United States

Objectives: In healthy, normal - weight adults there is a circadian rhythm in blood pressure (BP) and plasma catecholamines (epinephrine and norepinephrine): BP and the levels of norepinephrine and epinephrine decline during sleep, followed by a rapid increase during the early morning hours. Studies in adults with Type 2 diabetes (T2D) show blunted reductions in BP and plasma catecholamine levels during sleep that are associated with increased risks for heart failure, stroke, myocardial infarction, and sudden death. In this study, we explored if the circadian rhythm in catecholamines is disrupted in youth-onset T2D. We hypothesized that increased sympatho-adrenal activity during sleep differentiates youth with T2D from non-diabetic overweight/obese and lean youth of comparable age, pubertal status, and ethnicity. To that end, we measured urine catecholamines in fasting – morning-spot urines and 24-hour urine samples.
Methods: 56 non-diabetic adolescents with overweight/obesity (&quot;obese&quot;), 42 adolescents with T2D (&quot;T2D&quot;), and 43 normal-weight controls (&quot;lean&quot;); aged 12-21 years, were studied. None was diagnosed with hypertension, and none was on antihypertensive treatment. Weight, height, BMI, BMI%, and BP were extracted from medical charts. Body fat percent (BF%) was measured by TANITA. Stress levels were assessed using PHQ-2 and PHQ-9 questionnaires. Fractionated free urine catecholamines (epinephrine, norepinephrine, and dopamine) were analyzed by liquid chromatography/tandem mass spectrometry (LC/MS-MS) in both spot and 24-hour urines, normalized to urinary creatinine. The ratio of fasting morning urine catecholamines to 24-hour urine catecholamines was calculated to assess circadian variation in catecholamines. Group differences were assessed by Kruskal-Wallis or ANOVA.
Results: Groups were comparable for age (obese 14.8 +/- 1.9; T2D 15.7 +/- 2.1 and lean 14.9 +/- 1.9-yr), pubertal status, and ethnicity. Obese youth with and without T2D were predominantly female (T2D, 28 F, 14 M; obese 33 F, 23 M; lean, 17 F, 26 M); those with T2D had highest BF% (obese 37.3 +/- 9.5; T2D 42.9 +/- 9.9; lean 20.1 +/- 6.3 %; p=2.58e-22) and systolic blood pressure (obese 115.36 +/- 11.54; T2D 127.83 +/- 12.08 and lean 111.65 +/- 9.13 mmHg; p=5.49e-10).  Fractionated free urine catecholamines (epinephrine, norepinephrine and dopamine) were comparable across groups in 24-hour urines. However, fasting morning epinephrine levels and the ratio of fasting morning/24-hour epinephrine were higher in T2D (p=0.0035, and p=0.035, respectively) than in either lean or nondiabetic youth with obesity. There were no differences in morning urine catecholamines between lean controls and non-diabetic youth with obesity.
Conclusions: Our results suggest a disrupted circadian rhythm in catecholamines in youth-onset T2D, with a blunted overnight fall in epinephrine levels. Higher levels of epinephrine levels at night in youth with T2D might be associated with, or predispose to, hypertension and long-term cardiovascular complications.

ENDO 2022

Disrupted circadian rhythm in catecholamines in youth-onset Type 2 Diabetes

**Program**
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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.

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**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:
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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. 

