Probes for INS

Temporal transcription profiles of fetal testes with Sertoli cell ablation were examined in 4-day culture using a diphtheria toxin (DT)-dependent cell knockout system in AMH-TRECK transgenic (Tg) mice. RNA analysis revealed that ovarian-specific genes, including Foxl2, were ectopically expressed in DT-treated Tg testis explants initiated at embryonic days 12.5-13.5. FOXL2-positive cells were ectopically observed in two testicular regions-near the testicular surface epithelia and around its adjacent mesonephros. The surface FOXL2-positive cells, together with ectopic expression of Lgr5 and Gng13 (markers of ovarian cords), were derived from the testis epithelia/subepithelia, whereas another FOXL2-positive population was the 3βHSD-negative stroma near the mesonephros. In addition to high expression of Fgfr1/Fgfr2 and heparan sulfate proteoglycan (a reservoir for FGF ligand) in these two sites, exogenous FGF9 additives repressed DT-dependent Foxl2 upregulation in Tg testes. These findings imply retention of Foxl2 inducibility in the surface epithelia and peri-mesonephric stroma of the testicular parenchyma, in which certain paracrine signals, including FGF9 derived from fetal Sertoli cells, repress feminization in these two sites of the early fetal testis.

In addition to its renal and cardiovascular functions, angiotensin signalling is thought to be responsible for the increases in salt and water intake caused by hypovolaemia. However, it remains unclear whether these behaviours require angiotensin production in the brain or liver. Here, we use in situ hybridization to identify tissue-specific expression of the genes required for producing angiotensin peptides, and then use conditional genetic deletion of the angiotensinogen gene (Agt) to test whether production in the brain or liver is necessary for sodium appetite and thirst. In the mouse brain, we identified expression of Agt (the precursor for all angiotensin peptides) in a large subset of astrocytes. We also identified Ren1 and Ace (encoding enzymes required to produce angiotensin II) expression in the choroid plexus, and Ren1 expression in neurons within the nucleus ambiguus compact formation. In the liver, we confirmed that Agt is widely expressed in hepatocytes. We next tested whether thirst and sodium appetite require angiotensinogen production in astrocytes or hepatocytes. Despite virtually eliminating expression in the brain, deleting astrocytic Agt did not reduce thirst or sodium appetite. Despite markedly reducing angiotensinogen in the blood, eliminating Agt from hepatocytes did not reduce thirst or sodium appetite, and in fact, these mice consumed the largest amounts of salt and water after sodium deprivation. Deleting Agt from both astrocytes and hepatocytes also did not prevent thirst or sodium appetite. Our findings suggest that angiotensin signalling is not required for sodium appetite or thirst and highlight the need to identify alternative signalling mechanisms. KEY POINTS: Angiotensin signalling is thought to be responsible for the increased thirst and sodium appetite caused by hypovolaemia, producing elevated water and sodium intake. Specific cells in separate brain regions express the three genes needed to produce angiotensin peptides, but brain-specific deletion of the angiotensinogen gene (Agt), which encodes the lone precursor for all angiotensin peptides, did not reduce thirst or sodium appetite. Double-deletion of Agt from brain and liver also did not reduce thirst or sodium appetite. Liver-specific deletion of Agt reduced circulating angiotensinogen levels without reducing thirst or sodium appetite. Instead, these angiotensin-deficient mice exhibited an enhanced sodium appetite. Because the physiological mechanisms controlling thirst and sodium appetite continued functioning without angiotensin production in the brain and liver, understanding these mechanisms requires a renewed search for the hypovolaemic signals necessary for activating each behaviour.

