United States

Well-treated persons living with HIV (PLWH) are predisposed to fat redistribution/visceral adiposity, potentially due to anti-retroviral therapy (ART) use and/or the virus itself, and demonstrate a 2-fold higher risk of cardiovascular disease (CVD) compared to those without HIV. Visceral adipose tissue (VAT) is a metabolically unfavorable ectopic fat depot, which is highly inflamed and dysfunctional. Fat redistribution leading to VAT accumulation is related to increased CVD risk. Current gold standard measures of VAT are obtained using CT and MRI, but are not used clinically. The visceral adiposity index (VAI) is a simple tool combining biochemical measures with anthropometrics that can be easily evaluated by the clinician. VAI may be a surrogate measure of VAT and indeed has been shown to relate to insulin resistance, type 2 diabetes mellitus, metabolic syndrome and CVD among the general population. In this study, we evaluated VAI in PLWH and its relation to VAT, anthropometric measures and other cardiometabolic parameters.
45 PLWH on stable ART with no known CVD were recruited as part of a clinical trial to evaluate CVD indices and were included if they were virologically controlled (HIV viral load &lt;200 copies/mL) and demonstrated increased abdominal VAT (VAT area&gt;110cm²) on CT. For purposes of this study, baseline fasting biochemical, radiologic and anthropometric data were used. VAI was calculated using the standard sex-specific formulas which incorporate waist circumference (WC), BMI, triglycerides and HDL. Presence of coronary plaque was assessed using coronary CT angiography or coronary PET scans. Linear regression was performed to assess relationships with VAI. Non-normally distributed variables were log-transformed for analyses.
Participants were predominantly male (73%), Caucasian (53%), and non-Hispanic (84%) with mean age 55 ± 7 years. Participants had a long duration of HIV and ART use (20 ± 8 and 15 [12,19] years, respectively). The majority of PLWH were obese (BMI 31.9 ± 5.8 kg/m²) with VAT 189 [127,267]cm² and VAI 4.9 [2.8,7.3]. VAI correlated strongly with VAT (r=0.59, P&lt;0.0001), anthropometric measures (BMI r=0.36, P=0.02; WC r=0.43, P=0.004; WHR r=0.33, P=0.03) and ALT (r=0.32, P=0.03), and did not relate to HIV-specific parameters and other metabolic parameters (blood pressure, HbA1c). Participants with coronary plaque tended to have a higher VAI compared to those without coronary plaque (log VAI 0.7 ± 0.3 vs. 0.5 ± 0.3, P=0.056).
These data show VAI, an easily obtained measure, is strongly correlated with abdominal VAT area measured by CT and may be a useful biomarker for visceral adiposity in HIV. Furthermore, VAI may be related to ALT and coronary plaque, which could help identify those PLWH at risk for fatty liver disease (another ectopic fat depot) and heart disease, respectively. Further studies are needed to assess the utility of VAI in evaluating metabolic disease in HIV.

ENDO 2022

Visceral adiposity index as a measure of cardiometabolic disease in persons living with HIV

**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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**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.

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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. 