Objectives: In healthy, normal - weight adults there is a circadian rhythm in blood pressure (BP) and plasma catecholamines (epinephrine and norepinephrine): BP and the levels of norepinephrine and epinephrine decline during sleep, followed by a rapid increase during the early morning hours. Studies in adults with Type 2 diabetes (T2D) show blunted reductions in BP and plasma catecholamine levels during sleep that are associated with increased risks for heart failure, stroke, myocardial infarction, and sudden death. In this study, we explored if the circadian rhythm in catecholamines is disrupted in youth-onset T2D. We hypothesized that increased sympatho-adrenal activity during sleep differentiates youth with T2D from non-diabetic overweight/obese and lean youth of comparable age, pubertal status, and ethnicity. To that end, we measured urine catecholamines in fasting – morning-spot urines and 24-hour urine samples.
Methods: 56 non-diabetic adolescents with overweight/obesity ("obese"), 42 adolescents with T2D ("T2D"), and 43 normal-weight controls ("lean"); aged 12-21 years, were studied. None was diagnosed with hypertension, and none was on antihypertensive treatment. Weight, height, BMI, BMI%, and BP were extracted from medical charts. Body fat percent (BF%) was measured by TANITA. Stress levels were assessed using PHQ-2 and PHQ-9 questionnaires. Fractionated free urine catecholamines (epinephrine, norepinephrine, and dopamine) were analyzed by liquid chromatography/tandem mass spectrometry (LC/MS-MS) in both spot and 24-hour urines, normalized to urinary creatinine. The ratio of fasting morning urine catecholamines to 24-hour urine catecholamines was calculated to assess circadian variation in catecholamines. Group differences were assessed by Kruskal-Wallis or ANOVA.
Results: Groups were comparable for age (obese 14.8 +/- 1.9; T2D 15.7 +/- 2.1 and lean 14.9 +/- 1.9-yr), pubertal status, and ethnicity. Obese youth with and without T2D were predominantly female (T2D, 28 F, 14 M; obese 33 F, 23 M; lean, 17 F, 26 M); those with T2D had highest BF% (obese 37.3 +/- 9.5; T2D 42.9 +/- 9.9; lean 20.1 +/- 6.3 %; p=2.58e-22) and systolic blood pressure (obese 115.36 +/- 11.54; T2D 127.83 +/- 12.08 and lean 111.65 +/- 9.13 mmHg; p=5.49e-10).  Fractionated free urine catecholamines (epinephrine, norepinephrine and dopamine) were comparable across groups in 24-hour urines. However, fasting morning epinephrine levels and the ratio of fasting morning/24-hour epinephrine were higher in T2D (p=0.0035, and p=0.035, respectively) than in either lean or nondiabetic youth with obesity. There were no differences in morning urine catecholamines between lean controls and non-diabetic youth with obesity.
Conclusions: Our results suggest a disrupted circadian rhythm in catecholamines in youth-onset T2D, with a blunted overnight fall in epinephrine levels. Higher levels of epinephrine levels at night in youth with T2D might be associated with, or predispose to, hypertension and long-term cardiovascular complications.

technical paper

Context: Growth hormone (GH) plays a critical role in glucose homeostasis and linear growth during childhood. GH secretion is controlled by multiple neurotransmitters and hormones. Sleep is also a powerful stimulus for GH secretion. In children, GH pulses occur after sleep onset in association with slow-wave sleep (SWS), but it is unclear if undisrupted sleep is required for normal GH secretion. Given the rising incidence of pediatric sleep disorders such as obstructive sleep apnea (OSA) that cause sleep fragmentation, it is critical to determine whether sleep fragmentation during childhood may dysregulate the somatotropic axis.
Objective: To quantify how undisrupted vs fragmented sleep affects GH secretion in children.
Methods: Pubertal subjects (10 M, 9 F; 9.1-14.4 yrs) were randomized to two overnight polysomnographic studies with frequent blood sampling (q10min) to measure GH: fourteen were healthy subjects studied with or without SWS disruption via auditory stimuli (3 sec, 1500 Hz tones, 40-100 dB followed by 18 sec of a 75 dB noise simulating a knock on the door) and five were subjects with OSA studied with or without prescribed continuous positive airway pressure (CPAP); the latter condition was expected to have sleep fragmentation due to untreated OSA. GH pulses were identified using sparse deconvolution. Studies without auditory stimuli or when using CPAP were combined and analyzed as 'undisrupted sleep'. The relationship between GH pulse rate per hour and sleep stage was evaluated using Poisson generalized estimating equations.
Results: In SWS disruption studies, an average of 77.51 ± 13.61 (range 23-190) auditory stimuli were delivered, causing a 40.02 ± 7.78% decrease in SWS in favor of lighter sleep stages. In subjects with OSA, CPAP withdrawal caused a slight increase in the apnea hypopnea index (1.08 ± 0.10 vs. 2.20 ± 1.70 events/hour of sleep, p=0.07) with no change in sleep stage durations. During undisrupted sleep, subjects had an average of 0.91 ± 0.067 GH pulses/hour with a mean pulse amplitude of 10.03 ± 1.00 ng/mL and mean basal secretion rate of 0.26 ± 0.10 ng/ml per min. GH pulses occurred more frequently during SWS (2.3 pulses/hour SWS, amplitude 9.92 ± 2.59 ng/mL) than during any other sleep stage (P < 0.001). Neither form of sleep disruption affected the distribution of GH pulses across sleep stages, GH pulse amplitude and frequency, nor basal GH secretion.
Conclusion: In children, nocturnal GH pulses are temporally associated with SWS. However, neither acute fragmentation of SWS nor nocturnal arousals from OSA altered basal or pulsatile GH secretion, indicating that somatotrophic axis activity is preserved in the face of acute sleep disruption in children.

