X-linked hypophosphatemia (XLH) is characterized by high serum fibroblast growth factor 23 (FGF23) levels, resulting in impaired 1,25 dihydroxyvitamin D (1,25D) production. Adults with XLH develop a painful mineralization of the tendon-bone attachment site (enthesis), called enthesopathy. Treatment of mice with XLH (Hyp) with 1,25D or an anti-FGF23 antibody, both of which increase 1,25D signaling, prevents enthesopathy. Therefore, we undertook studies to determine a role for impaired 1,25D action in enthesopathy development. Entheses from mice lacking vitamin D 1-alpha-hydroxylase (Cyp27b1) (C-/-) had a similar enthesopathy to Hyp mice, while deletion of Fgf23 in Hyp mice prevented enthesopathy and deletion of both Cyp27b1 and Fgf23 in mice resulted in enthesopathy, demonstrating that the impaired 1,25D action due to high FGF23 levels underlied XLH enthesopathy development. Like Hyp mice, enthesopathy in C-/- mice was observed by P14 and was prevented, but not reversed, with 1,25D therapy. Deletion of the vitamin D receptor in scleraxis-expressing cells resulted in enthesopathy, indicating 1,25D acted directly on enthesis cells to regulate enthesopathy development. These results showed that 1,25D signaling was necessary for normal post-natal enthesis maturation and played a role in XLH enthesopathy development. Optimizing 1,25D replacement in pediatric XLH patients is necessary to prevent enthesopathy.
Rakshya Rana, Jiana T. Baker, Melissa Sorsby, Supriya Jagga, Shreya Venkat, Shaza Almardini, Eva S. Liu
The adrenal glands synthesize and release essential steroid hormones such as cortisol and aldosterone, but many aspects of human adrenal gland development are not well understood. Here, we combined single-cell and bulk RNA sequencing, spatial transcriptomics, IHC, and micro-focus computed tomography to investigate key aspects of adrenal development in the first 20 weeks of gestation. We demonstrate rapid adrenal growth and vascularization, with more cell division in the outer definitive zone (DZ). Steroidogenic pathways favored androgen synthesis in the central fetal zone, but DZ capacity to synthesize cortisol and aldosterone developed with time. Core transcriptional regulators were identified, with localized expression of HOPX (also known as Hop homeobox/homeobox-only protein) in the DZ. Potential ligand-receptor interactions between mesenchyme and adrenal cortex were seen (e.g., RSPO3/LGR4). Growth-promoting imprinted genes were enriched in the developing cortex (e.g., IGF2, PEG3). These findings reveal aspects of human adrenal development and have clinical implications for understanding primary adrenal insufficiency and related postnatal adrenal disorders, such as adrenal tumor development, steroid disorders, and neonatal stress.
Ignacio del Valle, Matthew D. Young, Gerda Kildisiute, Olumide K. Ogunbiyi, Federica Buonocore, Ian C. Simcock, Eleonora Khabirova, Berta Crespo, Nadjeda Moreno, Tony Brooks, Paola Niola, Katherine Swarbrick, Jenifer P. Suntharalingham, Sinead M. McGlacken-Byrne, Owen J. Arthurs, Sam Behjati, John C. Achermann
Low-dose anti-thymocyte globulin (ATG) transiently preserves C-peptide and lowers HbA1c in individuals with recent-onset type 1 diabetes (T1D); however, the mechanisms of action and features of response remain unclear. Here, we characterized the post-hoc immunological outcomes of ATG administration and their potential use as biomarkers of metabolic response to therapy (i.e., improved preservation of endogenous insulin production). We assessed gene and protein expression, targeted gene methylation, and cytokine concentrations in peripheral blood following treatment with ATG (n=29), ATG plus granulocyte-colony stimulating factor (ATG/G-CSF, n=28), or placebo (n=31). Treatment with low-dose ATG preserved regulatory T cells (Tregs), as measured by stable methylation of FOXP3 Treg-specific demethylation region (TSDR) and increased proportions of CD4+FOXP3+ Tregs (p<0.001) identified by flow cytometry. While treatment effects were consistent across participants, not all maintained C-peptide. Responders exhibited a transient rise in IL-6, IP-10, and TNFα (p<0.05 for all) 2 weeks post-treatment and a durable CD4 exhaustion phenotype (increased PD-1+KLRG1+CD57- on CD4+ T cells [p=0.011] and PD1+CD4 TEMRA MFI [p<0.001] at 12 weeks, following ATG and ATG/G-CSF, respectively). ATG non-responders displayed higher proportions of senescent T cells (at baseline and post-treatment) and increased methylation of EOMES (i.e., less expression of this exhaustion marker). Altogether in these exploratory analyses, Th1 inflammation-associated serum, CD4 exhaustion transcript and cellular phenotyping profiles may be useful for identifying signatures of clinical response to ATG in T1D.
