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De novo lipogenesis (DNL) plays a role in the development of hepatic steatosis and non-alcoholic fatty liver disease (NAFLD). In rodent models of both health and lipodystrophy (LD), leptin decreases DNL. In human patients with LD, reduced adipose tissue results in adipokine deficiencies, including lower plasma leptin, which contributes to insulin resistance, dyslipidemia and ectopic accumulation of triglycerides (TG). The mechanisms by which leptin regulates serum and hepatic-TG are not well elucidated. Studying patients with LD before and after leptin therapy provides an important clinical model for understanding leptin’s effect on DNL. We hypothesized that leptin treatment in lipodystrophic patients would decrease DNL by decreasing insulin resistance and glycemia, resulting in reduced circulating and hepatic-TG.Leptin-naïve patients with LD (n=11) were treated with recombinant leptin (metreleptin) for 6 months. All measurements were performed after an 8-12 hr fast. The % of TG in TG-rich lipoproteins (TRLP-TG) derived from DNL (% DNL) was measured using body water labeling (oral D2O) of TG and mass spectrometry analysis. Absolute DNL was calculated as the product of TRLP-TG and % DNL. HbA1c and serum-TG were measured biochemically, hepatic-TG by MRI, and total body and hepatic insulin sensitivity measured during a hyperinsulinemic-euglycemic clamp.DNL decreased after metreleptin: % DNL from 22.8±6.8 to 9.1±5.1% (p=0.0008) and absolute DNL from 54.2±32.1 to 8.6±6.5 mg/dl (p=0.003). TRLP-TG decreased from (median [interquartile range]) 160 [107, 280] to 98 [66, 147] mg/dl (p=0.01). Total body and hepatic insulin sensitivity increased from 3.7 [3.0, 7.3] to 8.4 [5.1,10.6] mg/kgFFM/min (p=0.03) and from 61.0 [48.5, 69.3] to 84.7 [75.2, 107.6] % (p =0.01), respectively. HbA1c decreased from 8.6±1.8 to 7.1±1.4% (p=0.04), hepatic-TG decreased from 17.6±11.9 to 10.3±9.1% (p=0.02), and serum-TG from 386 [216, 686] to 223 [118, 497] mg/dl (p=0.06). DNL correlated negatively with insulin sensitivity both before (r=-0.73, p=0.03) and after (r=-0.85, p=0.004) metreleptin. DNL correlated positively with hepatic-TG before (r=0.70 p=0.03) and tended to correlate after metreleptin (r=0.65, p=0.06). The change in DNL correlated with change in serum-TG (r=0.77, p=0.04) but not the change in hepatic-TG (p=0.80).We show here for the first time that 6 months of metreleptin treatment in humans with LD decreased DNL by 84% and was associated with reductions in glycemia and improved peripheral and hepatic insulin sensitivity. These data indicate a strong link between metreleptin’s effects to increase clearance of blood glucose by peripheral tissues and reduce hepatic carbohydrate flux, resulting in DNL reductions. This led to lowered hepatic steatosis and dyslipidemia and suggests treatments that target multi-organ insulin resistance may lead to decreased NAFLD and cardiovascular risk.