Acute sleep disruption does not diminish pulsatile growth hormone secretion in pubertal children

Objectives: We have previously demonstrated that insulin resistance (IR) in youth is associated with elevated levels of branched-chain amino acids (BCAAs).  BCAAs compete with aromatic amino acids (AAA) including tryptophan, the precursor of serotonin, for uptake into β-cells and other tissues via the large neutral amino acid transporter. Serotonin has been reported to increase β-cell mass and glucose-dependent insulin secretion. In this study we have explored how BCAA, tryptophan (one of the AAA), and a subset of their metabolites are modulated in youth-onset T2D. Based on our prior studies in neuronal BCAA metabolism, we hypothesized that elevated BCAA could induce diversion of tryptophan metabolism towards production of kynurenine rather than serotonin in youth with T2D. To test this, we analyzed 24-hour urine samples and compared levels of byproducts of BCAAs and tryptophan metabolism in obese youth with T2D with those in non-diabetic obese and lean youth of comparable age, pubertal status and ethnicity.
Methods: 56 non-diabetic adolescents with overweight/obesity ("obese"), 42 adolescents with T2D ("T2D"), and 43 normal weight controls ("lean"), ages 12-21 years-old were studied. Weight, height, BMI, BMI% were extracted from medical charts. Body fat percent (BF%) was measured by TANITA. We also measured metabolites derived from BCAA catabolism, including the branched-chain ketoacids (BCKAs), and tryptophan metabolism, including intermediates of the serotonergic and kynurenine pathways, in spot and 24-hour urine samples by liquid chromatography/tandem-mass spectrometry (LC/MS-MS). Levels were normalized to urine creatinine. Group differences were assessed by Kruskal Wallis or ANOVA.
Results: Groups were comparable for age (obese 14.8 +/- 1.9; T2D 15.7 +/- 2.1 and lean 14.9 +/- 1.9-yr), pubertal status, and ethnicity. Youth with T2D were predominantly female (T2D, 28 F, 14 M; obese 33 F, 23 M and lean, 17 F, 26 M), and had highest BF% (obese 37.3 +/- 9.5; T2D 42.9 +/- 9.9; lean 20.1 +/- 6.3 %; p=2.58e-22). In 24-hour urine samples, BCKAs, tryptophan, and kynurenine levels were higher in T2D (p=0.0002, p= 0.0045 and p= 0.00009 respectively) than in either lean controls or nondiabetic youth with obesity; in contrast, there were no differences between lean controls and non-diabetic youth with obesity. The levels of 5-HIAA, the principal metabolite of serotonin, were comparable across groups; however, the ratio of kynurenine/tryptophan was higher (p= 0.0112) in youth with T2D and the ratios of 5-HIAA/ tryptophan (p=0.027) and 5-HIAA/Kynurenine (p=0.0067) were lower compared to the other two groups. Those ratios were comparable between lean controls and non-diabetic youth with obesity.
Conclusions: Increased BCKAs are accompanied by diversion of tryptophan metabolism from the serotonin pathway to the kynurenine pathway, suggesting perturbations in both BCAA and AAA metabolism in youth-onset T2D. These alterations could contribute to development of beta-cell dysfunction and progression to T2D in youth.

Branched-chain Amino Acid and Tryptophan Metabolism and the Pathogenesis of Youth-Onset Type 2 Diabetes Mellitus (T2D)

Type 1 diabetes (T1D) is a complex disease characterized by the loss of pancreatic beta-cells and lifelong dependence on insulin replacement therapy. As the worldwide incidence of T1D has increased and the prevalence of high-risk HLA haplotypes has declined among new diagnoses, a greater focus has been placed on environmental factors contributing to T1D pathogenesis.  Lactiplantibacillus plantarum 299v (Lp299v) supplement is reported to increase plasma and stool levels of anti-inflammatory short-chain fatty acids (SCFAs) and promote IL-10 signaling in colonic derived macrophages and T-cells.  Therefore, we investigated the effect of Lp299v supplement on T1D pathogenesis in diabetes-prone BioBreeding DRlyp/lyp rats.  Rats were weaned at 21 days of age (D21) onto a normal cereal diet (ND) or a gluten-free hydrolyzed casein diet (HCD), with or without daily Lp299v supplementation. All rats provided ND developed diabetes by D83 and supplementing ND with Lp299v did not significantly delay progression.  Rats provided HCD showed a significant delay in diabetes onset over ND, while progression was even more delayed in the HCD+Lp299v group, with 25% of animals remaining diabetes-free to D130 (study end).  Relative to the other groups, HCD+Lp299v rats exhibited significant increases in the plasma levels of the SCFAs propionate and butyrate (beneficial metabolites produced by intestinal bacteria). In addition, stool microbiota showed increased abundances of SCFA-producing taxa at D40.  Gene expression analysis of pancreatic islets was conducted at D40, prior to insulitis. Lp299v supplementation favorably modulated islet expression levels of pathways related to endoplasmic reticulum (ER) stress, and the major arms of the unfolded protein response (UPR), especially in combination with the HCD.  Notably, ER stress has been implicated in the formation of islet neoantigens that may underlie the initial loss of immune tolerance in T1D.  Lp299v supplement differentially modulated the UPR to favor the expression of the transcription factor Atf6, increase expression of transcripts related to cell survival/proliferation, and the ER-associated protein degradation (ERAD) pathway of the UPR.  This contrasted with HCD alone, which promoted a transcriptional program related to autophagy.  To colocalize and confirm activities detected in the transcriptional analyses to β-cells, pancreata of D40 rats were subjected to dual immunofluorescence staining for insulin and UPR-related proteins.  Consistent with elevated ERAD activity, significantly greater EIF4G1 and OS9 expression were observed in HCD+Lp299v islets. Phospho-PERK staining was significantly higher in islets of the HCD-alone group, consistent with the elevated expression of autophagy transcripts. Analysis of plasma proinsulin: C-peptide ratios revealed that Lp299v alone could improve islet function, as significant decreases were measured in all groups relative to ND.  While ongoing studies aim to define the bacterial metabolites underlying the favorable outcomes, these studies suggest that Lp299v supplementation improves proteostasis capacity in β-cells and slows T1D progression in DRlyp/lyp rats.