Neuropeptide FF (NPFF) group peptides belong to the evolutionary conserved RF-amide peptide family. While they have been assigned a role as pain modulators, their roles in other aspects of physiology have received much less attention. NPFF peptides and their receptor NPFFR2 have strong and localized expression within the dorsal vagal complex that has emerged as the key centre for regulating glucose homeostasis. Therefore, we investigated the role of the NPFF system in the control of glucose metabolism and the histochemical and molecular identities of NPFF and NPFFR2 neurons.We examined glucose metabolism in Npff-/- and wild type (WT) mice using intraperitoneal (i.p.) glucose tolerance and insulin tolerance tests. Body composition and glucose tolerance was further examined in mice after 1-week and 3-week of high-fat diet (HFD). Using RNAScope double ISH, we investigated the neurochemical identity of NPFF and NPFFR2 neurons in the caudal brainstem, and the expression of receptors for peripheral factors in NPFF neurons.Lack of NPFF signalling in mice leads to improved glucose tolerance without significant impact on insulin excursion after the i.p. glucose challenge. In response to an i.p. bolus of insulin, Npff-/- mice have lower glucose excursions than WT mice, indicating an enhanced insulin action. Moreover, while HFD has rapid and potent detrimental effects on glucose tolerance, this diet-induced glucose intolerance is ameliorated in mice lacking NPFF signalling. This occurs in the absence of any significant impact of NPFF deletion on lean or fat masses, suggesting a direct effect of NPFF signalling on glucose metabolism. We further reveal that NPFF neurons in the subpostrema area (SubP) co-express receptors for peripheral factors involved in glucose homeostasis regulation such as insulin and GLP1. Furthermore, Npffr2 is expressed in the glutamatergic NPFF neurons in the SubP, and in cholinergic neurons of the dorsal motor nucleus of the vagus (DMV), indicating that central NPFF signalling is likely modulating vagal output to innervated peripheral tissues including those important for glucose metabolic control.NPFF signalling plays an important role in the regulation of glucose metabolism. NPFF neurons in the SubP are likely to receive peripheral signals and mediate the control of whole-body glucose homeostasis via centrally vagal pathways. Targeting NPFF and NPFFR2 signalling may provide a new avenue for treating type 2 diabetes and obesity.

Amino acid transporters play an important role in supplying nutrition to cells and are associated with cell proliferation. L-type amino acid transporter 1 (LAT1) is highly expressed in many types of cancers and promotes tumor growth; however, how LAT1 affects tumor development is not fully understood.To investigate the role of LAT1 in intestinal tumorigenesis, mice carrying LAT1 floxed alleles that also expressed Cre recombinase from the promoter of gene encoding Villin were crossed to an ApcMin/+ background (LAT1fl/fl; vil-cre; ApcMin/+), which were subject to analysis; organoids derived from those mice were also analyzed.This study showed that LAT1 was constitutively expressed in normal crypt base cells, and its conditional deletion in the intestinal epithelium resulted in fewer Paneth cells. LAT1 deletion reduced tumor size and number in the small intestine of ApcMin/+ mice. Organoids derived from LAT1-deleted ApcMin/+ intestinal crypts displayed fewer spherical organoids with reduced Wnt/β-catenin target gene expression, suggesting a low tumor-initiation capacity. Wnt3 expression was decreased in the absence of LAT1 in the intestinal epithelium, suggesting that loss of Paneth cells due to LAT1 deficiency reduced the risk of tumor initiation by decreasing Wnt3 production.LAT1 affects intestinal tumor development in a cell-extrinsic manner through reduced Wnt3 expression in Paneth cells. Our findings may partly explain how nutrient availability can affect the risk of tumor development in the intestines.

Human intestinal organoids (HIOs) are a tissue culture model in which small intestine-like tissue is generated from pluripotent stem cells. By carrying out unsupervised hierarchical clustering of RNA-sequencing data, we demonstrate that HIOs most closely resemble human fetal intestine. We observed that genes involved in digestive tract development are enriched in both fetal intestine and HIOs compared to adult tissue, whereas genes related to digestive function and Paneth cell host defense are expressed at higher levels in adult intestine. Our study also revealed that the intestinal stem cell marker OLFM4 is expressed at very low levels in fetal intestine and in HIOs, but is robust in adult crypts. We validated our findings using in vivo transplantation to show that HIOs become more adult-like after transplantation. Our study emphasizes important maturation events that occur in the intestine during human development and demonstrates that HIOs can be used to model fetal-to-adult maturation.

