Probes for INS

The yolk sac and small intestine are 2 important organs responsible for the digestion and absorption of nutrients in chickens during the embryonic and posthatch periods, respectively. The peptide transporter PepT1 is expressed in both the yolk sac and small intestine and plays an important role in the transport of amino acids as short peptides. The objective of this study was to profile the spatial transcriptional patterns of PepT1 mRNA in the yolk sac and small intestine from embryonic and posthatch broilers. The distribution of PepT1 mRNA was investigated by in situ hybridization at embryonic (e) d 11, 13, 15, 17, 19 and day of hatch (doh) in the yolk sac and at e19, doh, and d 1, d 4, and d 7 posthatch in the small intestine. PepT1 mRNA was expressed in the endodermal cells of the yolk sac. PepT1 mRNA was barely detectable at e11, increased from e11 to e13, e15, and e17, and then gradually decreased from e19 to doh. In the small intestine, there was a rapid increase in expression of PepT1 mRNA in the enterocytes from e19 to doh, with expression relatively constant from d 1 to d 7. In addition, there was a differential increase in the heights of the villi in different parts of the small intestine from d 1 to 7, which may partially explain the temporal increase in PepT1 mRNA detected by qPCR. The villi in the duodenum showed the earliest increase in villus height and ultimately resulted in the highest villi at d 7. These results demonstrate that there are temporal changes in PepT1 mRNA expression in the yolk sac and the small intestine, which correspond with their expected role in nutrient uptake during the embryonic and posthatch periods.

The hormone fibroblast growth factor 23 (FGF23) is secreted from bone and is involved in phosphorus (P) metabolism. FGF23 mainly binds the FGF receptor, which interacts with αKlotho in the kidney or parathyroid and regulates Na-dependent phosphate co-transporter type IIa (NaPi-IIa) and type IIc (NaPi-IIc) expression, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) activity, and parathyroid hormone (PTH) secretion. In this study, we utilized hemi-nephrectomized rats fed a high-P diet (HP Nx), rats subjected to a partial nephrectomy (PN) and rats with doxorubicin-induced renal failure (DXR) as chronic kidney disease (CKD) animal models and analyzed the P metabolism and FGF23 expression in the kidneys in each CKD model. We cultured HK2 cells with a high level of P, 1,25(OH)2D3 or transforming growth factor-β1 (TGFβ1) to investigate the FGF23 expression mechanism. In both the HP Nx and PN rats, the blood FGF23 and PTH levels were increased. However, the 1,25(OH)2D3 level was increased in the HP Nx rats and decreased in the PN rats. In all three animal models, the mRNA expression of αKlotho, NaPi-IIa and NaPi-IIc was decreased, and the mRNA expression of TGFβ1, collagen1a1, osteopontin and FGF23 was elevated in the kidney. FGF23 protein and mRNA were expressed at high levels in the extended tubule epithelium, which was an osteopontin-positive region in the HP and PN rats. FGF23 and osteopontin mRNAs were expressed in HK2 cells incubated with TGFβ1; however, these levels were not altered in HK2 cells incubated with 1,25(OH)2D3 and high P levels in vitro. Altogether, FGF23 is expressed in the kidneys in CKD model rats. Following stimulation with TGFβ1, the injured renal tubular epithelial cells are strongly suspected to express both FGF23 and osteopontin. FGF23 produced in the kidney might contribute to P metabolism in subjects with CKD.

Corneal cool cells are sensitive to the ocular fluid status of the corneal surface and may be responsible for the regulation of basal tear production. Previously, we have shown that dry eye, induced by lacrimal gland excision (LGE) in rats, sensitized corneal cool cells to the TRPM8 agonist menthol and to cool stimulation. In the present study, we examined the effect of dry eye on the sensitivity of cool cells to the TRPV1 agonist capsaicin. Single-unit recordings in the trigeminal ganglion were performed 7-10 days after LGE. At a concentration of 0.3mM, capsaicin did not affect ongoing or cool-evoked activity in control animals yet facilitated ongoing activity and suppressed cool-evoked activity in LGE animals. At higher concentrations (3 mM), capsaicin continued to facilitate ongoing activity in LGE animals but suppressed ongoing activity in control animals. Higher concentrations of capsaicin also suppressed cool-evoked activity in both groups of animals, with an overall greater effect in LGE animals. In addition to altering cool-evoked activity, capsaicin enhanced the sensitivity of cool cells to heat in LGE animals. Capsaicin-induced changes were prevented by the application of the TRPV1 antagonist capsazepine. Using fluorescent in situ hybridization, TRPV1 and TRPM8 expression was examined in retrograde tracer identified corneal neurons. The co-expression of TRPV1 and TRPM8 in corneal neurons was significantly greater in LGE treated animals when compared to sham controls. These results indicate that LGE-induced dry eye increases TRPV1-mediated responses in corneal cool cells at least in part through the increased expression of TRPV1.

