhou L, Li L, et al. Shift from androgen to estrogen action causes abdominal muscle fibrosis, atrophy, and inguinal hernia in a transgenic male mouse model. Proceedings of the National Academy of Sciences. 2018;115(44): E10427-E10436.
2. Sazhin A, Zolotukhin I, Seliverstov E, et al. Prevalence and risk factors for abdominal wall hernia in the general Russian population. Hernia. 2019;23(6): 1237-1242.

A Unique Estrogen-Sensitive Fibroblast Population Drives Abdominal Muscle Fibrosis

Estrogens are steroid hormones that play a key role in a wide range of physiological and pathological processes. Estrogen receptor-alpha, ERɑ, functions primarily as a nuclear transcription factor (TF) through ligand-dependent activation by 17β-estradiol (E2), the predominant naturally-occurring estrogen. This results in dimerization, DNA binding to cis-regulatory elements called enhancers, and gene regulatory function of ERɑ. In vivo, E2 production and action fluctuates during reproductive cycles and possibly even in a circadian manner. In tissues and cells, E2-regulated gene expression is dynamic, with rapid induction of enhancer formation and target gene expression (or repression), followed by enhancer "decommissioning" and cessation of the gene regulatory effects. The effects of sustained E2 production and signaling, fluctuations in E2 levels, and removal of E2 on estrogen-regulated gene expression are unknown. Classical experiments exploring the gene regulatory effects of E2 involve stimulation of cultured cells throughout the duration of the experiment with a continuous dose of hormone, with the cells harvested at the end of the stimulation period. While this has allowed us to understand the molecular underpinnings of signal-dependent transcription, it has not allowed analysis of physiological fluctuations in estrogen signaling involving periods of stimulation and non-stimulation. In our studies, we are applying a combination of next generation sequencing techniques and fluorescence microscopy to investigate signal-dependent responses to fluctuating E2 levels using ERɑ-positive MCF-7 human breast cancer cells after E2 treatment and removal. Our initial results suggest that different E2 target genes respond differently to E2 withdrawal. Moreover, for some E2 traget genes, sustained E2 treatment may not be required for a complete E2 transcriptional response. With these studies, we hope to connect physiological aspects of E2 production, release, and action with the molecular mechanisms of E2-dependent gene expression - an aspect of our understanding of estrogen signaling that has been lacking.
This work is supported by grants from the NIH/NIDDK (DK058110) and CPRIT (RP160319) to W.L.K.

Connecting Fluctuations in Estrogen Production, Release, and Action with Hormone-Dependent Gene Expression in Breast Cancers

Imidacloprid (IMI) is a neonicotinoid pesticide used for pest control and seed treatments. It mimics nicotine and selectively targets insect nicotinic acetylcholine receptors to induce paralysis and death. Popularly used worldwide, IMI residue is readily detectable in crops, the environment, and human samples. Gastrointestinal symptoms in patients as well as defective gut barrier and hepatoxicity in animal models have been reported subsequent to IMI exposure. However, the mechanism underlying IMI toxicity in mammals is unclear. Pesticide exposure frequently activates xenobiotic nuclear receptors, like constitutive androstane receptor (CAR), to induce detoxification phase I and phase II genes. We found that IMI exposure in wild-type (WT) mice induced hepatic expression of Cyp2b10, a bona fide target of CAR. This data led us to investigate the role of CAR in alleviating IMI toxicity. We dosed wild-type (WT) and Car-/- mice with 50mg/kg BW IMI, twice daily for 21 days and collected serum, liver, and intestine tissues. The absence of CAR resulted in a three-fold increased accumulation of IMI within the liver as well as a 6% increase in body weight. Intriguingly, IMI exposure resulted in hepatic inflammatory pockets in WT mice but did not induce serum liver injury markers (ALT and AST) in either WT or Car-/- mice. We quantified the expression of genes involved in detoxification and oxidative stress within the liver and ileum using qRT-PCR. We found Car-/-mice had significantly blunted expression of Cyp-P450 enzymes in the ileum. To compare gut function, we measured organ coefficient and examined ileal histology using Alcian Blue/PAS staining. We found the absence of CAR altered small intestine (SI) morphology independent of IMI exposure suggesting a basal role of CAR in maintaining overall SI morphology. Car-/-mice also exhibited increased SI weight and length and shorter villi height. AB-PAS staining showed IMI reduced ileal mucin production in WT mice but not Car-/- mice. To further examine CAR's role in the xenobiotic metabolism in the ileum, we will compare microbial diversity and bile acid pools with and without IMI exposure. Our results suggest IMI is not overtly toxic, but the absence of xenobiotic nuclear receptor CAR allows increased accumulation of IMI in the liver and disrupts the structure and Cyp gene expression in the intestine.