Well-treated persons living with HIV (PLWH) are predisposed to fat redistribution/visceral adiposity, potentially due to anti-retroviral therapy (ART) use and/or the virus itself, and demonstrate a 2-fold higher risk of cardiovascular disease (CVD) compared to those without HIV. Visceral adipose tissue (VAT) is a metabolically unfavorable ectopic fat depot, which is highly inflamed and dysfunctional. Fat redistribution leading to VAT accumulation is related to increased CVD risk. Current gold standard measures of VAT are obtained using CT and MRI, but are not used clinically. The visceral adiposity index (VAI) is a simple tool combining biochemical measures with anthropometrics that can be easily evaluated by the clinician. VAI may be a surrogate measure of VAT and indeed has been shown to relate to insulin resistance, type 2 diabetes mellitus, metabolic syndrome and CVD among the general population. In this study, we evaluated VAI in PLWH and its relation to VAT, anthropometric measures and other cardiometabolic parameters.
45 PLWH on stable ART with no known CVD were recruited as part of a clinical trial to evaluate CVD indices and were included if they were virologically controlled (HIV viral load <200 copies/mL) and demonstrated increased abdominal VAT (VAT area>110cm²) on CT. For purposes of this study, baseline fasting biochemical, radiologic and anthropometric data were used. VAI was calculated using the standard sex-specific formulas which incorporate waist circumference (WC), BMI, triglycerides and HDL. Presence of coronary plaque was assessed using coronary CT angiography or coronary PET scans. Linear regression was performed to assess relationships with VAI. Non-normally distributed variables were log-transformed for analyses.
Participants were predominantly male (73%), Caucasian (53%), and non-Hispanic (84%) with mean age 55 ± 7 years. Participants had a long duration of HIV and ART use (20 ± 8 and 15 [12,19] years, respectively). The majority of PLWH were obese (BMI 31.9 ± 5.8 kg/m²) with VAT 189 [127,267]cm² and VAI 4.9 [2.8,7.3]. VAI correlated strongly with VAT (r=0.59, P<0.0001), anthropometric measures (BMI r=0.36, P=0.02; WC r=0.43, P=0.004; WHR r=0.33, P=0.03) and ALT (r=0.32, P=0.03), and did not relate to HIV-specific parameters and other metabolic parameters (blood pressure, HbA1c). Participants with coronary plaque tended to have a higher VAI compared to those without coronary plaque (log VAI 0.7 ± 0.3 vs. 0.5 ± 0.3, P=0.056).
These data show VAI, an easily obtained measure, is strongly correlated with abdominal VAT area measured by CT and may be a useful biomarker for visceral adiposity in HIV. Furthermore, VAI may be related to ALT and coronary plaque, which could help identify those PLWH at risk for fatty liver disease (another ectopic fat depot) and heart disease, respectively. Further studies are needed to assess the utility of VAI in evaluating metabolic disease in HIV.

technical paper

Background: While wireless mobile device (WMD) overuse might be perceived as normal behavior in people of all ages, the pathophysiology of WMD addiction is similar to that of substance addiction. Increasing prevalence of disorders related to screentime/sedentary lifestyle such as obesity, dyslipidemia, prediabetes, and type 2 diabetes mellitus calls for comprehensive and cost-effective interventions.
Objectives: We aimed to synthesize existing data on the neuroscience that underlies WMD addiction, its adverse health outcomes and suggest practical strategies to achieve life-technology balance at home and work.
Methods: Comprehensive search of databases (PubMed and MEDLINE) was conducted until May 2021, for smartphone, screentime and phone addiction. The data were collected as part of a population-based educational initiative, Phreedom (Freedom from smartPhone), to inspire individuals to achieve life-technology balance.
Results: From the initial 655 citations, 234 were included. The addictive use of WMDs was linked to the 'incentive sensitization theory of addiction': repeated exposure to potentially addictive stimuli (i.e., notifications) can cause persistent structural and functional brain changes in the prefrontal cortex. As a result, an individual's executive functions and cue-reactivity could be impaired. The hyper-reactive dopamine system could also drive this compulsive behavior in the form of a habit-loop of cue, craving, response, and reward. Each hour/day increase in screentime was associated with 0.05-0.07 increase in body mass index (BMI) (p < 0.001), likely due to food marketing, distracted eating, reduced satiety, and procrastination of physical activity. Overuse of WMDs was associated with insufficient and low-quality sleep, daytime tiredness, daytime sleepiness, depression, and daily cognitive failures. Nine of 10 Americans reported using a technological device in the hour before bed. The more interactive the device, the higher the difficulty falling asleep and unrefreshing sleep (p < 0.05). The younger the age at initial exposure and the longer the duration of exposure, the higher the odds of adverse health outcomes, such as illegal substance use. High users of screens were more likely to have been diagnosed with anxiety or depression, requiring medication. Practical strategies to disrupt the addictive dopamine habit-loop included WMD triage and uncluttering, eliminating redundant notifications, deleting time-consuming applications, modifying the digital environment at home and work, and replacing screentime with greentime. Other tactics could include using an alarm clock, wearing a wristwatch, specifying times to check emails and social media, refocusing teams to decrease reliance on text messages and lowering expectations of immediate responses.
Conclusions: While it would be irrational to suggest complete avoidance of all WMDs, moderation, balance, and real-life engagement together with strategies aimed at disrupting the dopamine habit loop will be critical components of any intervention plan. Educating patients about the neuroscience of WMD addiction and empowering them with tips to curb WMD use might impact their behavior substantially.