ENDO 2020

Leptin Decreases De Novo Lipogenesis in Lipodystrophic Patients

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ENDO 2021

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technical paper

We implemented single-cell RNA sequencing (scRNAseq) technology as a discovery tool to identify factors enriched in differentiated thyrotropes. Thyroid-stimulating hormone (TSH) is produced in the pars distalis of the anterior pituitary (AP) and primarily acts on the thyroid gland to regulate metabolism through T3/T4. However, TSH is also produced by cells in the pars tuberalis (PT), which is comprised of a thin layer of cells that extends rostrally from the pars distalis along the pituitary stalk to the median eminence in the hypothalamus. TSH produced by PT thyrotropes acts on hypothalamic tanycytes to regulate seasonal reproduction. PT thyrotropes likely descend from rostral tip thyrotropes that arise at e12.5 of mouse development, which transcribe the TSH beta subunit (Tshb) without detectable expression of the transcription factor POU1F1. POU1F1 is required for Tshb transcription in thyrotropes of the adenohypophysis, and it acts synergistically with GATA2 to drive cell fate. The molecular mechanisms driving Tshb expression independently of Pou1f1 in PT thyrotropes are unclear.Thyrotropes are the least abundant endocrine cell-type in the pituitary gland. We used genetic labeling and fluorescence-activated cell sorting (FACS) to enrich for thyrotropes for single-cell sequencing. We performed scRNAseq on 7-day-old GFP-positive pituitary cells from Tshb-Cre; R26-LSL-eYFP and intact whole pituitaries, recovering more than 15,000 cells altogether. We observe two distinct populations of cells expressing Tshb. The larger thyrotrope population has approximately twenty fold higher levels of Tshb and five fold higher Cga transcripts than the smaller population, and they are also distinguished by expression of Pou1f1, TSH-releasing hormone receptor (Trhr), and deiodinase 2 (Dio2), consistent with expectations for AP thyrotropes. The smaller thyrotrope population does not express Pou1f1, but those cells are characterized by expression of TSH receptor (Tshr) and melatonin receptor 1A (Mtnr1a), consistent with expectations for PT thyrotropes. They express mildly increased levels of Eya3 and Six1, although these genes are expressed in other cell-types including AP thyrotropes, stem cells, and gonadotropes. They have two-fold higher levels of Gata2 transcripts and uniquely express the transcription factor Sox14. SOX14 is a SoxB2 family transcription factor that counteracts the transcriptional activity of SoxB1 family members, such as Sox2. In conclusion, our scRNAseq has identified novel markers of PT thyrotropes and unveils novel insights into the similarities and differences in the development and function of pituitary thyrotrope subpopulations.

Single-Cell Sequencing Identifies Novel Regulators of Thyrotrope Populations and POU1F1-Independent Thyroid-Stimulating Hormone Expression

Glucocorticoid Production in the Nervous and Immune Systems: Evidence for a Local HPA Axis HomologThe hypothalamic-pituitary-adrenal (HPA) axis is a critical stress response system in vertebrates. The hypothalamus secretes corticotropin-releasing hormone (CRH), which binds its receptor (CRH-R1) in the anterior pituitary. The anterior pituitary then secretes adrenocorticotropic hormone (ACTH), which binds its receptor (MC2R) in the adrenal glands and stimulates secretion of glucocorticoids into the bloodstream. Glucocorticoids are critical modulators of neural and immune system development. During early development (postnatal day (PND) 2 to 12), mice show decreased adrenal glucocorticoid secretion at baseline and in response to stressors, termed the stress hyporesponsive period (SHRP) (1). Traditionally, glucocorticoids have been thought to be synthesized only in the adrenal glands. However, recent evidence demonstrates that glucocorticoids are also produced in extra-adrenal tissues, such as the brain and lymphoid organs (2). This may be of particular importance during the SHRP, as local production allows glucocorticoid modulation of specific tissues and cells, without general effects throughout the organism. Importantly, the factors that regulate local glucocorticoid production remain unknown. To study the regulation of local glucocorticoid production, we examined whether mediators of the HPA axis are locally expressed at baseline and in response to an immune stressor. We assessed systemic and local glucocorticoid levels in neonatal (PND5) C57BL/6J mice 4hr after an immune challenge with lipopolysaccharide (50µg/kg i.p.) or vehicle control. We examined blood, microdissected brain regions (prefrontal cortex, hippocampus, hypothalamus), and lymphoid organs (thymus, spleen, bone marrow). A panel of 7 steroids was measured via liquid chromatography tandem mass spectrometry (LC-MS/MS). Gene expression of Crh, Crh-R1, Pomc, and Mc2r was quantified via qPCR. Preliminary data indicate that corticosterone was 2-fold higher in tissues than in blood after an immune stressor. The thymus expressed all genes of interest, supporting the existence of a local HPA axis “homolog” in the thymus. Brain, spleen and bone marrow expressed a subset of the genes of interest. These exciting data demonstrate that all the mediators of the HPA axis are locally expressed within the thymus, likely to regulate thymocyte development and reactivity. Greater understanding of local glucocorticoid production will provide crucial insight into neural and immune development and function. Reference: (1) Sapolsky et al., Brain Res Rev. 1986 11(1):65-76. (2) Taves et al., Endocrinology. 2015 156(2):511-522.