Laura M. Jacobsen, Kirsten Diggins, Lori Blanchfield, James A. McNichols, Daniel J. Perry, Jason Brant, Xiaoru Dong, Rhonda Bacher, Vivian H. Gersuk, Desmond A. Schatz, Mark A. Atkinson, Clayton E. Mathews, Michael J. Haller, S. Alice Long, Peter S. Linsley, Todd M. Brusko
We previously reported that measles virus nucleocapsid protein (MVNP) expression in osteoclasts (OCLs) of Paget’s disease (PD) patients or targeted to the OCL lineage in transgenic (T-MVNP) mice increases IGF1 production in osteoclasts (OCL-IGF1) and develop PD-OCLs and pagetic bone lesions (PDLs). Conditional deletion of Igf1 in OCLs of T-MVNP mice fully blocked development of pagetic bone lesions (PDLs). In this manuscript we examined if osteocytes (OCys), key regulators of normal bone remodeling, contribute to PD. OCys in PDLs of patients and of T-MVNP mice expressed less sclerostin, and had increased RANKL expression compared to OCys in bones from WT mice or normal patients. To test if increased OCL-IGF1 is sufficient to induce PDLs and PD-phenotypes, we generated TRAP-Igf1 (T-Igf1) transgenic mice to characterize if increased IGF1 expression in the absence of MVNP in OCLs is sufficient to induce pagetic lesions and pagetic OCLs. We found that T-Igf1 mice at 16 months of age developed PD-OCLs, PDLs, and OCys with decreased sclerostin and increased RANKL similar to T-MVNP mice. Thus, pagetic phenotypes could be induced by OCLs expressing increased IGF1. OCL-IGF1 in turn increased RANKL production in OCys to induce PD-OCL and PDLs.
Kazuaki Miyagawa, Hirofumi Tenshin, Patrick L. Mulcrone, Jesus Delgado-Calle, Mark A. Subler, Jolene J. Windle, John M. Chirgwin, Garson David Roodman, Noriyoshi Kurihara
The mineralocorticoid aldosterone, secreted by the adrenal zona glomerulosa (ZG), is critical for life, maintaining ion homeostasis and blood pressure. Therapeutic inhibition of protein phosphatase 3 (Calcineurin (Cn)) results in inappropriately low plasma aldosterone levels despite concomitant hyperkalemia and hyperreninemia. We tested the hypothesis that Cn participates in the signal transduction pathway regulating aldosterone synthesis. Inhibition of Cn with tacrolimus abolished the potassium (K+)-stimulated expression of aldosterone synthase, encoded by CYP11B2, in the NCI-H295R human adrenocortical cell line as well as ex vivo in mouse and human adrenal tissue. ZG-specific deletion of the regulatory Cn subunit CnB1 diminished Cyp11b2 expression in vivo and disrupted K+-mediated aldosterone synthesis. Phosphoproteomic analysis identified Nuclear factor of activated T-cells, cytoplasmic 4 (NFATc4) as a target for Cn-mediated dephosphorylation. Deletion of NFATc4 impaired K+-dependent stimulation of CYP11B2 expression and aldosterone production while expression of a constitutively active form of NFATc4 increased expression of CYP11B2 in NCI-H295R cells. Chromatin immunoprecipitation revealed NFATc4 directly regulates CYP11B2 expression. Thus, calcineurin controls aldosterone production via the Cn-NFATc4 pathway. Inhibition of Cn-NFATc4 signaling may explain low plasma aldosterone levels and hyperkalemia in patients treated with tacrolimus and the Cn-NFATc4 pathway may provide novel molecular targets to treat primary aldosteronism.
