Genetic defects of GNAS, the imprinted gene encoding the stimulatory G protein α-subunit, are responsible for multiple diseases. Abnormal GNAS imprinting causes pseudohypoparathyroidism type 1B (PHP1B), a prototype of mammalian end-organ hormone resistance. Hypomethylation at the maternally methylated GNAS A/B region is the only shared defect in PHP1B patients. In autosomal dominant (AD) PHP1B kindreds, A/B hypomethylation is associated with maternal microdeletions at either the GNAS NESP55 differentially methylated region or the STX16 gene located ~170 kb upstream. Functional evidence is meager regarding the causality of these microdeletions. Moreover, the mechanisms linking A/B methylation and these putative imprinting control regions (ICRs), NESP-ICR and STX16-ICR, remain unknown. Here, we generated a human embryonic stem cell model of AD-PHP1B by introducing ICR deletions using CRISPR/Cas9. Using this model, we showed that NESP-ICR is required for methylation and transcriptional silencing of A/B on the maternal allele. We also found that SXT16-ICR is a long-range enhancer of NESP55 transcription, which originates from maternal NESP-ICR. Furthermore, we demonstrated that STX16-ICR is an embryonic stage-specific enhancer enabled by the direct binding of pluripotency factors. Our findings uncover an essential GNAS imprinting control mechanism and advance the molecular understanding of the PHP1B pathogenesis.
Yorihiro Iwasaki, Cagri Aksu, Monica Reyes, Birol Ay, Qing He, Murat Bastepe
How phosphate levels are detected in mammals is unknown. The bone-derived hormone fibroblast growth factor 23 (FGF23) lowers blood phosphate by reducing kidney phosphate reabsorption and 1,25(OH)2D production, but phosphate does not directly stimulate bone FGF23 expression. Using PET scanning and LC-MS, we show that phosphate increases kidney-specific glycolysis and synthesis of glycerol-3-phosphate (G-3-P), which then circulates to bone to trigger FGF23 production. Further, we find that glycerol-3-phosphate dehydrogenase 1 (Gpd1), a cytosolic enzyme that synthesizes G-3-P and oxidizes NADH to NAD+, is required for phosphate-stimulated G-3-P and FGF23 production and prevention of hyperphosphatemia. In proximal tubule cells, we find that phosphate availability is substrate-limiting for glycolysis and G-3-P production, and that increased glycolysis and Gpd1 activity are coupled through cytosolic NAD+ recycling. Finally, we show that the type II sodium-dependent phosphate co-transporter Npt2a, which is expressed exclusively in the proximal tubule, confers kidney specificity to phosphate-stimulated G-3-P production. Importantly, exogenous G-3-P stimulates FGF23 production when Npt2a or Gpd1 are absent, confirming that it is the key circulating factor downstream of glycolytic phosphate sensing in the kidney. Together, these findings place glycolysis at the nexus of mineral and energy metabolism and identify a kidney-bone feedback loop that controls phosphate homeostasis.
Wen Zhou, Petra Simic, Iris Y. Zhou, Peter Caravan, Xavier Vela Parada, Donghai Wen, Onica L. Washington, Maria Shvedova, Kerry A. Pierce, Clary B. Clish, Michael Mannstadt, Tatsuya Kobayashi, Marc N. Wein, Harald Jüppner, Eugene P. Rhee
Adaptation of the islet β-cell insulin secretory response to changing insulin demand is critical for blood glucose homeostasis, yet the mechanisms underlying this adaptation are unknown. Here, we have shown that nutrient-stimulated histone acetylation plays a key role in adapting insulin secretion through regulation of genes involved in β-cell nutrient sensing and metabolism. Nutrient regulation of the epigenome occurred at sites occupied by the chromatin-modifying enzyme Lysine-specific demethylase 1 (Lsd1) in islets. β-cell-specific deletion of Lsd1 led to insulin hypersecretion, aberrant expression of nutrient response genes, and histone hyperacetylation. Islets from mice adapted to chronically increased insulin demand exhibited shared epigenetic and transcriptional changes. Moreover, we found that genetic variants associated with type 2 diabetes were enriched at LSD1-bound sites in human islets, suggesting that interpretation of nutrient signals is genetically determined and clinically relevant. Overall, these studies revealed that adaptive insulin secretion involves Lsd1-mediated coupling of nutrient state to regulation of the islet epigenome.