Probiotic Supplementation with Lactiplantibacillus Plantarum 299v Modulates β-Cell Endoplasmic Reticulum Stress and Prevents Type 1 Diabetes in Biobreeding Rats

Background: Peripubertal hyperandrogenemia (HA) often represents a forerunner to adolescent/adult polycystic ovary syndrome (PCOS), but risk factors for peripubertal HA are unclear. Studies have associated obesity with HA throughout puberty (e.g., McCartney, JCEM 2007;92: 430-436, Knudsen, Obesity 2010;18: 2118-2124). However, these studies were performed in academic centers and could have been influenced by recruitment bias. We therefore investigated if there was a difference in calculated free testosterone (T) levels between girls with healthy weight and those with obesity in a nationally-representative U.S. sample.
Methods: We analyzed data for 1,196 girls aged 6–18 years from the National Health and Nutrition Examination Survey (NHANES) 2013-2016 databases, utilizing a cross-sectional study design. We calculated free T from total T and sex hormone-binding globulin (Vermuelen equation). We divided girls into 3 groups by age, based on presumed pubertal stages: "Pre-/Early" (6–9 years), "Middle" (10–14 years), and "Late" (15–18 years). Each individual was also categorized as healthy-weight (BMI-for-age percentile 5-85) or obese (BMI-for-age percentile ≥95). We analyzed mean free T concentrations by weight status (healthy-weight vs. obese) for each age group by calculating population estimates. We also performed survey-weighted regression analysis to assess the relationships between weight status and age category (independent variables) and free T (outcome).
Results: Among Pre-/Early girls, free T was 0.33 pg/ml (0.28-0.38) [mean (95% CI)] for healthy-weight vs. 0.86 pg/ml (0.67-1.05) for obese. Among Middle girls, free T was 2.53 pg/ml (2.27-2.78) for healthy-weight vs. 4.35 pg/ml (3.75-4.95) for obese. Among Late girls, free T was 3.33 pg/ml (2.96-3.70) for healthy-weight vs. 5.64 pg/ml (4.93-6.36) for obese. (Notably, in each age group, the 95% CIs for free T did not overlap between healthy-weight vs. obese subgroups). Mean free T increased with advancing age in both healthy-weight and obese groups, especially between Pre-/Early and Middle age ranges (Pre-/Early-to-Middle differences were 2.20 pg/ml for healthy-weight and 3.49 pg/ml for obese). Our regression model accounted for 45% of the variance in free T (R²=0.45). Both weight status (obese vs. healthy-weight) and age group were independent predictors of free T concentrations (p<0.0001 for both).  When adding an age-in-months-by-BMI-percentile interaction term, the model R² value was 0.47, with age category, weight category, and the interaction term all being independent predictors of free T (p<0.001 for all).  Conclusion: We conclude that obesity is associated with higher free T levels in U.S. girls aged 6–18 years. Age also predicts free T, likely reflecting increases related to pubertal maturation, and free T differences according to weight status are more prominent in older girls. This study in a nationally-representative sample of U.S. girls supports previous analyses of girls studied at academic centers, further suggesting an important relationship between obesity and peripubertal androgen excess.