Abstract Aims Intestinal stem cell (ISC) markers such as LGR5, ASCL2, EPHB2 and OLFM4 and their clinical implications have been extensively studied in colorectal cancers (CRCs). However, little is known about their expression in precancerous lesions of CRCs. Here, we investigated the expression and distribution of ISC markers in serrated polyps and conventional adenomas. Methods and results RT-PCR analysis revealed that all ISC markers were significantly upregulated in conventional adenomas with low grade dysplasia (CALGs) compared with other lesions. RNA in situ hybridization confirmed that CALGs exhibited strong and diffuse expression of all ISC markers, which indicate a stem cell-like phenotype. However, normal colonic mucosa hyperplastic polyps and sessile serrated adenomas harbored LGR5+ cells that were confined to the crypt base and demonstrated an organized expression of ISC markers. Notably, in traditional serrated adenomas, expression of LGR5 and ASCL2 was localized to the ectopic crypts as in the normal crypts, but expression of EPHB2 and OLFM4 was distributed in a diffuse manner, which is suggestive of a progenitor-like features. Conclusions The expression and distribution profile of ISC markers possibly provides insights into the organization of stem and progenitor-like cells in each type of precancerous lesion of CRC

Leigh syndrome is a mitochondrial disease characterized by neurodegeneration, neuroinflammation, and early death. Mice lacking NDUFS4, a mitochondrial complex I subunit (Ndufs4 KO mice), have been established as a good animal model for studying human pathology associated with Leigh syndrome. As the disease progresses, there is an increase in neurodegeneration and neuroinflammation, thereby leading to deteriorating neurological symptoms, including motor deficits, breathing alterations, and eventually, death of the animal. However, despite the magnitude of neuroinflammation associated with brain lesions, the role of neuroinflammatory pathways and their main cellular components have not been addressed directly as relevant players in the disease pathology. Here, we investigate the role of microglial cells, the main immune cells of the CNS, in Leigh-like syndrome pathology, by pharmacologically depleting them using the colony-stimulating factor 1 receptor antagonist PLX3397. Microglial depletion extended lifespan and delayed motor symptoms in Ndufs4 KO mice, likely by preventing neuronal loss. Next, we investigated the role of the major cytokine interleukin-6 (IL-6) in the disease progression. IL-6 deficiency partially rescued breathing abnormalities and modulated gliosis but did not extend the lifespan or rescue motor decline in Ndufs4 KO mice. The present results show that microglial accumulation is pathogenic, in a process independent of IL-6, and hints toward a contributing role of neuroinflammation in the disease of Ndufs4 KO mice and potentially in patients with Leigh syndrome.

The majority of colorectal cancers (CRCs) present with early mutations in tumor suppressor gene APC. APC mutations result in oncogenic activation of the Wnt pathway, which is associated with hyperproliferation, cytoskeletal remodeling, and a global increase in mRNA translation. To compensate for the increased biosynthetic demand, cancer cells critically depend on protein chaperones to maintain proteostasis, although their function in CRC remains largely unexplored. In order to investigate the role of molecular chaperones in driving CRC initiation, we captured the transcriptomic profiles of murine wild type and Apc-mutant organoids during active transformation. We discovered a strong transcriptional upregulation of Hspb1, which encodes small heat shock protein 25 (HSP25). We reveal an indispensable role for HSP25 in facilitating Apc-driven transformation, using both in vitro organoid cultures and mouse models, and demonstrate that chemical inhibition of HSP25 using brivudine reduces the development of premalignant adenomas. These findings uncover a hitherto unknown vulnerability in intestinal transformation that could be exploited for the development of chemopreventive strategies in high-risk individuals.