Matthew Wortham, Fenfen Liu, Austin R. Harrington, Johanna Y. Fleischman, Martina Wallace, Francesca Mulas, Medhavi Mallick, Nicholas K. Vinckier, Benjamin R. Cross, Joshua Chiou, Nisha A. Patel, Yinghui Sui, Carolyn McGrail, Yesl Jun, Gaowei Wang, Ulupi S. Jhala, Roland Schüle, Orian S. Shirihai, Mark O. Huising, Kyle J. Gaulton, Christian M. Metallo, Maike Sander
Alice E. Hughes, Elisa De Franco, Rachel M. Freathy, Sarah E. Flanagan, Andrew T. Hattersley
Glucose homeostasis can be improved after bariatric surgery that alters bile flow and stimulate gut hormone secretion, in particular FGF15/19. FGFR1 expression in AGRP expressing cells is required for bile acids's ability to improve glucose control. We show that the mouse Agrp gene has 3 promoter/enhancer regions that direct transcription of each of their own AGRP transcripts. One of these Agrp promoters/enhancers, Agrp B, is regulated by bile acids. We generated an Agrp B knock-in FLP/knockout allele. AGRP B expressing cells are found in endocrine cells the pars tuberalis (PT) and co-expressDAGLB (an endocannabinoid biosynthetic enzyme), distinct from PT thyrotropes. AGRP B expression is also found in the folliculostellate cells of the pituitary's anterior lobe. Mice without AGRP B are protected from high fat feeding induced glucose intolerance but not excess weight gain. Chemogenetic inhibition of AGRP B cells improves glucose tolerance by enhancing glucose stimulated insulin secretion. Inhibition of the AGRP B cells also caused weight loss. The improved glucose tolerance and reduced body weight persisted up to 6 weeks after cessation of the DREADD mediated inhibition, suggesting the presence of a biological switch for glucose homeostasis that is regulated by long term stability of food availability.
Shun-Mei Liu, Bruno Ifebi, Fred Johnson III, Alison Xu, Jacquelin Ho, Yunlei Yang, Gary J. Schwartz, Young-Hwan Jo, Streamson Chua
Type 2 diabetes (T2D) is caused by insufficient insulin secretion from pancreatic β-cells. To identify candidates contributing to T2D pathophysiology, we studied human pancreatic islets from ~300 individuals. We found 395 differentially expressed genes (DEGs) in islets from individuals with T2D, including, to our knowledge, novel (OPRD1, PAX5, TET1) and previously identified (CHL1, GLRA1, IAPP) candidates. A third of the identified islet expression changes may predispose to diabetes, as they associated with HbA1c in individuals not previously diagnosed with T2D. Most DEGs were expressed in human β-cells based on single-cell RNA-sequencing data. Additionally, DEGs displayed alterations in open chromatin and associated with T2D-SNPs. Mouse knock-out strains demonstrated that T2D-associated candidates regulate glucose homeostasis and body composition in vivo. Functional validation showed that mimicking T2D-associated changes for OPRD1, PAX5, and SLC2A2 impaired insulin secretion. Impairments in Pax5-overexpressing β-cells were due to severe mitochondrial dysfunction. Finally, we discovered PAX5 as a potential transcriptional regulator of many T2D-associated DEGs in human islets. Overall, we identified molecular alterations in human pancreatic islets contributing to β-cell dysfunction in T2D pathophysiology.
Karl Bacos, Alexander Perfilyev, Alexandros Karagiannopoulos, Elaine Cowan, Jones K. Ofori, Ludivine Bertonnier-Brouty, Tina Rönn, Andreas Lindqvist, Cheng Luan, Sabrina Ruhrmann, Mtakai Ngara, Åsa Nilsson, Sevda Gheibi, Claire L. Lyons, Jens O. Lagerstedt, Mohammad Barghouth, Jonathan L.S. Esguerra, Petr Volkov, Malin Fex, Hindrik Mulder, Nils Wierup, Ulrika Krus, Isabella Artner, Lena Eliasson, Rashmi B. Prasad, Luis Rodrigo Cataldo, Charlotte Ling
Insulin and IGF-1 receptors (IR/IGF1R) are highly homologous and share similar signaling systems, but each has a unique physiological role, with IR primarily regulating metabolic homeostasis and IGF1R regulating mitogenic control and growth. Here, we showed that replacement of a single amino acid at position 973, just distal to the NPEY motif in the intracellular juxtamembrane region, from leucine, which is highly-conserved in IRs, to phenylalanine, the highly-conserved homologous residue in IGF1Rs, resulted in decreased IRS-1-PI3K-Akt-mTORC1 signaling and increased of Shc-Gab1-MAPK-cell cycle signaling. As a result, cells expressing L973F-IR exhibited decreased insulin-induced glucose uptake, increased cell growth and impaired receptor internalization. Mice with knockin of the L973F-IR showed similar alterations in signaling in vivo, and this leaded to decreased insulin sensitivity, a modest increase in growth and decreased weight gain when challenged with high-fat diet. Thus, leucine973 in the juxtamembrane region of the IR acts as a crucial residue differentiating IR signaling from IGF1R signaling.