Glucocorticoid Production in the Nervous and Immune Systems: Evidence for a Local HPA Axis Homolog

IntroductionCritical illness is hallmarked by high plasma cortisol without elevated ACTH. This is partly explained by low cortisol-binding proteins and reduced binding affinity and by suppressed cortisol metabolism, 3 peripheral drivers of increased free cortisol (corticosterone (CORT) in rodents).1 We hypothesized that lack of elevated plasma ACTH is explained by CORT-induced feed-back inhibition either due to suppressed CRH or AVP pituitary signaling or to direct glucocorticoid receptor (GR)-mediated action within corticotrophs. To test this hypothesis, we documented alterations in ACTH precursors and key enzymes controlling ACTH maturation and secretion in a mouse model of sepsis-induced critical illness.MethodsC57Bl/6 mice were randomly allocated to a healthy control group or to 4 critically ill groups sacrificed after increasing illness duration (30 hours (H), 3 days (D), 5D or 7D). Critical illness was induced by sepsis brought about by cecal-ligation and puncture followed by fluid-resuscitation and antibiotics treatment. The study was continued until 15 surviving animals per time cohort were reached (n=120).We quantified pituitary pro-opiomelanocortin (POMC) gene/protein expression and POMC plasma concentrations, pituitary POMC intracellular trafficking and cleavage via intracellular POMC sorting/trafficking receptor Carboxypeptidase E (CPE) and prohormone convertase 1 (PC1/3) gene/protein expression. Gene expression of Annexin A1, an inhibitor of mature ACTH secretion, was quantified as marker of GR-mediated CORT-induced feedback inhibition at corticotroph level.ResultsPlasma CORT concentrations were median 3-fold increased during critical illness (p<0.001 for all time cohorts) in the face of normal (for 30H, 3D and 5D cohorts) to low (7D time cohort; p=0.01) plasma ACTH concentrations. Plasma POMC concentrations were higher in critically ill than in control mice (p=0.05). POMC gene expression (but not protein, P=0.8) was a median 55% higher in critically ill mice than in controls (p<0.05 for all time cohorts). In contrast, pituitary mature ACTH protein concentration was median 61% lower in critically ill than in control mice (p<0.01). CPE gene expression was only increased in 30H time cohort (p<0.001). PC1/3 gene and protein expression were positively correlated (R2 0.1; p=0.001) and were reduced (by 37% and 43%, respectively) during the entire course of critical illness (p<0.01). Annexin A1 gene expression was increased during critical illness (p<0.05 for all time cohorts).ConclusionSuppressed CRH or AVP signaling and GR-mediated action within corticotrophs explained lack of elevated plasma ACTH in critical illness, as indicated by impaired POMC processing and ACTH maturation. However, increased POMC gene expression suggests ongoing corticotroph activation, the driver of which needs to be identified.1. Teblick A et al. Nat Rev Endocrinol 2019

Sepsis-Induced Critical Illness in Mice Alters Key Regulators of ACTH Production and Secretion Within the Anterior Pituitary Gland