Examining the role of Constitutive Androstane Receptor during Imidacloprid exposure

Bile acids are increasingly becoming recognized as hormones, and their main endogenous receptor is the farnesoid X receptor (FXR). In order to investigate whether FXR is necessary to maintain proliferation of the bile ducts in response to injury, we challenged male and female wild-type (WT) and Fxr knockout (FxrKO) mice with 0.1% 1,4 dihydro 2,4,6 trimethyl 3,5 pyridinediacarboxylic acid diethyl ester (DDC) in the diet for two weeks. DDC is an inhibitor of ferrochelatase, the final enzyme of heme biosynthesis, resulting in a buildup of bile plugs in the liver causing an inflammatory response called ductular reaction. We found that deletion of Fxr led to a dramatic reduction in the ductular reaction independent of the sex. Then we tested if the inhibition of ferrochelatase was compensated for in FxrKO mice by analyzing the expression of genes in the heme biosynthesis pathway. Although we find a decrease in basal Alad gene expression in FxrKO mice in both sexes, upon DDC treatment, expression does not decrease as much as seen in the WT mice. Intriguingly, we also noted sex-specific changes. Cpox expression was increased in male FxrKO mice, but FxrKO mice showed reduced suppression of Cpox expression when treated with DDC. Conversely, Ppox expression was reduced in male FxrKO mice compared to WT mice, and both male WT and FxrKO mice had a comparable reduction in Ppox expression upon DDC treatment. Female FxrKO mice showed a significant decrease in Fech expression basally that did not change with DDC treatment. These results suggest that FXR may contribute towards regulating expression of genes in the heme biosynthesis pathway. However, these observations do not explain the lack of ductular reaction. Since PGC1α was previously shown to control heme biosynthesis through its downstream targets, FOXO1 and NRF1, we examined their transcript expression in our study to determine if the pathway is playing a role in our observations. We examined this pathway and observed a significant decrease in Foxo1 expression in both sexes of WT and FxrKO mice basally but only a reduction in Nrf1 expression upon DDC treatment. We are currently investigating other potential regulators of heme biosynthesis and ductular reaction, such as Klf1 which regulates erythropoiesis and plays a role in ductular reaction and the nuclear receptors Rev-Erbα/β that use heme as a ligand, to determine how FXR is regulating heme biosynthesis.

Determining Mechanisms of Farnesoid X Receptor Regulation of Heme Biosynthesis and Ductular Reaction

Objectives: Although 25(OH)D is generally considered the best marker for assessing vitamin D body stores, its role as a marker during acute illness is less well established. Indeed, acute inflammatory insult may reduce circulating 25(OH)D. The objective was to examine serum 25(OH)D levels during the evolution of acute COVID-19 pneumonia.
Material and methods: This pilot study was undertaken as a prospective cohort study. Patients with severe COVID-19, defined as clinical signs of pneumonia and respiratory rate > 30 breaths/minute or severe respiratory distress or oxygen saturation < 90% on room air, were admitted to the internal medicine department between 1 November and 31 December. Blood samples were taken on admission (day 0) and every 24 hours for the subsequent four days (days 1-4). Patients were not supplemented with vitamin D preparations during the monitoring period. All patients received 6 milligrams of dexamethasone daily during the monitoring period.
Results: 22 patients (6 females, 16 males; median age 60.6 years) were included. On admission, 59% of patients were 25(OH)D sufficient (>30 ng/ml), and 41% of patients had 25(OH)D inadequacy (<30 ng/ml) according to the existing guidelines. A significant fall in mean 25(OH)D concentration from admission to day 2 (first 48 h) was observed (30,7 ng/ml vs. 26,4 ng/ml; p<0.0001). No subsequent significant fall in 25(OH)D concentration was observed between day 2 and 3 (26, 4 ng/ml vs. 25,9 ng/ml; p=0.2300) and day 3 and day 4 (25,8 ng/ml vs. 25,9 ng/ml; p=0.7026). The absolute 25(OH)D change between hospital admission and day 4 was 4.8 ng/mL (p<0.0001) and was not associated with mortality or the need for high flow oxygen (p=0.2113 and p=0.6467, respectively). On day 4, the number of patients with 25(OH)D inadequacy (<30 ng/ml) increased by 18% (p=0.0180).
Conclusions: Serum concentration of 25(OH)D decreases significantly during the first 48 hours after hospital admission in acutely ill COVID-19 patients and should be therefore interpreted with caution. Whether low 25(OH)D in COVID-19 reflects tissue level vitamin D deficiency or represents only a laboratory phenomenon remains to be elucidated in prospective randomized trials of vitamin D supplementation.