Obesity is Associated with Adolescent Hyperandrogenemia in Girls Aged 6-18, National Health and Nutrition Examination Survey (NHANES) 2013-2016

PCOS confers an increased risk for obesity-related comorbidities. It has been challenging to identify precise risk genes associated with the pathogenesis of PCOS. A novel variant located within MYO10 intron 2 (rs9312937) was identified in a PCOS GWAS in the genetically isolated Finnish and related Estonian populations. We identified PCOS risk variants at the MYO10 locus in additional PCOS populations. We hypothesized that PCOS risk variants at the locus were associated with metabolic traits.
Women from Iceland (n=1347) and Boston (n=591) were diagnosed with PCOS using the Rotterdam criteria and NIH criteria, respectively, with a non-overlapping replication cohort (n=586) identified using an algorithm incorporating ICD codes and natural language processing. Controls included population controls from Iceland (effective n=1200), well-phenotyped controls from Boston (n=441) and a cohort with no PCOS diagnoses or features in the electronic medical record (EMR; n=749). We performed an association study to identify variants in the MYO10 locus associated with PCOS. We also examined the association of variants in MYO10 and 15 independent phenotypic traits in women of European ethnicity from the Boston PCOS cohort. Data were log-normalized and analyzed using linear regression, then corrected for the false discovery rate using the Benjamini-Hochberg procedure.
Two novel risk variants for PCOS were identified within intron 2 of MYO10, rs31506-T (p = 1.6X10-05, OR 0.83 [95% CI 0.77-0.91]) and rs253336-A (p = 2.4X10-06, 0.82 [0.75-0.89]). The rs31506 variant is in moderate linkage disequilibrium with the novel variant rs9312937 (r² ≥ 0.4) and rs253336 (r² ≥ 0.4). The variant rs31506 maps to enhancer sites in pancreas, osteoblast and fetal adrenal cells, whereas rs253336 maps to methylation sites in adipose stem cells and ovary (pubs.broadinstitute.org/mammals/haploreg). Neither variant had eQTLs, and expression was highest in lung, thyroid, neurons, breast and ovary (gtexportal.org).
Among cases and controls, the variant rs253336 was associated with BMI (β=0.015±0.0057; p=0.01), waist circumference (β=0.011±0.0047; p=0.02), hip circumference β=0.010±0.0035; p=0.003) and triglycerides (β=0.028±0.013; p=0.03). The variant rs31506 was also associated with BMI (β=0.0065±0.0027; p=0.02), waist circumference (β=0.0051±0.0022; p=0.02), hip circumference (β=0.0039±0.0017; p=0.02), HDL (β=-0.0083±0.0037; p=0.02) and triglycerides (β=0.018±0.0063; p=0.003). The association between rs253336 and hip circumference, and rs31506 and triglyceride levels remained significant after correction. There was no association between the variants and BMI in the subjects identified through the EMR.
We identified two novel risk variants for PCOS in intron 2 of MYO10 (rs31506 and rs253336), a gene recently implicated within a risk locus for PCOS. We also demonstrated that the variants are associated with hip circumference and triglyceride levels. Previous data demonstrate increased expression of MYO10 in omental adipose of obese type 2 diabetics and an association with lean body mass. The data suggest a potential role for MYO10 in the metabolic etiology of PCOS.