Wireless Mobile Device Overuse and Obesity: Understanding the Neuroscience, Implications and Interventions

Background/Hypothesis:
Leptin is positively associated with adiposity at birth and may vary by sex. Whether cord blood leptin is associated with long term adiposity is unclear. We hypothesized that cord blood leptin is positively associated with adiposity measures in peripubertal children and that the association differs by sex.

Methods:
Data from mother-child pairs in an ethnically and racially diverse prospective birth cohort were used for analysis. Leptin was measured in 990 stored cord blood samples using Millipore ELISA assays. Adiposity measures from children (mean age 11.5 ± SD 1.1 years) included body mass index (BMI), sum of three skinfolds (SSF), waist circumference (WC), and fat mass and body fat percent by air-displacement plethysmography (ADP/BOD POD). Leptin levels were log transformed to improve normality. Linear and logistic regression were used to evaluate associations between cord blood leptin and childhood adiposity outcomes for continuous and categorical variables, respectively. Statistical models accounted for covariates: Model 1 [child age, sex, field center and maternal pregnancy variables (age, parity, smoking status, drinking status, gestational age at oral glucose tolerance test (OGTT), mean arterial pressure at OGTT, family history of diabetes)] and Model 2 [Model 1+ maternal mid-pregnancy BMI + maternal gestational diabetes (present/absent)]. In Model 1, statistical interaction terms were also included to evaluate whether associations between cord blood leptin and adiposity outcomes varied by sex. Statistical significance was determined by p<0.05 for all statistical tests.

Results:
Cord blood leptin was positively associated with all childhood adiposity outcomes. Associations did not vary by child sex. Associations were attenuated in Model 2, but remained statistically significant. For log-cord blood leptin higher by one SD, continuous childhood adiposity outcomes were higher as follows: BMI: Model 1, beta coefficient 0.57 kg/m2 (95% CI 0.29-0.86), Model 2, 0.31 kg/m2 (95% CI 0.03-0.59); fat mass: 1.18 kg (0.66-1.71), 0.76 kg (0.23-1.29); body fat percent: 1.69% (1.02-2.36), 1.23% (0.56-1.91); SSF: 2.98 mm (1.56-4.41), 1.99 mm (0.56-3.43); and WC: 1.52 cm (0.75-2.29), 0.78 cm (0.02-1.54). For log-cord blood leptin higher by one SD, associations with dichotomous childhood adiposity outcomes were as follows: overweight/obesity: Model 1, odds ratio (OR): 1.37 (95% 1.18-1.60), Model 2, OR 1.24 (95% CI 1.06-1.45); obesity: 1.52 (1.22-1.91), 1.34 (1.06-1.70); body fat percent >85th percentile: 1.49 (1.23-1.81), 1.34 (1.10-1.64); and WC >85th percentile: 1.38 (1.15-1.66), 1.22 (1.01-1.48). Leptin was associated with SSF >85th percentile in Model 1 1.32 (1.10-1.59) but not Model 2. Leptin was not associated with child fat-free mass.

Conclusion:
Cord blood leptin is positively associated with peripubertal offspring adiposity, independent of maternal BMI and gestational diabetes. The relationship between cord blood leptin and peri-pubertal adiposity did not differ by sex.

Cord Blood Leptin and Childhood Adiposity Outcomes in the Hyperglycemia and Adverse Pregnancy Outcome Follow-up Study

