Pituitary develops from oral ectoderm in contact with adjacent ventral hypothalamus. Impairment in this process results in congenital pituitary hypoplasia (CPH); however, there have been no human disease models for CPH thus far, prohibiting the elucidation of the underlying mechanisms. In this study, we established a disease model of CPH using patient-derived induced pluripotent stem cells (iPSCs) and 3D organoid technique, in which oral ectoderm and hypothalamus develop simultaneously. Interestingly, patient iPSCs with a heterozygous mutation in the orthodenticle homeobox 2 (OTX2) gene showed increased apoptosis in the pituitary progenitor cells, and the differentiation into pituitary hormone–producing cells was severely impaired. As an underlying mechanism, OTX2 in hypothalamus, not in oral ectoderm, was essential for progenitor cell maintenance by regulating LHX3 expression in oral ectoderm via FGF10 expression in the hypothalamus. Convincingly, the phenotype was reversed by the correction of the mutation, and the haploinsufficiency of OTX2 in control iPSCs revealed a similar phenotype, demonstrating that this mutation was responsible. Thus, we established an iPSC-based congenital pituitary disease model, which recapitulated interaction between hypothalamus and oral ectoderm and demonstrated the essential role of hypothalamic OTX2.
Ryusaku Matsumoto, Hidetaka Suga, Takashi Aoi, Hironori Bando, Hidenori Fukuoka, Genzo Iguchi, Satoshi Narumi, Tomonobu Hasegawa, Keiko Muguruma, Wataru Ogawa, Yutaka Takahashi
The mineralocorticoid aldosterone is produced in the adrenal zona glomerulosa (ZG) under the control of the renin–angiotensin II (AngII) system. Primary aldosteronism (PA) results from renin-independent production of aldosterone and is a common cause of hypertension. PA is caused by dysregulated localization of the enzyme aldosterone synthase (Cyp11b2), which is normally restricted to the ZG. Cyp11b2 transcription and aldosterone production are predominantly regulated by AngII activation of the Gq signaling pathway. Here, we report the generation of transgenic mice with Gq-coupled designer receptors exclusively activated by designer drugs (DREADDs) specifically in the adrenal cortex. We show that adrenal-wide ligand activation of Gq DREADD receptors triggered disorganization of adrenal functional zonation, with induction of Cyp11b2 in glucocorticoid-producing zona fasciculata cells. This result was consistent with increased renin-independent aldosterone production and hypertension. All parameters were reversible following termination of DREADD-mediated Gq signaling. These findings demonstrate that Gq signaling is sufficient for adrenocortical aldosterone production and implicate this pathway in the determination of zone-specific steroid production within the adrenal cortex. This transgenic mouse also provides an inducible and reversible model of hyperaldosteronism to investigate PA therapeutics and the mechanisms leading to the damaging effects of aldosterone on the cardiovascular system.
Matthew J. Taylor, Matthew R. Ullenbruch, Emily C. Frucci, Juilee Rege, Mark S. Ansorge, Celso E. Gomez-Sanchez, Salma Begum, Edward Laufer, David T. Breault, William E. Rainey
Diabetes is a common complication of cystic fibrosis (CF) that affects approximately 20% of adolescents and 40% to 50% of adults with CF. The age-at-onset of CF-related diabetes (marked by clinical diagnosis and treatment initiation) is an important measure of the disease process. DNA variants associated with age-at-onset of CFRD reside in and near SLC26A9. Deep sequencing of the SLC26A9 gene in 762 individuals with CF revealed that two common DNA haplotypes formed by the risk variants account for the association with diabetes (high risk, P-value: 4.34E-3; low risk, P-value: 1.14E-3). Single-cell RNA (scRNA) sequencing indicated that SLC26A9 is predominantly expressed in pancreatic ductal cells, and frequently co-expressed with CFTR along with transcription factors that have binding sites 5′ of SLC26A9. These findings replicated upon re-analysis of scRNA data from 4 independent studies. DNA fragments derived from the 5′ region of SLC26A9 bearing variants from the low risk haplotype generated 12% to 20% higher levels of expression in PANC-1 and CFPAC-1 cells compared to the high risk haplotype (P-values: 2.00E-3 to 5.15E-9). Taken together, our findings indicate that an increase in SLC26A9 expression in ductal cells of the pancreas delays the age-at-onset of diabetes, thereby suggesting a CFTR-agnostic treatment for a major complication of CF.