Serum 25-Hydroxyvitamin D Concentration Significantly Decreases in COVID-19 Patients With Pneumonia During the First 48 Hours After Hospital Admission

Whilst hormonal therapies have proved effective in targeting ER positive breast cancer, intrinsic and acquired resistance to therapy has meant that the disease remains deadly. To identify potential drivers of transcriptional and epigenetic heterogeneity in breast cancer, we performed detailed single-cell multi-ome experiments +/- estrogen stimulation. Our results identified that the transcription factor KLF4 is highly associated with heterogeneity in our experimental models. KLF4 is one of four Yamanaka pluripotency factors that can reprogram cells to a stem like state and is also a pioneer factor, that possesses the ability to bind to condensed chromatin. Silencing of KLF4 significantly reduced transcriptional heterogeneity in multiple models. Importantly, we also found that KLF4 acts as a pioneer factor for ER. ER binding, transcriptional activity and chromatin accessibility at ER binding sites were lost upon KLF4 silencing. Using proteomics based interactome profiling, we find multiple other signaling pathway rely on KLF4. Together, our results suggest that KLF4 maintains plasticity in ER+ breast cancer cells and also functions as a pivotal factor for ER to function.

KLF4 drives estrogen driven tumor heterogeneity in breast cancer

Background: More than 1 in 4 men will undergo inguinal hernia repair during their lifetime. Age is a significant risk factor for inguinal hernias in men with increased incidence in older age. Sex steroid hormone changes are observed with a higher ratio of serum estradiol (E2) to testosterone (T) as men age. We previously demonstrated that a shift from androgen to estrogen action caused inguinal hernia formation in a transgenic male mouse model with increased estrogen action in lower abdominal muscle tissues. However, age-related changes in serum E2 levels in men are conflicting, as some studies have reported increases but others have noted unchanged or even decreased E2 levels with advancing age.
Method: This study was performed on 212 men with inguinal hernias, aged 20 to 90 years, stratified at 10-year intervals of age (20-30, 31-40, 41-50, 51-60, 61-70, 71-80, 81-90 years). We assessed total T (TT), total E2 (TE2), dehydroepiandrosterone (DHEA), sex hormone-binding globulin (SHBG), and gonadotropins (FSH and LH). Body mass index (BMI) was calculated using height and weight.
Results: The BMI distribution was balanced for men with inguinal hernias among all age groups. TE2 was low in 23.7%, normal in 65.8%, and high in 10.5% of men 50 years or younger. In contrast, the percentage of patients with high TE2 was increased 2.6-fold in men over the age of 50 (20.1% in low, 52.3% in normal, and 27.6% in high). TT was normal in all men 50 years and younger but was low in 3.5% and normal in 96.5% of men over the age of 50. We found that serum TE2 levels and the ratio of TE2/TT positively correlated with age (p=0.001). In addition, serum TE2 levels were significantly higher in the advanced age groups (71-80 and 81-90 years) than in the young age group (20-40 years). Serum TT levels decreased but did not reach significance in the advanced age groups compared to the young age group. Remarkably the ratio of E2/T was higher in the advanced age groups than in the young age group (20-30 years). Serum DHEA levels in men over the age of 50 were significantly higher than in men 50 years or younger. SHBG levels were higher in men over 70 years than men 50 years or younger (p< 0.01). Serum levels of FSH and LH in men over 60 years were 3-fold greater than in men 50 or younger (p<0.01).
Conclusions: Our findings suggest a correlation of increased estrogen action in the formation of inguinal hernias at an advanced age. This points to a potential utilization of inhibitors of estrogen synthetase or estrogen antagonists in a subset of elderly inguinal hernia patients with increased circulating estrogen as a non-surgical intervention.

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