OBJECTIVES Cortisol is known to promote adipocyte differentiation and maturation, and prolonged exposure to excess cortisol contributes to the development of obesity and metabolic dysregulation. The intracellular 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) enzyme catalyzes the conversion of inactive cortisone to active cortisol. Recently, we demonstrated reduced brain 11β-HSD1 levels in vivo with increasing body mass index (BMI), using positron emission tomography (PET) radioligands [¹¹C]- and [¹⁸F]AS2471907(1). PET imaging can simultaneously quantify uptake in multiple organs (e.g., liver/brain). [¹⁸F]AS2471907 has high uptake and specific binding in the brain and liver(2). Liver and brain 11β-HSD1 measured by PET imaging may help elucidate the roles of cortisol activation in the setting of metabolic dysregulation. Thus, we performed PET imaging studies to examine 11β-HSD1 levels in the liver and brain.
METHODS Nine individuals (5F/4M) with a range of BMIs (22.6-34.4 kg/m²) underwent a 90 min PET/CT acquisition with arterial plasma sampling after injection of [¹⁸F]AS2471907. Regions-of-interest (ROI) for the liver were manually drawn on a summed PET image (60-90 min). Seventeen brain ROIs were selected from the anatomical automatic labeling (AAL) template and applied to the dynamic PET images to generate time-activity-curves (TACs). Brain volume of distribution (VT, mL/cm³), the target tissue to plasma ratio of radioligand at equilibrium, was estimated for each ROI using the multilinear analysis-1 method with plasma input function. Mean whole-brain VT values were calculated by averaging all ROIs. Given the appearance of irreversible kinetics in the liver, Ki (min-1), the rate of irreversible tracer uptake, was calculated using the Patlak method.
RESULTS Qualitative brain and liver uptake was assessed by examining a summed PET image (SUV 60‑90min). The parent fraction in plasma was 88±1% at 90 min, indicating negligible plasma radiolabeled-metabolites contributing the measured signal in the liver. Kinetic modeling estimates demonstrated decreasing whole brain VT with increasing BMI (R²=0.53), similar to our previously published study examining only brain in a larger cohort(1). Patlak methods provided good estimates of Ki. In an opposite manner from the brain, correlations seen in the liver using Ki were positively correlated with BMI (R²=0.57).
CONCLUSIONS These preliminary studies suggest obesity is associated with increased 11β-HSD1 levels in the liver but decreased 11β-HSD1 levels in the brain. Clinically, liver specific 11β-HSD1 inhibitors may prove beneficial in treating metabolic sequalae of obesity such non-alcoholic fatty liver disease (NAFLD) by inhibiting activity of higher 11β‑HSD1 levels. Further studies in NAFLD and obesity are necessary to determine appropriate populations that would benefit from 11β-HSD1 inhibitors and longitudinal in vivo PET imaging to help inform development of future therapeutics.
REFERENCES:
[1] Bini, et al, Molecular Imaging and Biology, 2020.
[2] Bini, et al, IEEE Trans on Radiation and Plasma Medical Sciences, 2021

Quantifying Liver and Brain Levels of 11β-hydroxysteroid Dehydrogenase Type 1 in Obesity Using Positron Emission Tomography Imaging

Introduction:
Pituitary abscesses are rare, representing less than 1% of pituitary lesions. Patients may present with pituitary hypofunction, headache, and/or vision changes, often without systemic or biochemical signs of infection, with diagnosis only made intraoperatively.
Cases:
A 74-year-old man with history of diabetes and coronary artery disease presented with headache and left-eye ptosis. MRI revealed a 1.8 cm hypoenhancing cystic, hemorrhagic sellar mass extending into the left cavernous sinus. Pre-operative labs showed AM cortisol 1.1 ug/dL, testosterone 43 ng/dL (reference range 250-1100), FSH 15.3 mIU/mL, LH 4.5 mIU/mL consistent with adrenal insufficiency and hypogonadism. He underwent transsphenoidal pituitary resection (TSR) during which frank pus was demonstrated after incision into the dura overlying the pituitary. Surgical pathology showed necrosis and granulation-type reaction with acute inflammatory cell infiltrates. Gram stain and methenamine silver stains did not reveal definite micro-organisms while tissue bacterial cultures showed normal skin flora. He remained hemodynamically stable without fevers throughout the hospitalization and was discharged on empiric antibiotics and hydrocortisone. 
A 74-year-old female with history of a pituitary macroadenoma was admitted for worsening headaches. Labs were consistent with her known history of secondary adrenal insufficiency and hypogonadotropic hypogonadism. Repeat MRI showed characteristics suggestive of hemorrhage within a 1.8 cm intrasellar mass with inhomogeneous and peripheral enhancement after contrast. She underwent TSR and intraoperatively frank pus was encountered. Surgical pathology showed sinonasal mucosa with chronic inflammation. There was no growth in aerobic, fungal, or AFB cultures; anaerobic cultures grew rare commensal bacteria. She was discharged with a course of antibiotics and remains on hydrocortisone. 
A 42-year-old male presented with headache and was found to have a 2.5 cm sellar mass with rim enhancement elevating and compressing the optic chiasm on MRI. Labs were significant for AM cortisol 4.9 ug/dL and testosterone 8 ng/dL, FSH 0.4 mIU/mL, LH <0.2 mIU/mL consistent with adrenal insufficiency and hypogonadism. He underwent TSR and was found to have purulent debris in the sella. Pathology showed epithelial tissue fragments insufficient for diagnosis, and cultures grew few skin and oropharyngeal contaminants. He was discharged on hydrocortisone with a course of antibiotics. 
Conclusion:
We describe three patient cases presenting with headache and varying degrees of hypopituitarism, found to have purulent material emanating from the sella during transsphenoidal resection. None presented with clinical or biochemical systemic signs of infection. MRI imaging was variable, ranging from cystic, hemorrhagic features to peripheral enhancement. Surgical pathology and cultures were unrevealing for a definitive infectious source or other cystic lesion such as Rathke's cleft cyst. This case series demonstrates the potential nonspecific presentation of pituitary abscesses including negative cultures and pathology. It is important to keep this rare diagnosis in the differential for patients presenting with pituitary disease.