Anh-Thu N. Lam, Melis A. Aksit, Briana Vecchio-Pagan, Celeste A. Shelton, Derek L. Osorio, Arianna F. Anzmann, Loyal A. Goff, David C. Whitcomb, Scott M. Blackman, Garry R. Cutting
In order to determine whether the glucose-alanine cycle regulates rates of hepatic mitochondrial oxidation in humans, we applied positional isotopomer NMR tracer analysis (PINTA) to assess rates of hepatic mitochondrial oxidation and pyruvate carboxylase flux in healthy volunteers following both an overnight (12 hours) and a 60-hour fast. Following the 60-hour fast, rates of endogenous glucose production and mitochondrial oxidation decreased, whereas rates of hepatic pyruvate carboxylase flux remained unchanged. These reductions were associated with reduced rates of alanine turnover, assessed by [3-13C]alanine, in a subgroup of participants under similar fasting conditions. In order to determine whether this reduction in alanine turnover was responsible for the reduced rates of hepatic mitochondrial oxidation, we infused unlabeled alanine into another subgroup of 60-hour fasted subjects to increase rates of alanine turnover, similar to what was measured after a 12-hour fast, and found that this perturbation increased rates of hepatic mitochondrial oxidation. Taken together, these studies demonstrate that 60 hours of starvation induce marked reductions in rates of hepatic mitochondrial oxidation, which in turn can be attributed to reduced rates of glucose-alanine cycling, and reveal a heretofore undescribed role for glucose-alanine in the regulation of hepatic mitochondrial oxidation in humans.
Kitt Falk Petersen, Sylvie Dufour, Gary W. Cline, Gerald I. Shulman
Palmitic acid esters of hydroxy stearic acids (PAHSAs) are bioactive lipids with antiinflammatory and antidiabetic effects. PAHSAs reduce ambient glycemia and improve glucose tolerance and insulin sensitivity in insulin-resistant aged chow- and high-fat diet–fed (HFD-fed) mice. Here, we aimed to determine the mechanisms by which PAHSAs improve insulin sensitivity. Both acute and chronic PAHSA treatment enhanced the action of insulin to suppress endogenous glucose production (EGP) in chow- and HFD-fed mice. Moreover, chronic PAHSA treatment augmented insulin-stimulated glucose uptake in glycolytic muscle and heart in HFD-fed mice. The mechanisms by which PAHSAs enhanced hepatic insulin sensitivity included direct and indirect actions involving intertissue communication between adipose tissue and liver. PAHSAs inhibited lipolysis directly in WAT explants and enhanced the action of insulin to suppress lipolysis during the clamp in vivo. Preventing the reduction of free fatty acids during the clamp with Intralipid infusion reduced PAHSAs’ effects on EGP in HFD-fed mice but not in chow-fed mice. Direct hepatic actions of PAHSAs may also be important, as PAHSAs inhibited basal and glucagon-stimulated EGP directly in isolated hepatocytes through a cAMP-dependent pathway involving Gαi protein–coupled receptors. Thus, this study advances our understanding of PAHSA biology and the physiologic mechanisms by which PAHSAs exert beneficial metabolic effects.
Peng Zhou, Anna Santoro, Odile D. Peroni, Andrew T. Nelson, Alan Saghatelian, Dionicio Siegel, Barbara B. Kahn
The parathyroid hormone receptor (PTH1R) mediates the biologic actions of parathyroid hormone (PTH) and parathyroid hormone related protein (PTHrP). Here, we showed that salt inducible kinases (SIKs) are key kinases that control the skeletal actions downstream of PTH1R and that this GPCR, when activated, inhibited cellular SIK activity. Sik gene deletion led to phenotypic changes that were remarkably similar to models of increased PTH1R signaling. In growth plate chondrocytes, PTHrP inhibited SIK3 and ablation of this kinase in proliferating chondrocytes rescued perinatal lethality of PTHrP-null mice. Combined deletion of Sik2/Sik3 in osteoblasts and osteocytes led to a dramatic increase in bone mass that closely resembled the skeletal and molecular phenotypes observed when these bone cells express a constitutively active PTH1R that causes Jansen’s metaphyseal chondrodysplasia. Finally, genetic evidence demonstrated that class IIa HDACs were key PTH1R-regulated SIK substrates in both chondrocytes and osteocytes. Taken together, our findings established that SIK inhibition is central to PTH1R action in bone development and remodeling. Furthermore, this work highlighted the key role of cAMP-regulated salt inducible kinases downstream of GPCR action.