Secreted metalloproteases have diverse roles in the formation, remodeling, and the destruction of extracellular matrix. Recessive mutations in the secreted metalloprotease ADAMTS17 cause ectopia lentis and short stature in humans with Weill-Marchesani-like syndrome and primary open angle glaucoma and ectopia lentis in dogs. Little is known about this protease or its connection to fibrillin microfibrils, whose major component, fibrillin-1, is genetically associated with ectopia lentis and alterations in height. Fibrillin microfibrils form the ocular zonule and are present in the drainage apparatus of the eye. We show that recombinant ADAMTS17 has unique characteristics and an unusual life cycle. It undergoes rapid autocatalytic processing in trans after its secretion from cells. Secretion of ADAMTS17 requires O-fucosylation and its autocatalytic activity does not depend on propeptide processing by furin. ADAMTS17 binds recombinant fibrillin-2 but not fibrillin-1 and does not cleave either. It colocalizes to fibrillin-1 containing microfibrils in cultured fibroblasts and suppresses fibrillin-2 (FBN2) incorporation in microfibrils, in part by transcriptional downregulation of Fbn2 mRNA expression. RNA in situ hybridization detected Adamts17 expression in specific structures in the eye, skeleton and other organs, where it may regulate the fibrillin isoform composition of microfibrils.

GPR81 is a receptor for the metabolic intermediate lactate with an established role in regulating adipocyte lipolysis. Potentially novel GPR81 agonists were identified that suppressed fasting plasma free fatty acid levels in rodents and in addition improved insulin sensitivity in mouse models of insulin resistance and diabetes. Unexpectedly, the agonists simultaneously induced hypertension in rodents, including wild-type, but not GPR81-deficient mice. Detailed cardiovascular studies in anesthetized dogs showed that the pressor effect was associated with heterogenous effects on vascular resistance among the measured tissues: increasing in the kidney while remaining unchanged in hindlimb and heart. Studies in rats revealed that the pressor effect could be blocked, and the renal resistance effect at least partially blocked, with pharmacological antagonism of endothelin receptors. In situ hybridization localized GPR81 to the microcirculation, notably afferent arterioles of the kidney. In conclusion, these results provide evidence for a potentially novel role of GPR81 agonism in blood pressure control and regulation of renal vascular resistance including modulation of a known vasoeffector mechanism, the endothelin system. In addition, support is provided for the concept of fatty acid lowering as a means of improving insulin sensitivity.

Hey2 gene mutations in both humans and mice have been associated with multiple cardiac defects. However, the currently reported localization of Hey2 in the ventricular compact zone cannot explain the wide variety of cardiac defects. Furthermore, it was reported that, in contrast to other organs, Notch doesn't regulate Hey2 in the heart. To determine the expression pattern and the regulation of Hey2, we used novel methods including RNAscope and a Hey2 CreERT2 knockin line to precisely determine the spatiotemporal expression pattern and level of Hey2 during cardiac development. We found that Hey2 is expressed in the endocardial cells of the atrioventricular canal and the outflow tract, as well as at the base of trabeculae, in addition to the reported expression in the ventricular compact myocardium. By disrupting several signaling pathways that regulate trabeculation and/or compaction, we found that, in contrast to previous reports, Notch signaling and Nrg1/ErbB2 regulate Hey2 expression level in myocardium and/or endocardium, but not its expression pattern: weak expression in trabecular myocardium and strong expression in compact myocardium. Instead, we found that FGF signaling regulates the expression pattern of Hey2 in the early myocardium, and regulates the expression level of Hey2 in a Notch1 dependent manner.