Mesut Berber, Sining Leng, Agnieszka Wengi, Denise V. Winter, Alex Odermatt, Felix Beuschlein, Johannes Loffing, David T. Breault, David Penton
Fatty acid binding protein 4 (FABP4) is a lipid chaperone secreted from adipocytes upon stimulation of lipolysis. Circulating FABP4 levels strongly correlate with obesity and metabolic pathologies in experimental models and humans. While adipocytes have been presumed to be the major source of hormonal FABP4, this question has not been addressed definitively in vivo. We generated mice with Fabp4 deletion in cells known to express the gene; adipocytes (Adipo-KO), endothelial cells (Endo-KO), myeloid cells (Myeloid-KO), and the whole body (Total-KO) to examine the contribution of these cell types to basal and stimulated plasma FABP4 levels. Unexpectedly, baseline plasma FABP4 was not significantly reduced in Adipo-KO mice, whereas Endo-KO mice showed ~87% reduction versus wildtype controls. In contrast, Adipo-KO mice exhibited ~62% decreased induction of FABP4 responses to lipolysis, while Endo-KO mice showed only mildly decreased induction, indicating that adipocytes are the main source of increases in FABP4 during lipolysis. We did not detect any myeloid contribution to circulating FABP4. Surprisingly, despite the nearly intact induction of FABP4, Endo-KO mice showed blunted lipolysis-induced insulin secretion, identical to Total-KO mice. We conclude that the endothelium is the major source of baseline hormonal FABP4 and is required for the insulin response to lipolysis.
Karen E. Inouye, Kacey J. Prentice, Alexandra Lee, Zeqiu B. Wang, Carla Dominguez-Gonzalez, Mu Xian Chen, Jillian K. Riveros, M. Furkan Burak, Grace Y. Lee, Gokhan S. Hotamisligil
Growing evidence indicates that the glucagon-like-peptide-1 (GLP-1) system is involved in the neurobiology of addictive behaviors and GLP-1 analogues may be used for the treatment of alcohol use disorder (AUD). Semaglutide is a long-acting GLP-1 analogue with compelling characteristics for clinical translation. The goal of this study was to examine the effects of semaglutide on biobehavioral correlates of alcohol use in rodents, using psychopharmacology and electrophysiology experiments. A drinking-in-the-dark procedure was used to test the effects of semaglutide on binge-like drinking in male and female mice. We also tested the effects of semaglutide on both binge-like and dependence-induced alcohol drinking in male and female rats. Finally, the acute effects of semaglutide on GABA neurotransmission were examined by recording spontaneous inhibitory postsynaptic currents (sIPSCs) from central nucleus of the amygdala (CeA) and infralimbic cortex (ILC) neurons. Results showed that semaglutide dose-dependently reduced binge-like alcohol drinking in mice; a similar effect was observed on the intake of other caloric/non-caloric solutions. Semaglutide also reduced binge-like and dependence-induced alcohol drinking in rats. In alcohol-naïve rats, an acute application of semaglutide increased sIPSC frequency in CeA and ILC neurons, suggesting enhanced GABA release, while in alcohol-dependent rats, semaglutide did not significantly alter overall CeA and ILC GABA transmission. In conclusion, the GLP-1 analogue semaglutide decreased alcohol intake across different drinking models and species and modulated central GABA neurotransmission in rodents, providing support for clinical testing of semaglutide as a potential novel pharmacotherapy for AUD.
Vicky Chuong, Mehdi Farokhnia, Sophia Khom, Claire L. Pince, Sophie K. Elvig, Roman Vlkolinsky, Renata C.N. Marchette, George F. Koob, Marisa Roberto, Leandro F. Vendruscolo, Lorenzo Leggio
Fasting is associated with increased susceptibility to hypoglycemia in people with type 1 diabetes, thereby making it a significant health risk. To date, the relationship between fasting and insulin-induced hypoglycemia has not been well characterized, so our objective was to determine whether insulin-independent factors, such as counterregulatory hormone responses, are adversely impacted by fasting in healthy control subjects. Counterregulatory responses to insulin-induced hypoglycemia were measured in twelve healthy people during two metabolic studies. During one study, subjects ate breakfast and lunch, after which they underwent a two-hour bout of insulin-induced hypoglycemia (FED). During the other study, subjects remained fasted prior to hypoglycemia (FAST). As expected, hepatic glycogen concentrations were lower in FAST, and associated with diminished peak glucagon levels and reduced endogenous glucose production (EGP) during hypoglycemia. Accompanying lower EGP in FAST was a reduction in peripheral glucose utilization, and a resultant reduction in the amount of exogenous glucose required to maintain glycemia. These data suggest that whereas a fasting-induced lowering of glucose utilization could potentially delay the onset of insulin-induced hypoglycemia, subsequent reductions in glucagon levels and EGP are likely to encumber recovery from it. As a result of this diminished metabolic flexibility in response to fasting, susceptibility to hypoglycemia could be enhanced in patients with type 1 diabetes under similar conditions.