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Background: Patients with congenital adrenal hyperplasia (CAH) due to classic 21-hydroxylase deficiency have poor health outcomes related to inadequate glucocorticoid (GC) replacement. We compared disease control of adults with classic CAH treated with a modified release hydrocortisone (MRHC), which replicates physiological diurnal cortisol secretion, versus standard GC therapy.Methods: 6 month, open label, study in 122 patients randomised either to treatment with MRHC (Chronocort®, Diurnal Ltd, Cardiff, UK) twice daily at ~ 0700h & ~2300h, or to remain on their standard GC regimen (hydrocortisone, prednisolone, prednisone, dexamethasone). Patients had 24-hr profiling of serum 17-hydroxyprogesterone (17-OHP) at baseline and for dose titration at 4 and 12 weeks. The primary efficacy endpoint was the change from baseline to 24 weeks in the natural logarithm of the mean of the 24-hr standard deviation score (SDS) profile for 17-OHP.Results: Both groups achieved improved hormonal control at 24 weeks. The mean 24-hour 17-OHP SDS was significantly lower on MRHC compared to standard GC at 4 weeks (p = 0.0074) and 12 weeks (p = 0.019), but not at 24 weeks. In post-hoc analyses at 24 weeks, MRHC treatment showed a greater reduction in 17-OHP SDS compared to standard GC in the morning, 0700-1500h (p = 0.0442) and a greater reduction in log transformed 17-OHP 24 hour AUC (p=0.0251). Defining a morning 17-OHP <1200ng/dl (<36 nmol/L) as good control, for patients not controlled at baseline 85% were well controlled at 24 weeks with MHRC versus 50% on standard GC. For patients controlled at baseline 100% were controlled at 24 weeks on MHRC versus 84% with standard GC (p = 0.0018). The variability of 17-OHP over 24 hours was significantly reduced in the MRHC group compared to standard GC: the ratio of amplitude at 24 weeks divided by amplitude at baseline was for MRHC, 0.361 [95% CI: 0.235, 0.651], and standard GC, 0.917 [0.773, 1.366]; (p = 0.0001).There were no adrenal crises on MRHC and fewer stress doses despite similar incidence of inter-current illness to the standard GC group which had 3 adrenal crises. MRHC was associated with patient reported benefit including restoration of menstruation in 4 patients on MRHC and 1 on standard GC and two partner pregnancies in patients on MRHC and none on standard GC.Discussion: This is the largest randomised controlled trial of GC treatment in CAH and showed that intensification of therapy could improve control of the androgen-precursor, 17-OHP, and that this hormonal control was superior in the morning with MRHC. MRHC reduced the fluctuations in 17-OHP such that in the majority of patients the 17-OHP profile was within the reference range throughout 24 hours, providing consistent and optimal disease control.Conclusion: Diurnal cortisol replacement with a MRHC improves the biochemical control of classic CAH with a twice-daily therapeutic regimen.

A Phase 3 Study of a Modified-Release Hydrocortisone in the Treatment of Congenital Adrenal Hyperplasia