Transdifferentiation of hypertrophic chondrocytes into bone-forming osteoblasts has been reported, yet the underlying molecular mechanism remains incompletely understood. SHP2 is an ubiquitously expressed cytoplasmic protein tyrosine phosphatase. SHP2 loss-of-function mutations in chondroid cells are linked to metachondromatosis in humans and mice, suggesting a crucial role for SHP2 in the skeleton. However, the specific role of SHP2 in skeletal cells has not been elucidated. To approach this question, we ablated SHP2 in collagen 2α1(Col2α1)-Cre- and collagen 10α1(Col10α1)-Cre-expressing cells, predominantly proliferating and hypertrophic chondrocytes, using "Cre-loxP"-mediated gene excision. Mice lacking SHP2 in Col2α1-Cre-expressing cells die at mid-gestation. Postnatal SHP2 ablation in the same cell population caused dwarfism, chondrodysplasia and exostoses. In contrast, mice in which SHP2 was ablated in the Col10α1-Cre-expressing cells appeared normal but were osteopenic. Further mechanistic studies revealed that SHP2 exerted its influence partly by regulating the abundance of SOX9 in chondrocytes. Elevated and sustained SOX9 in SHP2-deficient hypertrophic chondrocytes impaired their differentiation to osteoblasts and impaired endochondral ossification. Our study uncovered an important role of SHP2 in bone development and cartilage homeostasis by influencing the osteogenic differentiation of hypertrophic chondrocytes and provided insight into the pathogenesis and potential treatment of skeletal diseases, such as osteopenia and osteoporosis.

The pituitary gonadotrophins and testosterone are the main hormonal regulators of spermatogenesis, but estradiol is also known to play a role in the process. The hormonal responses in the testis are partially mediated by somatic Sertoli cells that provide nutritional and physical support for differentiating male germ cells. Hydroxysteroid (17β) dehydrogenase 1 (HSD17B1) is a steroidogenic enzyme that especially catalyzes the conversion of low potent 17keto-steroids to highly potent 17beta-hydroxysteroids. In this study, we show that Hsd17b1 is highly expressed in Sertoli cells of fetal and newborn mice, and HSD17B1 knockout males present with disrupted spermatogenesis with major defects, particularly in the head shape of elongating spermatids. The cell–cell junctions between Sertoli cells and germ cells were disrupted in the HSD17B1 knockout mice. This resulted in complications in the orientation of elongating spermatids in the seminiferous epithelium, reduced sperm production, and morphologically abnormal spermatozoa. We also showed that the Sertoli cell–expressed HSD17B1 participates in testicular steroid synthesis, evidenced by a compensatory up-regulation of HSD17B3 in Leydig cells. These results revealed a novel role for HSD17B1 in the control of spermatogenesis and male fertility, and that Sertoli cells significantly contribute to steroid synthesis in the testis.

Colorectal traditional serrated adenomas (TSAs) are often associated with precursor polyps, including hyperplastic polyps and sessile serrated adenoma/polyps. To elucidate the molecular mechanisms involved in the progression from precursor polyps to TSAs, the present study analyzed 15 precursor polyp-associated TSAs harboring WNT pathway gene mutations. Laser microdissection-based sequencing analysis showed that BRAF or KRAS mutations were shared between TSA and precursor polyps in all lesions. In contrast, the statuses of WNT pathway gene mutations were different between the 2 components. In 8 lesions, RNF43, APC, or CTNNB1 mutations, were exclusively present in TSA. RNF43 mutations were shared between the TSA and precursor components in 3 lesions; however, they were heterozygous in the precursor polyps whereas homozygous in the TSA. In 4 lesions with PTPRK-RSPO3 fusions, RNA in situ hybridization demonstrated that overexpression of RSPO3, reflecting PTPRK-RSPO3 fusion transcripts, was restricted to TSA components. Consistent with the results of the genetic and in situ hybridization analyses, nuclear β-catenin accumulation and MYC overexpression were restricted to the TSA component in 13 and 12 lesions, respectively. These findings indicate that the WNT pathway gene alterations are acquired during the progression from the precursor polyps to TSAs and that the activation of the WNT pathway plays a critical role in the development of TSA rather than their progression to high-grade lesions.