Culture Negative Pituitary Abscesses: A Case Series

A male patient presented with a unique complex phenotype including panhypopituitarism, short stature with failure to respond to GH [IGF-1 generation test: no response to 2.8 mg/m²/day GH], sensorineural deafness, hypoparathyroidism, retinal dystrophy, and intellectual disability. He was 127cm tall at the age of 21 and was completely prepubertal (LH 0.8 U/L, FSH 2.6 U/L, Testosterone <0.69 nmol/L) with a bone age of 12 years. A trial of recombinant IGF-1 failed to improve growth. He died at the age of 26 years following a collapse of unknown etiology.
Exome sequencing and subsequent variant calling on this patient revealed a novel heterozygous de novo missense variant (c.6395C>T, p.A2132V) at a highly conserved residue in FASN encoding Fatty Acid Synthase. The variant was not present in any control database, including the GnomAD browser. Human embryonic brain expression analysis using in situ hybridisation, revealed FASN mRNA transcript staining in the diencephalon and hypothalamus at Carnegie stages (CS) 16, 19, 20 and 23, and in Rathke's pouch (primordium of the anterior pituitary) at CS16 only.
Fatty acid synthase (FASN), a crucial multienzyme with seven different catalytic activities, converts acetyl-CoA and malonyl-CoA into long-chain saturated fatty acids such as palmitate, in the presence of NADPH. Murine null mutant Fasn-/- embryos die before implantation and Fasn+/- embryos at various stages in the early postnatal period. Fasn is expressed in multiple tissues including the brain, parathyroids, liver and adrenal.
Biochemical investigation of the patient revealed high triglyceride concentrations (3.81-6.49 mmol/L; NR 0.38-2) at all time points during an 18 hour fast. He mobilised free fatty acids but did not generate an increase in 3-hydroxybutyrate, suggesting a defect in fatty acid oxidation or ketone body synthesis. He maintained normoglycaemia with normal hormone responses to fasting, except for an undetectable IGF-1 (<25 ng/ml) and IGFBP-3 (1.17 mg/L).
We measured de novo fatty acid synthesis in cultured patient and control fibroblast cells with 13C-Glucose, with and without the C75 fatty acid synthase inhibitor. Cell pellets were collected at three time-points, and 13C-fatty acids derived from 13C-glucose were analysed by mass spectrometry. Preliminary data showed that more C13-glucose was incorporated into de novo synthesised palmitic acid in control cells, suggesting reduced fatty acid synthesis in the patient.
Our data suggest that FASN p.A2132V is pathogenic and contributed significantly to the complex patient phenotype, consistent with recent studies suggesting that neural stem/progenitor cells (NSPCs) in the rodent brain are governed by Fasn-dependent de novo lipogenesis for proliferation.

A novel missense variant in the gene encoding Fatty Acid Synthase (FASN) associated with a unique multi-system disorder including hypopituitarism and hypoparathyroidism.