The arcuate nucleus (ArcN) is an integrative hub for the regulation of energy balance, reproduction, and arterial pressure (AP), all of which are influenced by Angiotensin II (AngII); however, the cellular mechanisms and downstream neurocircuitry are unclear. Here we show that ArcN AngII increases AP in female rats via two phases, both of which are mediated via activation of AngII type 1 receptors (AT1aR): initial vasopressin-induced vasoconstriction, followed by slowly developing increases in sympathetic nerve activity (SNA) and heart rate (HR). In male rats, ArcN AngII evoked a similarly slow increase in SNA, but the initial pressor response was variable. In females, the effects of ArcN AngII varied during the estrus cycle, with significant increases in SNA, HR, and AP occurring during diestrus and estrus, but only increased AP during proestrus. Pregnancy markedly increased the expression of AT1aR in the ArcN with parallel substantial AngII-induced increases in SNA and MAP. In both sexes, the sympathoexcitation relied on suppression of tonic ArcN sympathoinhibitory Neuropeptide Y inputs, and activation of pro-opiomelanocortin (POMC) projections, to the paraventricular nucleus (PVN). Few or no NPY or POMC neurons expressed the AT1aR, suggesting that AngII increases AP and SNA at least in part indirectly via local interneurons, which express tyrosine hydroxylase (TH) and VGat (i.e. GABAergic). ArcN TH neurons release GABA locally, and central AT1aR and TH neurons mediate stress responses; therefore, we propose that TH AT1aR neurons are well situated to locally coordinate the regulation of multiple modalities within the ArcN in response to stress.SIGNIFICANCEThe arcuate nucleus (ArcN) is an integrative hub for the regulation of energy balance, reproduction, and arterial pressure (AP), all of which are influenced by Angiotensin II (AngII). Here we show that ArcN AngII activates AT1aR to increase AP in male and female rats by slowly increasing sympathetic nerve activity. In females, ArcN AngII also evoked an initial pressor response mediated by vasopressin-induced vasoconstriction. Pregnant and estrus females responded more than males, in association with higher ArcN AT1aR expression. AT1aR were identified in ArcN interneurons that express tyrosine hydroxylase (TH) and GABA. Since brain AT1aR and TH mediate stress responses, ArcN AT1aR TH neurons are well situated to locally coordinate autonomic, hormonal, and behavioral responses to stress.

The catalytic enzymes tankyrase 1 and 2 (TNKS1/2) alter protein turnover by poly-ADP-ribosylating target proteins, which earmark them for degradation by the ubiquitin-proteasomal system. Prominent targets of the catalytic activity of TNKS1/2 include AXIN proteins, resulting in TNKS1/2 being attractive biotargets for addressing of oncogenic WNT/β-catenin signaling. Although several potent small molecules have been developed to inhibit TNKS1/2, there are currently no TNKS1/2 inhibitors available in clinical practice. The development of tankyrase inhibitors has mainly been disadvantaged by concerns over biotarget-dependent intestinal toxicity and a deficient therapeutic window. Here we show that the novel, potent, and selective 1,2,4-triazole-based TNKS1/2 inhibitor OM-153 reduces WNT/β-catenin signaling and tumor progression in COLO 320DM colon carcinoma xenografts upon oral administration of 0.33-10 mg/kg twice daily. In addition, OM-153 potentiates anti-programmed cell death protein 1 (anti-PD-1) immune checkpoint inhibition and antitumor effect in a B16-F10 mouse melanoma model. A 28-day repeated dose mouse toxicity study documents body weight loss, intestinal damage, and tubular damage in the kidney after oral-twice daily administration of 100 mg/kg. In contrast, mice treated oral-twice daily with 10 mg/kg show an intact intestinal architecture and no atypical histopathologic changes in other organs. In addition, clinical biochemistry and hematologic analyses do not identify changes indicating substantial toxicity. The results demonstrate OM-153-mediated antitumor effects and a therapeutic window in a colon carcinoma mouse model ranging from 0.33 to at least 10 mg/kg, and provide a framework for using OM-153 for further preclinical evaluations. Significance: This study uncovers the effectiveness and therapeutic window for a novel tankyrase inhibitor in mouse tumor models.

Shank3 is an excitatory postsynaptic scaffolding protein implicated in multiple brain disorders, including autism spectrum disorders (ASD) and Phelan-McDermid syndrome (PMS). Although previous neurobiological studies on Shank3 and Shank3-mutant mice have revealed diverse roles of Shank3 in the regulation of synaptic, neuronal and brain functions, whether Shank3 expression in specific cell types distinctly contributes to mouse phenotypes remains largely unclear. In the present study, we generated two Shank3-mutant mouse lines (exons 14-16) carrying global and GABA neuron-specific deletions and characterized their electrophysiological and behavioral phenotypes. These mouse lines show similar decreases in excitatory synaptic input onto dorsolateral striatal neurons. In addition, the abnormal social and locomotor behaviors observed in global Shank3-mutant mice are strongly mimicked by GABA neuron-specific Shank3-mutant mice, whereas the repetitive and anxiety-like behaviors are only partially mimicked. These results suggest that GABAergic Shank3 (exons 14-16) deletion has strong influences on striatal excitatory synaptic transmission and social and locomotor behaviors in mice.