Shigeki Nishimori, Maureen J. O'Meara, Christian Castro, Hiroshi Noda, Murat Cetinbas, Janaina da Silva Martins, Ugur Ayturk, Daniel J. Brooks, Michael Bruce, Mizuki Nagata, Wanida Ono, Christopher J. Janton, Mary L. Bouxsein, Marc Foretz, Rebecca Berdeaux, Ruslan I. Sadreyev, Thomas J. Gardella, Harald Jüppner, Henry M. Kronenberg, Marc N. Wein
Acyl-ghrelin administration increases food intake, body weight, and blood glucose. In contrast, mice lacking ghrelin or ghrelin receptors (GHSRs) exhibit life-threatening hypoglycemia during starvation-like conditions, but do not consistently exhibit overt metabolic phenotypes when given ad libitum food access. These results, and findings of ghrelin resistance in obese states, imply nutritional state dependence of ghrelin’s metabolic actions. Here, we hypothesized that liver-enriched antimicrobial peptide-2 (LEAP2), a recently characterized endogenous GHSR antagonist, blunts ghrelin action during obese states and postprandially. To test this hypothesis, we determined changes in plasma LEAP2 and acyl-ghrelin due to fasting, eating, obesity, Roux-en-Y gastric bypass (RYGB), vertical sleeve gastrectomy (VSG), oral glucose administration, and type 1 diabetes mellitus (T1DM) using humans and/or mice. Our results suggest that plasma LEAP2 is regulated by metabolic status: its levels increased with body mass and blood glucose and decreased with fasting, RYGB, and in postprandial states following VSG. These changes were mostly opposite of those of acyl-ghrelin. Furthermore, using electrophysiology, we showed that LEAP2 both hyperpolarizes and prevents acyl-ghrelin from activating arcuate NPY neurons. We predict that the plasma LEAP2/acyl-ghrelin molar ratio may be a key determinant modulating acyl-ghrelin activity in response to body mass, feeding status, and blood glucose.
Bharath K. Mani, Nancy Puzziferri, Zhenyan He, Juan A. Rodriguez, Sherri Osborne-Lawrence, Nathan P. Metzger, Navpreet Chhina, Bruce Gaylinn, Michael O. Thorner, E. Louise Thomas, Jimmy D. Bell, Kevin W. Williams, Anthony P. Goldstone, Jeffrey M. Zigman
Serine rich splicing factor 3 (SRSF3) plays a critical role in liver function and its loss promotes chronic liver damage and regeneration. As a consequence, genetic deletion of SRSF3 in hepatocytes caused progressive liver disease and ultimately led to hepatocellular carcinoma. Here we show that SRSF3 is decreased in human liver samples with non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), or cirrhosis that was associated with alterations in RNA splicing of known SRSF3 target genes. Hepatic SRSF3 expression was similarly decreased and RNA splicing dysregulated in mouse models of NAFLD and NASH. We showed that palmitic acid-induced oxidative stress caused conjugation of the ubiquitin like NEDD8 protein to SRSF3 and proteasome mediated degradation. SRSF3 was selectively neddylated at lysine11 and mutation of this residue (SRSF3-K11R) was sufficient to prevent both SRSF3 degradation and alterations in RNA splicing. Finally prevention of SRSF3 degradation in vivo partially protected mice from hepatic steatosis, fibrosis and inflammation. These results highlight a neddylation-dependent mechanism regulating gene expression in the liver that is disrupted in early metabolic liver disease and may contribute to the progression to NASH, cirrhosis and ultimately hepatocellular carcinoma.