Risk Variants at the MYO10 Locus are Associated with Metabolic Traits

Introduction and Purpose: Polycystic Ovary Syndrome (PCOS) is the most common endocrine disorder in women of reproductive age. Notably, PCOS women with hyperandrogenism have a pronounced increased risk for cardio-metabolic comorbidities. The gut microbiome is responsible for fermentation of indigestible fiber to produce short-chain fatty acids (SCFA), which help maintain metabolic function and reduce gut inflammation. Alteration in the gut microbiome, or dysbiosis, affects overall metabolic homeostasis and contributes to pathogenesis of metabolic diseases. While dysbiosis has been observed in women with PCOS, the molecular mechanisms of hyperandrogenemia-induced dysbiosis and its contribution to SCFA excretion and gut inflammation have not been studied in PCOS.
Methods: Four-week old C57BL/6N female mice were implanted subcutaneously with dihydrotestosterone (DHT, 8.0 mg) or vehicle silastic tubes (n=8/grp). Animals were treated day 90 and the gastrointestinal tract was collected. Expression levels of mRNA were assessed by RT-qPCR. Fecal microbiota was assessed by 16S rRNA gene sequencing and profiled using MicrobiomeAnalyst; SCFA concentrations were assessed by LC-MS.
Results: DHT-treated female mice had significantly increased lean mass (21.35 ± 0.84 vs 19.25 ± 0.55 g, p<0.05) and increased fat mass (5.18 ± 1.45 vs 2.73 ± 0.64 g, p<0.05) compared to the vehicle counterparts. DHT-treated female mice showed increased HbA1C (5.18 ± 1.45 vs 2.73 ± 0.64 %, p<0.05). Hyperandrogenemic females showed altered gastrointestinal morphology, having decreased small intestine (33.03±2.05 vs 28.69±2.604 cm, p<0.05) and colon length (7.88 ± 0.64 vs 6.60 ± 0.55 cm, p<0.05) as well as increased immune cell infiltration assessed by histology. DHT-treated female mice have an increase in the relative percent abundance of Proteobacteria (2.04 ± 0.62 vs 0.35 ± 0.23%, p<0.05) and in the ratio of Firmicutes/Bacteriodetes (1.92 ± 0.47 vs 1.11 ± 0.17, p<0.05). Fecal concentrations of short-chain fatty acids were higher in DHT-females for butyric acid (92.33 vs 404.8 ng/mg feces, p<0.05), acetic acid (571.8 vs 1592 ng/mg feces, p<0.05), and propionic acid (80.59 vs 167.3 ng/mg feces, p<0.05). However, expression of the SCFA receptor Ffar2 in the colon was upregulated by DHT (1.31 ± 0.15-fold, p<0.05), indicating the possibility of a compensation to ameliorate increased SCFA excretion. The mRNA expression of lipopolysaccharide induced inflammation markers were significantly upregulated by DHT in the proximal colon (Litaf, 1.69 ± 0.29-fold; Tlr4 1.27 ± 0.15-fold, p<0.05) compared with vehicles.
Conclusions: Androgen-mediated gut dysbiosis may contribute to altered glucose handling, adiposity, and metabolic homeostasis in PCOS. Increased elimination of SCFA in DHT-treated female mice may indicate less efficient absorption, which can contribute to colonic inflammation, altered gastrointestinal morphology, and increased gut permeability. Together, our results highlight the potential role of androgen-mediated gut dysbiosis to influence metabolic dysfunction.

Contribution of Hyperandrogenemia to Dysbiosis, Gut Inflammation, and Dysregulation of Short-Chain Fatty Acid Homeostasis in Mouse Model of Polycystic Ovary Syndrome

Objective: Adolescent polycystic ovary syndrome (PCOS) is a heterogenous clinical syndrome characterized by hyperandrogenism and irregular menses. Heterogeneous features of the syndrome include the presence/absence of obesity, degree of insulin resistance, and clinical and/or biochemical hyperandrogenism. In adult women with PCOS, discrete metabolic and reproductive subgroups can be identified. A recent study described distinct phenotypes using unsupervised hierarchical cluster analysis: a metabolic subgroup characterized by higher BMI, glucose, and insulin levels, and a reproductive subgroup demonstrating higher LH and SHBG levels. We hypothesized that non-obese and obese adolescent girls with PCOS would exhibit similar distinct phenotypes at the time of diagnosis.

Methods: Data were extracted from a multi-site PCOS registry including seven academic centers in the US with the following inclusion criteria: PCOS confirmed per 2018 international guidelines, diagnosis before age 18 years, and at least one follow-up visit. Data collected included demographics, medical and family history, physical characteristics including hirsutism, acanthosis nigricans, and laboratory measures such as HbA1c, ALT, lipid profiles and reproductive hormones (androgens, LH, FSH, SHBG). Hormone levels were normalized to the upper limit provided by the measuring laboratory. Patients were divided by obesity status, defined as obese (OB) with BMI ≥ 95th percentile for age and sex and non-obese (NO) < 85th percentile for age and sex. Differences in continuous variables were assessed with unpaired t-tests using GraphPad Prism.

Results: Complete diagnostic data from 390 patients from 7 different sites were included for analysis, with 284 OB (Age 15.3±1.5 years, BMI 36.1±5.6 kg/m²) and 68 NO (Age 15.8±1.3 years, BMI 22.6±3.4 kg/m²). OB presented with more severe acanthosis (p<0.0001) and higher HbA1c (OB 5.5±0.5, NO 5.2±0.3; %, p<0.0001), ALT (OB 35±30, NO 22±17; IU/L, p=0.0006), and triglycerides (OB 145±90, NO 110,±88; mg/dL, p=0.002). OB also presented with higher free testosterone (OB 219±188% vs NO 158±125% above upper limit of normal; p=0.003), while NO presented with higher total testosterone (OB 121±68 %, NO 152±88 % above upper limit of normal; p=0.003), androstenedione (OB 87±37 %, NO 125±62 above upper limit of normal; p=0.002), and LH (OB 9.7±4.9, NO 15.2±10.4; mIU/mL, p=0.0001). No differences in Ferriman Gallwey score or number of menses in the 6 months prior to diagnosis were detected.