Hirofumi Nagao, Weikang Cai, Bruna Brasil Brandão, Nicolai J. Wewer Albrechtsen, Martin Steger, Arijeet K. Gattu, Hui Pan, Jonathan M. Dreyfuss, F. Thomas Wunderlich, Matthias Mann, C. Ronald Kahn
Disorders of isolated mineralocorticoid deficiency causing potentially life-threatening salt-wasting crisis early in life have been associated with gene variants of aldosterone biosynthesis or resistance, but in some patients no such variants are found. WNT/β-catenin signaling is crucial for differentiation and maintenance of the aldosterone producing adrenal zona glomerulosa (zG). We describe a highly consanguineous family with multiple perinatal deaths or infants presenting at birth with failure to thrive, severe salt-wasting crises associated with isolated hypoaldosteronism, nail anomalies, short stature, and deafness. Whole exome sequencing revealed a homozygous splice variant in the R-SPONDIN receptor LGR4 gene (c.618-1G>C) regulating WNT signaling. The resulting transcripts affected protein function and stability, and resulted in loss of Wnt/β-catenin signaling in vitro. The impact of LGR4 inactivation was analyzed by adrenal cortex specific ablation of Lgr4, using Lgr4Flox/Flox mated with Sf1:Cre mice. Inactivation of Lgr4 within the adrenal cortex in the mouse model caused decreased WNT signaling, aberrant zonation with deficient zG and reduced aldosterone production. Thus, human LGR4 mutations establish a direct link between LGR4 inactivation and decreased canonical WNT signaling with abnormal zG differentiation and endocrine function. Therefore, variants in WNT signaling and its regulators should systematically be considered in familial hyperreninemic hypoaldosteronism.
Cécily Lucas, Kay-Sara Sauter, Michael Steigert, Delphine Mallet, James Wilmouth Jr., Julie Olabe, Ingrid Plotton, Yves Morel, Daniel Aeberli, Franca Wagner, Hans Clevers, Amit V. Pandey, Pierre Val, Florence Roucher-Boulez, Christa E. Fluck
Multiple genetic loci have been reported for progeroid syndromes. However, the molecular defects in some extremely rare forms of progeria have yet to be elucidated. Here we report a 21-year-old man of Chinese origin who had a novel autosomal recessive form of progeria, characterized by severe dwarfism, mandibular hypoplasia, hyperopia and partial lipodystrophy. Analyses of exome sequencing data of the entire family revealed only one rare homozygous missense variant, (c.86C>T; p.Pro29Leu), in TOMM7 in the proband, while the parents and two unaffected siblings were heterozygous for the variant. TOMM7, a nuclear gene, encodes a translocase in the outer mitochondrial membrane. The TOMM complex constitutes the outer membrane pore for import of several preproteins into mitochondria. Proteomics analyses of mitochondria from cultured fibroblasts of the proband, as compared to control fibroblasts, revealed increases in several proteins involved in oxidative phosphorylation, but reduced abundance of proteins involved in the phospholipid metabolism. We also observed elevated basal and maximal oxygen consumption rates in the fibroblasts from the proband as compared to control fibroblasts. We conclude that altered mitochondrial protein import due to loss of function bi-allelic variant in TOMM7 can cause severe growth retardation and progeroid features.
Abhimanyu Garg, Wee-Teik Keng, Zhenkang Chen, Adwait Amod Sathe, Chao Xing, Pavithira Devi Kailasam, Yanqiu Shao, Nicholas P. Lesner, Claire B. Llamas, Anil K. Agarwal, Prashant Mishra
As a highly regenerative organ, the intestine is a promising source for cellular reprogramming to replace lost pancreatic β-cells in diabetes. Gut enterochromaffin cells can be converted to insulin-producing cells by FoxO1 ablation, but their numbers are limited. In this study we report that insulin-immunoreactive cells with Paneth/goblet cell features are present in human fetal intestine. Accordingly, lineage tracing experiments show that upon genetic or pharmacologic FoxO1 ablation the Paneth/goblet lineage can also undergo conversion to the insulin lineage. We designed a screening platform in gut organoids to accurately quantitate β-like cell reprogramming and fine-tune a combination treatment to increase the efficiency of the conversion process in mice and human adult intestinal organoids. We identified a triple blockade of FOXO1, Notch, and TGFβ that, when tested in insulin-deficient STZ or NOD diabetic animals resulted in near-normalization of glucose levels, associated with the generation of intestinal insulin-producing cells. The findings illustrate a therapeutic approach to replace insulin treatment in diabetes.
Wen Du, Junqiang Wang, Taiyi Kuo, Liheng Wang, Wendy M. McKimpson, Jinsook Son, Hitoshi Watanabe, Takumi Kitamoto, Yun-Kyoung Lee, Remi J. Creusot, Lloyd E. Ratner, Kasi McCune, Ya-Wen Chen, Brendan H. Grubbs, Matthew E. Thornton, Jason Fan, Nishat Sultana, Bryan S. Diaz, Iyshwarya Balasubramanian, Nan Gao, Sandro Belvedere, Domenico Accili