Shana O. Warner, Yufei Dai, Nicole Sheanon, Michael V. Yao, Rebecca L. Cason, Shahriar Arbabi, Shailendra B. Patel, Diana M. Lindquist, Jason J. Winnick
Elevation of glucagon levels and increase in alpha cell proliferation is associated with states of hyperglycemia in diabetes. A better understanding of the molecular mechanisms governing glucagon secretion could have major implications in understanding abnormal responses to hypoglycemia in diabetes patients and provide novel avenues for diabetes management. Our previous studies have highlighted the role of nutrient signaling via mTOR complex 1 (mTORC1) regulation that controls glucagon secretion and alpha cell mass and that hyperglucagonemia can improve glucose homeostasis by diminishing glucagon action in the liver. However, it is unclear if short-term effects of mTORC1 activation are sufficient to induce glucagon secretion without changes in alpha cell mass and whether short-term hyperglucagonemia reduces liver glucagon action in a reversible manner. Using mice with inducible induction of the regulator of the mTORC1 complex (Rheb) in alpha cells (αRhebTg), we showed that short-term activation of mTORC1 signaling is sufficient to induce hyperglucagonemia as a result of increased glucagon secretion. Hyperglucagonemia in the αRhebTg was also associated with an increase in alpha cell size and mass expansion. This model allowed us to identify the effects of chronic and short-term hyperglucagonemia on glucose homeostasis by regulating glucagon signaling in the liver. Short-term hyperglucagonemia impaired glucose tolerance, which was reversible over time. Decrease in liver glucagon effects in αRhebTg mice was associated with reduced expression of the glucagon receptor (GCGR) and genes involved in gluconeogenesis, amino acid metabolism, and urea production. However, only genes regulating gluconeogenesis returned to baseline upon improvement of glycemia. Overall, these studies demonstrate that hyperglucagonemia exerts a biphasic response on glucose metabolism: short-term hyperglucagonemia leads to glucose intolerance, whereas chronic exposure to glucagon generates decrease on hepatic glucagon action along with improved glucose tolerance.
Camila Lubaczeuski, Nadejda Bozadjieva-Kramer, Ruy A. Louzada, George K. Gittes, Gil Leibowitz, Ernesto Bernal-Mizrachi
BACKGROUND Elevated circulating branched chain amino acids (BCAAs), measured at a single time point in middle life, are strongly associated with an increased risk of developing type 2 diabetes mellitus (DM). However, the longitudinal patterns of change in BCAAs through young adulthood and their association with DM in later life are unknown.METHODS We serially measured BCAAs over 28 years in the Coronary Artery Risk Development in Young Adults (CARDIA) study, a prospective cohort of apparently healthy Black and White young adults at baseline. Trajectories of circulating BCAA concentrations from years 2–30 (for prevalent DM) or years 2–20 (for incident DM) were determined by latent class modeling.RESULTS Among 3,081 apparently healthy young adults, trajectory analysis from years 2–30 revealed 3 distinct BCAA trajectory groups: low-stable (n = 1,427), moderate-stable (n = 1,384), and high-increasing (n = 270) groups. Male sex, higher body mass index, and higher atherogenic lipid fractions were more common in the moderate-stable and high-increasing groups. Higher risk of prevalent DM was associated with the moderate-stable (OR = 2.59, 95% CI: 1.90–3.55) and high-increasing (OR = 6.03, 95% CI: 3.86–9.43) BCAA trajectory groups in adjusted models. A separate trajectory group analysis from years 2–20 for incident DM after year 20 showed that moderate-stable and high-increasing trajectory groups were also significantly associated with higher risk of incident DM, after adjustment for clinical variables and glucose levels.CONCLUSION BCAA levels track over a 28-year span in most young adults, but serial clinical metabolomic measurements identify subpopulations with rising levels associated with high risk of DM in later life.FUNDING This research was supported by the NIH, under grants R01 HL146844 (JTW) and T32 HL069771 (MRC). The CARDIA study is conducted and supported by the NIH National Heart, Lung, and Blood Institute in collaboration with the University of Alabama at Birmingham (HHSN268201800005I and HHSN268201800007I), Northwestern University (HHSN268201800003I), the University of Minnesota (HHSN268201800006I), and Kaiser Foundation Research Institute (HHSN268201800004I).
Konrad T. Sawicki, Hongyan Ning, Norrina B. Allen, Mercedes R. Carnethon, Amisha Wallia, James D. Otvos, Issam Ben-Sahra, Elizabeth M. McNally, Janet K. Snell-Bergeon, John T. Wilkins
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