Background/objective: Insulin-like-growth-factor-I (IGF-I) levels in the lower or upper normal range have been proposed as a biomarker of risk for later disease in healthy adults, specifically cardiovascular disease and cancer. In addition, concern has been raised about the frequently observed supraphysiological IGF-I levels in non-growth-hormone-deficient children treated with growth hormone (GH). However, whether a single IGF-I measurement is a valid indicator of cumulative lifetime exposure to IGF-I and thus disease risk is not established. We aimed to evaluate intra-individual longitudinal tracking of IGF-I and IGF-binding-protein-3 (IGFBP-3) levels and to estimate cumulative lifetime exposure to IGF-I in healthy and GH-treated individuals. We hypothesized that individuals follow a certain IGF-I trajectory throughout life and that GH therapy in childhood does not increase lifetime IGF-I exposure substantially.
Methods: We performed a combined cross-sectional (n=5,326) and longitudinal (n=1,133) study of 6,459 healthy participants (49% male) aged 0­­­–76 years recruited as part of six Danish population-based studies, resulting in a total of 9,963 serum samples. In addition, we included 238 samples from nine short children born small-for-gestational-age (SGA) before, during and after GH treatment.
Serum samples were analyzed for IGF-I and IGFBP-3 with the IDS-iSYS immunoassay and anthropometric measures were obtained. Intra-individual tracking was determined by intraclass correlation coefficients (ICC) derived from a linear mixed model with IGF-I (SDS) or IGFBP-3 (SDS) as dependent variable and subject as random effect, unadjusted and adjusted for BMI-changes. Cumulative lifetime exposure to IGF-I was estimated by calculating area under the curve of the predicted SD trajectory from 0-76 years.
Results: Sex- and age-specific reference curves for IGF-I and IGFBP-3 were established. For IGF-I, ICCs were 0.50 (95% CI: 0.47–0.53) and 0.53 (0.50–0.56) for male and female participants, respectively. ICCs for IGFBP-3 were 0.52 (0.49–0.55) for male participants and 0.59 (0.56–0.62) for female. Cumulative lifetime IGF-I exposure was significantly higher in female (mean ± SD, 12,723 ± 3,691) than in male participants (12,563 ± 3,393); p=0.02. The SGA patients had a mean (range) GH-treatment duration of 9.2 years (5.2–11.9). Treatment caused an increase in estimated cumulative lifetime IGF-I exposure of 1,759 ± 556 shifting them from a mean estimated lifetime exposure without treatment of 9,512 ± 1,889 to 11,271 ± 1,689 with treatment. This corresponded to a rise in IGF-I trajectory (SDS) of 0.55 SD ± 0.18, from -0.89 SD ± 0.57 to -0.35 SD ± 0.49.
Conclusion: Our results suggest that IGF-I and IGFBP-3 levels are tracking throughout life and that a single measurement reliably reflects lifetime exposure. We, for the first time, estimated lifetime exposure to IGF-I in healthy individuals and show that pediatric GH therapy only slightly increases lifetime exposure and not beyond levels commonly found in the reference population.

Tracking and Cumulative Lifetime Exposure to Circulating IGF-I in 6,459 Healthy Individuals and in SGA Children Treated with GH

Splicing variants in the exon-intron boundaries of the GH1 gene have been reported to cause autosomal dominant growth hormone deficiency (GHD type 2). However, whether variants in the intron consensus sequence (XGGG repeats) have an important role in GH1 gene splicing has not been established. Two siblings (proband 1 and 2) were diagnosed with growth hormone deficiency due to poor linear growth at age 4 years (height SDS -2.2 and – 1.6, respectively). Peak serum GH levels with provocative testing were 4.39 ng/ml and 2.77 ng/ml, respectively. They had no other pituitary hormone deficiencies and their pituitary MRIs were normal. Both children are being treated with recombinant human GH (rhGH) with excellent responses in linear growth. Their mother also had a history of GHD in childhood and received rhGH from ages 4 to 13 years. After GH treatment, she reached a normal adult height. The remainder of her pituitary function was normal. Exome sequencing was performed on the probands, their affected mother, and unaffected maternal grandparents to identify the underlying genetic cause. Exome sequencing revealed that the affected mother carried a de novo variant in intron 3 of the GH1 gene (c.291+34G>A, NM_000515.5), and the intronic variant was passed on to the two probands. The variant is not found in the general population according to gnomAD. No other variants in GH1 or other genes were detected to cosegregate with GHD in this family. The intronic variant was located in one of the two intron consensus regions in intron 3 and was predicted by ESEfinder to create a new splicing enhancer (SF2/ASF) binding site. Because intron consensus sequence repeats are important for spliceosome assembly, the variant is predicted to cause aberrant pre-mRNA splicing but not affecting the donor or acceptor site. Previously, a variant in the intron consensus sequence, c.291+28G>A, was found to cause exon 3 skipping, presumably generating a small amount of 22 kDa GH but a large amount of 17 kDa GH. Small deletions in intron 3 (c.291+28_45del and c.291+56_77del) have also been reported to cause exon 3 skipping, categorized as GHD type 2. In vitro studies with the new variant will be required to confirm exon 3 skipping as the mechanism of GHD in this family. This novel intronic variant which creates a new splicing enhancer-binding site supports the previous finding that the intron consensus sequence plays a significant role in the splicing of GH1 pre-mRNA and results in autosomal dominant GHD. As other variants in intron consensus sequence sites may cause GHD type 2, we recommend genetic testing in patients with GHD include the intron consensus sequence regions.