Deepak Kumar, Manasi Das, Consuelo Sauceda, Lesley G. Ellies, Karina Kuo, Purva Parwal, Mehak Kaur, Lily Jih, Gautam K. Bandyopadhyay, Douglas Burton, Rohit Loomba, Olivia Osborn, Nicholas J.G. Webster
Prostate cancer (PC) is initially dependent on androgen receptor (AR) signaling for survival and growth. Therapeutics designed to suppress AR activity serve as the primary intervention for advanced disease. However, supraphysiological androgen (SPA) concentrations can produce paradoxical responses leading to PC growth inhibition. We sought to discern the mechanisms by which SPA inhibits PC and to determine if molecular context associates with anti-tumor activity. SPA produced an AR-mediated, dose-dependent induction of DNA double-strand breaks (DSBs), G0/G1 cell cycle arrest and cellular senescence. SPA repressed genes involved in DNA repair and delayed the restoration of damaged DNA which was augmented by PARP1 inhibition. SPA-induced DSBs were accentuated in BRCA2-deficient PCs, and combining SPA with PARP or DNA-PKcs inhibition further repressed growth. Next-generation sequencing was performed on biospecimens from PC patients receiving SPA as part of ongoing Phase II clinical trials. Patients with mutations in genes mediating homology-directed DNA repair were more likely to exhibit clinical responses to SPA. These results provide a mechanistic rationale for directing SPA therapy to PCs with AR amplification or DNA repair deficiency, and for combining SPA therapy with PARP inhibition.
Payel Chatterjee, Michael T. Schweizer, Jared M. Lucas, Ilsa Coleman, Michael D. Nyquist, Sander B. Frank, Robin Tharakan, Elahe Mostaghel, Jun Luo, Colin C. Pritchard, Hung-Ming Lam, Eva Corey, Emmanuel S. Antonarakis, Samuel R. Denmeade, Peter S. Nelson
BACKGROUND In the Joslin Medalist Study (Medalists), we determined whether significant associations exist between β cell function and pathology and clinical characteristics.METHODS Individuals with type 1 diabetes (T1D) for 50 or more years underwent evaluation including HLA analysis, basal and longitudinal autoantibody (AAb) status, and β cell function by a mixed-meal tolerance test (MMTT) and a hyperglycemia/arginine clamp procedure. Postmortem analysis of pancreases from 68 Medalists was performed. Monogenic diabetes genes were screened for the entire cohort.RESULTS Of the 1019 Medalists, 32.4% retained detectable C-peptide levels (>0.05 ng/mL, median: 0.21 ng/mL). In those who underwent a MMTT (n = 516), 5.8% responded with a doubling of baseline C-peptide levels. Longitudinally (n = 181, median: 4 years), C-peptide levels increased in 12.2% (n = 22) and decreased in 37% (n = 67) of the Medalists. Among those with repeated MMTTs, 5.4% (3 of 56) and 16.1% (9 of 56) had waxing and waning responses, respectively. Thirty Medalists with baseline C-peptide levels of 0.1 ng/mL or higher underwent the clamp procedure, with HLA–/AAb– and HLA+/AAb– Medalists being most responsive. Postmortem examination of pancreases from 68 Medalists showed that all had scattered insulin-positive cells; 59 additionally had few insulin-positive cells within a few islets; and 14 additionally had lobes with multiple islets with numerous insulin-positive cells. Genetic analysis revealed that 280 Medalists (27.5%) had monogenic diabetes variants; in 80 (7.9%) of these Medalists, the variants were classified as “likely pathogenic” (rare exome variant ensemble learner [REVEL] >0.75).CONCLUSION All Medalists retained insulin-positive β cells, with many responding to metabolic stimuli even after 50 years of T1D. The Medalists were heterogeneous with respect to β cell function, and many with HLA+ diabetes risk alleles also had monogenic diabetes variants, indicating the importance of genetic testing for clinically diagnosed T1D.FUNDING Funding for this work was provided by the Dianne Nunnally Hoppes Fund; the Beatson Pledge Fund; the NIH, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK); and the American Diabetes Association (ADA).
Marc Gregory Yu, Hillary A. Keenan, Hetal S. Shah, Scott G. Frodsham, David Pober, Zhiheng He, Emily A. Wolfson, Stephanie D’Eon, Liane J. Tinsley, Susan Bonner-Weir, Marcus G. Pezzolesi, George Liang King