Conclusions: Metabolic and reproductive phenotypes differed in OB and NO adolescent girls at the time of PCOS diagnosis. The reproductive phenotype in NO was characterized by increased LH, total testosterone and androstenedione levels. In contrast, OB girls had a phenotype characterized by insulin resistance-driven hyperandrogenemia established by lower SHBG and higher free testosterone levels. Our data are consistent with findings reported for adult women with two divergent phenotypes: an insulin resistance-driven metabolic subtype and a primarily reproductive subtype, potentially driven by neuroendocrine mechanisms.

Heterogeneity of Clinical Presentation of Adolescent PCOS Is Attributable to Distinct Subtypes

During the SARS-COV2 pandemic (COVID-19) many studies have examined changes in women's stress, depression/anxiety, reproduction, plus post-vaccination cycle/flow changes¹. Although there are retrospective basal temperature data², we lack menstrual cycle and ovulation information during the pandemic. This analysis compared two independent, similar-design, convenience/community-based, single-cycle cohort studies 13 years apart: Menstruation Ovulation Study (MOS³, n=301, 2007-8) and MOS2 (n=112) during the pandemic. With MOS as a control, and given evidence subclinicalovulatory disturbances (SOD; short luteal phase/anovulatory menstrual cycles of normal lengths) occur before changed cycle lengths4 5, we hypothesized MOS2 would have increased SOD but similar cycle lengths as MOS. Methods: In both studies, recruitment of menstruating women ages 19-35 years, not using systemic or combined hormonal contraceptives (CHC) used posters/eblasts/social media. In MOS, ovulation was assessed by 3-fold increased follicular (FP)-to-luteal (LP/premenstrual) urinary progesterone (PdG); in MOS2, by validated Quantitative Basal Temperature© (QBT)6 7 (normal LP=10+ days). We performed the same interviewer-administered (CaMos©) questionnaire for demographics, SES, and reproduction, measured anthropomorphic variables, plus collected daily Menstrual Cycle Diary© (Diary) for all. FP and LP/premenstrual PdG or salivary progesterone (Ps) samples were respectively collected. Participants in MOS2 were not different from MOS in: average age 29, menarche age 12.5, BMI 24, living situation and education (≥75% university graduates). Cohorts also differed: MOS2 women were less likely to to be White (56% vs 76%), work fulltime, ever use CHC (68% vs 79%) or to be parous (8% vs 20%); they were younger at starting CHC (17.9 vs 18.6 years). Results: MOS2 and MOS had similar cycle (30.3 vs 29.9 days, P =.306) and flow lengths (median 6.0 days; P=.055). MOS2 recorded significantly more SOD cycles (>50% anovulatory) vs MOS³ (63% vs 10%; P<.001). MOS2 Diary analyses by Principal Components Analysis showed significantly increased anxiety/depression/frustration (negative moods) and "outside stresses" plus sleep problems and headaches vs MOS (all P<.001). Discussion: This is the first evidence that ovulatory disturbances without cycle length changes may be associated with the multidimensional stresses women experience during the pandemic. Increased SOD may also relate to greater nulliparity, younger CHC teen use and more non-White women in MOS2, as well higher prevalence of negative moods, outside stresses and sleep problems. Salivary progesterone, cortisol and estradiol levels remain pending. Prevalent SOD cycles, if persistent/recurrent, risk increased infertility, bone loss⁵, early heart attacks, and breast and endometrial cancers⁸. In this 'experiment of nature,' the pandemic was associated with disturbed ovulation rather than menstrual cycles. ¹Edelman AB Ob Gyn 2022; ²Nguyen JK PLOS ONE 2021; ³Prior JC IJERPH 2018; ⁴Prior JC NEJM 1990; ⁵Li D Epidiol Rev 2014; ⁶Prior JC Clin Invest. Med 1990; ⁷Bedford JL Eur J ObGyn Reprod Biol 2009; ⁸Prior JC DDDM 2020.

Epidemic of Subclinical Ovulatory Disturbances During SARS-COV2 Pandemic—an Experiment of Nature