A novel variant in an intron consensus sequence associated with familial growth hormone deficiency

Mammalian genomes contain thousands of genes for long non-coding RNA (lncRNAs) that affect the expression of other genes through diverse mechanisms. The lncRNA transcripts are longer than 200 nucleotides and are often capped, spliced and polyadenylated, but they are not translated into proteins. Nuclear lncRNAs modify chromatin structure and transcription in trans or cis by interacting with the DNA, forming R-loops, and recruiting regulatory proteins. Not much is known about the role of lncRNA in pituitary gland differentiation and function, but there are several examples that provide a strong premise for investigating this class of transcripts more deeply. For example, the lncRNA MEG3 is a tumor suppressor that is silenced in non-functioning pituitary adenomas; and lncRNAs have been implicated in regulating the function of key transcription factors such as POU1F1, PITX1, and PITX2. To develop a research resource for the study of lncRNA, we used cell-type specific cre transgenes to tag individual adult pituitary cell types with fluorescent markers and enriched for thyrotropes, gonadotropes and somatotropes using fluorescence activated cell sorting. We determined the transcriptome of each cell population using RNA sequencing and mined the data for lncRNA. We detected hundreds of lncRNA in adult pituitary cells, and some were cell-type specific, and located in immediate proximity of genes that encode pituitary hormones or lineage-specific transcription factors. We discovered that the lncRNA Nr5a1os is enriched in gonadotropes, while Foxl2os is enriched in gonadotropes and thyrotropes. The lncRNA Platr9 is located on the opposite strand near Cga, which encodes the alpha subunit of thyrotropin and gonadotropin. We also mined transcriptome data from pituitary glands collected at embryonic days 12.5 and 14.5 and identified 351 unique lncRNAs, many of which are exclusively expressed during development. Among these is Six3os1, which is located upstream of Six3, a gene associated with holoprosencephaly and hypopituitarism. The location of many of these lncRNAs suggests that they may have a cis-acting regulatory role in pituitary development or physiological function. This research resource sets the foundation for examining the actions of lncRNAs on expression of nearby genes during development and in response to physiological demand. Future research will elucidate their mechanisms of action, determining whether they recruit regulatory proteins to the chromatin and/or form R-loops in these regions, and how this affects expression of the target gene(s). We expect these studies to further our understanding of pituitary biology.

Long Non-coding RNAs in Pituitary Differentiation and Endocrine Function

Introduction: Hormone-producing pituitary cell lineages are derived from a population of embryonic precursors expressing SOX2. These cells maintain multipotency into early postnatal life, acting as the resident population of pituitary stem cells (PSCs) and contributing extensively to all the endocrine cell lineages. In addition to this direct contribution to pituitary turnover, paracrine signalling from PSCs has been shown to be important for cell proliferation of neighbouring progenitors (PMC7803373). It is not known if SOX2+ PSCs are involved in the regulation of additional cell attributes during normal physiology and if there is functional heterogeneity among the SOX2+ PSC population.
Experimental Methods: We have carried out single-cell RNA-Sequencing of SOX2+ PSCs from Sox2Egfp/+ mouse pituitaries at three postnatal stages from P3 to P56 and used computational approaches to analyse their molecular signatures. A novel conditional mouse model expressing a constitutively active mutant form of the RNA binding factor BRF1 (R26stop-mBRF1) has been used to attenuate the expression of several cytokines and chemokines in SOX2+ cells embryonically and postnatally (PMC4589897).
Results: We show that the SOX2+ PSC population consists of three subgroups (SC1, SC2 and SC3). We reveal that SC1-SC2 express abundant cytokines and secreted factors, suggesting paracrine function. In contrast, SC3 is characterised by robust expression of Lef1, is identified as a committing PSC cluster, and its presence diminishes with age. Key markers of PSC clusters SC1-SC2 include the RNA binding factor BRF1. We show that BRF1 is highly expressed in PSCs and validate its expression by immunohistochemistry in both mouse and human pituitaries. Secondly, we show that the dysregulation of BRF1 in embryonic SOX2+ cells using the Hesx1-Cre driver (PMC3461924) results in pituitary hypoplasia and severe hypopituitarism due to a failure of the PIT1 and SF1 cell-lineage committed progenitors to terminally differentiate into hormone-producing cells. Additionally, there is a significant reduction of the stem cell compartment, manifested by lower numbers of SOX2/SOX9+ stem cells. This phenotype is recapitulated when using a Sox2-CreERT2 driver (PMID24094324). The differentiation failure can be rescued in vitro through co-culture of mutant cells with wild-type stem cells, as well as in vivo, in mutant pituitaries where activation of constitutively active BRF1 is restricted to few SOX2+ PSCs in a mosaic manner. Finally, we identify key ligands underlying this differentiation phenotype and demonstrate a partial restoration of terminal differentiation in the mutant, when cultured in the presence of these ligands.
Conclusion: We provide evidence indicating the presence of functionally distinct groups of SOX2+ pituitary stem cells and reveal a critical role for a PSC subset in the development of the stem cell compartment and in driving terminal differentiation of committed progenitors.