Introduction: Classic congenital adrenal hyperplasia due to 21-hydroxylase deficiency (21OHD CAH) is a rare autosomal recessive disease that results in impaired cortisol synthesis and excess androgen production. Compounds that inhibit adrenocorticotropic hormone (ACTH) release could reduce adrenal androgen production and thus the amounts of exogenous glucocorticoids needed to decrease these androgen levels. This study evaluated the effect of crinecerfont (NBI-74788), a novel, non-steroidal, and selective corticotropin-releasing factor-1 (CRF1) receptor antagonist on adrenal androgens and precursors in adults with 21OHD CAH.Methods: This open-label, multiple-dose study enrolled men and women (18-50 years old) with 21OHD CAH. A sequential-cohort design evaluated 4 crinecerfont oral dosing regimens: 50 mg QHS (Cohort 1); 100 mg QHS (Cohort 2); 100 mg QD (Cohort 3); and 100 mg alternative dosing (Cohort 4). Each regimen was administered for 14 consecutive days. ACTH, 17-hydroxy-progesterone (17OHP), and androstenedione (A4) were measured serially over a 24-hour period, at baseline and after 14 days of dosing.Results: Analyses included 23 participants: Cohort 1 (4 women, 4 men: mean age 31.1 years); Cohort 2 (5 women, 2 men: mean age 32.9 years); and Cohort 3 (3 women, 5 men: mean age 30.9 years). Cohort 4 is ongoing. At baseline, median plasma ACTH, serum 17OHP, and serum A4 levels were as follows: Cohort 1 (ACTH, 151 pg/mL; 17OHP, 5352 ng/dL; A4, 270 ng/dL); Cohort 2 (ACTH, 232 pg/mL; 17OHP, 12821 ng/dL; A4, 597 ng/dL); and Cohort 3 (ACTH, 470 pg/mL; 17OHP, 6451 ng/dL; A4, 299 ng/dL). After 14 days of once-daily crinecerfont 50 mg, Cohort 1 patients had median percent reductions from baseline in plasma ACTH (-54%), serum 17OHP (-60%), and serum A4 (-21%). Median percent reductions were generally larger with 100 mg in Cohort 2 (ACTH, -67%; 17OHP, -75%; A4, -47%) and Cohort 3 (ACTH, -69%; 17OHP, -55%; A4, -43%), consistent with a dose-related response. Adverse events were mostly mild; no clinically significant findings from routine laboratory tests, vital signs, or electrocardiograms were noted.Conclusions: Results of this Phase 2 study of crinecerfont, a novel, orally administered, selective CRF1-receptor antagonist, indicated clinically meaningful reductions of elevated ACTH, 17OHP, and A4 in adults with 21OHD CAH after 14 days of treatment. Further studies are warranted to evaluate the effects of chronic crinecerfont therapy on maintenance of adrenal steroid production, clinical endpoints of disordered steroidogenesis, and reductions in GC exposure in both adult and pediatric patients with 21OHD CAH.

The Effects of Crinecerfont (NBI-74788), a Novel CRF1 Receptor Antagonist, on Adrenal Androgens and Precursors in Patients with Classic Congenital Adrenal Hyperplasia: Results from A Multiple-Dose Phase 2 Study

Background: The management of youth presenting with delayed puberty is challenging because it can be difficult to predict which children will eventually progress through puberty and which children will not. We have previously shown that exogenous administration of the neuropeptide kisspeptin, which stimulates GnRH release, can be used to probe the integrity of the reproductive endocrine axis. We hypothesized that responses to kisspeptin could predict outcomes for individuals with pubertal delay. Methods: We conducted a longitudinal study of 16 children (3 girls and 13 boys) with delayed or stalled puberty who had undergone stimulation testing with kisspeptin and GnRH. Participants were followed with serial physical examinations and laboratory studies every six months for evidence of progression through puberty. Inhibin B was measured in boys. A subset of participants underwent exome sequencing. Results: “Kisspeptin responders” who had responded to kisspeptin with a rise in LH of 0.8 mIU/mL or greater all subsequently progressed through puberty (n = 8). In contrast, “kisspeptin nonresponders” who had exhibited LH responses to kisspeptin ≤0.4 mIU/mL all reached age 18 years without developing physical signs of puberty (n = 8). Thus, responses to kisspeptin accurately predicted later pubertal outcomes (p = 0.0002), with sensitivity and specificity of 100% (95% CI 74-100%). Moreover, the kisspeptin-stimulation test outperformed overnight LH measurements, GnRH-stimulated LH, inhibin B, and genetic testing in predicting pubertal outcomes. Conclusion: The kisspeptin-stimulation test can be used to reveal future reproductive endocrine potential in prepubertal children and is a promising novel tool for predicting pubertal outcomes for children with delayed puberty. Trial registration: ClinicalTrials.gov NCT01438034

Leptin Decreases De Novo Lipogenesis in Lipodystrophic Patients

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