In orthotopic xenografts and a PDX, our anticancer drug ErSO eradicates primary and metastatic therapy-resistant estrogen receptor alpha (ERα) positive breast cancer, and induces near-complete regression of ovarian cancer. The mechanism by which ErSO induces necrosis and kills ER positive cancer cells was unknown. From genome-wide CRISPR-Cas9 screens in MCF-7 and T47D cells with negative selection against first-generation BHPI and second-generation ErSO, and follow-on experiments, we identified the Ca2+ activated, plasma membrane Na+ channel TRPM4 as the executioner protein that BHPI and ErSO use to induce necrosis. Notably, in 6 ERα+ breast and ovarian cancer cell lines, knockout of TRPM4 completely abolished the ability of ErSO to induce death of cancer cells. Moreover, TRPM4 mRNA and protein were dramatically down-regulated in breast cancer cells selected for resistance to BHPI and ErSO. Furthermore, in a mouse xenograft, while ErSO induced near complete regression of orthotopic MCF-7-ERY537S-luciferase tumors, ErSO had no effect on the TRPM4 knockout tumors, which continued their robust growth. Since necrosis, but not most other death pathways, activates immune cells, inducing immunogenic cell death, this provides a new avenue for enhancing cancer immunotherapy. Importantly, medium from ErSO-treated wild type MCF-7 cells, but not medium from TRPM4 knockout cells, robustly activates human THP-1 monocytes and greatly increases their migration. BHPI and ErSO-induced initial anticipatory unfolded protein response (a-UPR) activation results in elevated cytosolic Ca2+, opening the plasma membrane TRPM4 channel, eliciting a rapid influx of external Na+, accompanying Cl- to balance the charge, and water to maintain osmolality. This swells the cells, causing osmotic stress. Importantly, it is the osmotic stress that sustains UPR hyperactivation, leading to ATP depletion, which contributes to membrane rupture and rapid necrotic cell death and to near complete inhibition of protein synthesis that ultimately kills any surviving cancer cells . Suggesting a broad role of TRPM4 in the actions of necrosis inducing anticancer drugs, TRPM4 knockout also inhibited necrosis induced by unrelated anticancer therapies, the mitochondrial targeting oncolytic peptide, LTX-315 and the Ca2+ channel targeting agent, Englerin A. Since increasing expression TRPM4 by viral transduction results in progressively increased sensitivity of ER positive breast cancer cells to killing by ErSO, this enables identification of breast cancer patients whose elevated TRPM4 levels make them most likely to benefit from this novel therapy. The TRPM4 pathway is a new mechanism for sustained lethal activation of the UPR and for targeting ER positive breast and ovarian cancer.

How Strong and Sustained Activation of the Estrogen Receptor-mediated Anticipatory Unfolded Protein Response Kills Breast and Ovarian Cancer Cells

Recurrence of metastatic breast cancer stemming from acquired endocrine and chemotherapy resistance remains a health burden for women with luminal (ER+) breast cancer. ER+ tumor cells can remain viable but quiescent for years to decades. Contributing factors to metastatic spread include the survival and expansion of breast cancer stem cells (CSCs). Breast CSCs are poorly proliferative and frequently exist as a minority population in recurrent tumors. Our objective is to define novel signaling pathways that govern therapy resistance in ER+ breast cancer. We previously showed that cytoplasmic complexes composed of steroid receptor (SR) co-activators, PELP1 and SRC-3, modulate breast CSC expansion through upregulation of the HIF-activated metabolic target genes PFKFB3 and PFKFB4. PELP1 interacts with PFKFB proteins, and inhibition of PFKFB3 and PFKFB4 kinase activity blocks PELP1-induced tumorspheres and protein-protein interactions with SRC-3. PFKFB inhibitors in combination with ER targeted therapies blocked proliferation and tumorsphere formation in multiple models of advanced breast cancer, including paclitaxel (TaxR) and tamoxifen (TamR) resistant models and ER+ patient-derived organoids (PDxO). Chemotherapy and endocrine therapy resistant models contained increased levels of cytoplasmic PELP1, PELP1/SRC-3 complexes, and phenocopied PELP1/SRC-3/PFKFB biology and CSC behavior. To better understand CSC-mediated pathways in the context of therapy resistance, we performed RNA-seq on 3D-cultured TaxR tumorspheres to enrich for CSCs. Pathway analysis revealed that TaxR tumorspheres upregulate stem (Sox2-Oct4-Nanog), HIF, and progesterone mediated pathways. Progesterone receptor (PR) is a known contributor to CSC populations, and we observed increased PR mRNA and protein expression in TaxR cells grown as tumorspheres. Additionally, in vivo emergence of circulating tumor cell (CTC) populations was observed in ER+ TaxR mammary intraductal (MIND) xenografts. Together, our data suggest that PELP1, SRC-3, and PFKFBs cooperate to drive ER+ tumor cells that include CSCs and CTCs. Our findings are applicable to therapy resistant models, indicating that SR co-activator complexes are a key mediator of resistance in ER+ breast cancer and may cooperate with PR to promote therapy resistant CSCs. Identifying non-ER pharmacological targets offers a useful approach to blocking metastatic escape from standard of care ER/estrogen (E2)-targeted strategies to overcome endocrine and chemotherapy resistance in ER+ breast cancer.

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