BRF1-Mediated Paracrine Signalling by a Subset of SOX2-Expressing Stem Cells is Required for Normal Development of the Stem Cell Compartment and Terminal Differentiation of Pituitary Committed Progenitors

Context: Surgical removal is the primary treatment option for pituitary adenomas. However, pituitary surgery is frequently incomplete because of invasion of extrasellar cerebral structures. In particular, the vicinity with the carotid artery hinders the radical surgical excision of the adenoma portion invading the cavernous sinus. Our objective was to study the molecular bases of the cavernous sinus invasion by pituitary adenomas.
Methods: We obtained a unique tissue collection of 19 invasive pituitary adenomas with a sample from the intrasellar portion and a sample from the portion invading the cavernous sinus for each adenoma. We used RNA-sequencing to compare the gene expression patterns of the invading and intrasellar portions. The implication of one differentially expressed candidate gene in the invasive behavior was first analyzed in vitro using Transwell Assay to study the impact of its pharmacologic inhibition on cell migration and invasion. These experiments were conducted in murine lactosomatotroph GH3 cells and gonadotroph LbT2 cell. To further study the role of this gene on tumor growth and behavior in vivo, we elaborated a model of invasive pituitary adenomas by stereotactic injection of murine somatotroph GC cell into the lobes of the pituitary gland of female Wistar Furth rats. Twelve rats (10-12 week-old) received 20.000 GC cells in each pituitary lobe. After surgery, rats were monitored weekly, tumor development was assessed fortnightly by 3 successive 7Tesla MRI and analyzed by Horos software. Rats were sacrificed 7 weeks after GC cells injection. Six of the 12 rats were treated during this period with the pharmacological inhibitor of the candidate gene.
Results: RNA-sequencing identified 159 up-regulated genes and 11 down-regulated genes in the invasive adenoma portion. In vitro pharmacological inhibition of the candidate gene decreased cell migration and invasion in GH3 cells (p=0.0205 and p=0.0038) and LbT2 cell (p=0.0345 and p=0.0131). Amongst the 12 injected animals, 11 (92%) rats developed invasive pituitary tumors. Tumor growth was rapid, causing death from intracranial hypertensions before the theoretical end of the study protocol in 7 animals. Pharmacological inhibition tended to slow tumor growth from 30.3 mm³/week to 7.8 mm³/week (p=0.12) and decreased cumulative mortality (83% in untreated animals versus 33% in treated animals, p=0.08).
Conclusion: We described the molecular signature associated with the invasive behavior of pituitary adenomas and identified a therapeutic target, which is related to pituitary cells migration and invasion in functional in vitro studies. Pharmacologic inhibition of this target tended to decrease tumor growth and mortality from intracranial hypertension in vivo, however larger numbers of animals are necessary to confirm this observation. Our original approach of orthotopic cell injection into rat pituitaries resulting in tumor development provides a new tool for molecular studies of pituitary tumorigenesis and for the screening of new therapeutic agents.

Molecular determinants of cavernous sinus invasion by pituitary adenomas: functional in vitro studies and in vivo investigations in a rat model of invasive pituitary tumorigenesis obtained by stereotactic pituitary GC cells injection

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