BACKGROUND:

Visceral hypersensitivity, an important cause of abdominal pain in disorders such as IBD and IBS, presents with a poorly understood pathophysiology and limited treatment options. Several members of the Mas-related G protein-coupled receptor family (Mrgprs) have become promising targets in pain research. The potential link between the murine Mrgpr C11 (Mrgprc11) and gut nociception is currently uninvestigated. Therefore, we explored the expression and functional role of Mrgprc11 in the gut nociceptive innervation.

METHODS:

Mrgprc11 expression was evaluated in DRG neurons innervating the mouse colon using in situ hybridization and immunohistochemistry. Visceromotor responses to colorectal distension (CRD) assessed the effect of the Mrgprc11 agonist, BAM(8-22), on colonic pain sensitivity in healthy mice. Moreover, we determined pERK1/2-immunoreactivity in the thoracolumbar spinal cord after noxious CRD. Finally, from a translational point of view, we looked for expression of the human counterpart of Mrgprc11, MRGPRX1, in human thoracolumbar DRGs.

KEY RESULTS:

In situ hybridization and immunohistochemistry revealed Mrgprc11 expression in colonic DRG neurons. Intracolonic administration of BAM(8-22) significantly increased colonic pain sensitivity in an Mrgprc11-dependent manner, and led to a significantly increased degree of neuronal activation in the splanchnic spinal cord upon noxious stimulation. Furthermore, MRGPRX1 expression was also detected in human thoracolumbar DRG neurons. CONCLUSIONS & INFERENCES: Our findings established a novel function for Mrgprc11 in the gut nociceptive innervation and propose the receptor as a new player in visceral hypersensitivity. Given the presence of MRGPRX1 in human DRG neurons, our study warrants future research on its therapeutic potential in abdominal pain disorders.

Apelin binds to the G protein-coupled apelin receptor (APJ; gene name aplnr) to modulate diverse physiological systems including cardiovascular function, and hydromineral and metabolic balance. Recently a second endogenous ligand for APJ, named apela, has been discovered. We confirm that apela activates signal transduction pathways (ERK activation) in cells expressing the cloned rat APJ. Previous studies suggest that exogenous apela is diuretic, attributable wholly or in part to an action on renal APJ. Thus far the cellular distribution of apela in the kidney has not been reported. We have utilized in situ hybridization histochemistry to reveal strong apela labelling in the inner medulla (IM), with lower levels observed in the inner stripe of the outer medulla (ISOM), of rat and mouse kidneys. This contrasts with renal aplnr expression where the converse is apparent, with intense labelling in the ISOM (consistent with vasa recta labelling) and low-moderate hybridization in the IM, in addition to labelling of glomeruli. Apelin is found in sparsely distributed cells amongst more prevalent aplnr-labelled cells in extra-tubular regions of the medulla. This expression profile is supported by RNA-Seq data that shows that apela, but not apelin or aplnr, is highly expressed in microdissected rat kidney tubules. If endogenous tubular apela promotes diuresis in the kidney it could conceivably do this by interacting with APJ in vasculature, or via an unknown receptor in the tubules. The comparative distribution of apela, apelin and aplnr in the rodent kidney lays the foundation for future work on how the renal apelinergic system interacts.

We and others have reported that taste cells in taste buds express many peptides in common with cells in the gut and islets of Langerhans in the pancreas. Islets and taste bud cells express the hormones glucagon and ghrelin, the same ATP-sensitive potassium channel (KATP) responsible for depolarizing the insulin secreting beta (β) cell during glucose-induced insulin secretion, as well as the propeptide processing enzymes PC1/3 and PC2. Given the common expression of functionally specific proteins in taste buds and islets, it is surprising that no one has investigated whether insulin is synthesized in taste bud cells until now. Using immunofluorescence, we demonstrate the presence of insulin in mouse, rat and human taste bud cells. We further prove that insulin is synthesized in individual taste buds and not taken up from the parenchyma by: detection of the post-processing insulin molecule C-peptide and green fluorescence protein (GFP) in taste cells of both insulin 1- and insulin 2-GFP mice, and the presence of the mouse insulin transcript by in situ hybridization (ISH). In addition to our cytology data we measured the level of insulin transcript by qRT-PCR in the anterior and posterior lingual epithelium. These analyses show insulin is translated in the circumvallate and foliate papillae in the posterior but only insulin transcript was detected in the anterior fungiform papillae of rodent tongue. Thus, some taste cells are insulin synthesizing cells generated from a continually replenished source of precursor cells in adult mammalian lingual epithelium.