Vitamin D3 Promotes Not Motor Coordination but Motor Skill Learning without Influence on Muscle Function.

Although motor coordination or motor skill learning are improved by taking vitamin D in the animal experiment, muscle function have not been estimated. Here we examined the effect of vitamin D3 administration on motor coordination and motor skill learning, muscle strength, and muscle volume in mice fed a vitamin D deficient diet. In mice fed a vitamin D deficient diet, serum calcium and 25(OH)D3 concentrations were measured. We then conducted Rotarod test, beam walking assay, micro-CT analysis, and forelimb grip strength test. Administration of vitamin D3 elongated the retention time in the Rotarod test in a time dependent manner. In contrast, the time to reach a beam goal box in beam walking assay was not changed in mice administered with vitamin D3, compared to the control. Oral administration of vitamin D3 did not affect muscle strength nor muscle volume. Oral administration of vitamin D3 promotes not motor coordination but motor skill learning and does not affect muscle function.

Ngn3-Positive Cells Arise from Pancreatic Duct Cells.

The production of pancreatic ß cells is the most challenging step for curing diabetes using next-generation treatments. Adult pancreatic endocrine cells are thought to be maintained by the self-duplication of differentiated cells, and pancreatic endocrine neogenesis can only be observed when the tissue is severely damaged. Experimentally, this can be performed using a method named partial duct ligation (PDL). As the success rate of PDL surgery is low because of difficulties in identifying the pancreatic duct, we previously proposed a method for fluorescently labeling the duct in live animals. Using this method, we performed PDL on neurogenin3 (Ngn3)-GFP transgenic mice to determine the origin of endocrine precursor cells and evaluate their potential to differentiate into multiple cell types. Ngn3-activated cells, which were marked with GFP, appeared after PDL operation. Because some GFP-positive cells were aligned proximally to the duct, we hypothesized that Ngn3-positive cells arise from the pancreatic duct. Therefore, we next developed an in vitro pancreatic duct culture system using Ngn3-GFP mice and examined whether Ngn3-positive cells emerge from this duct. We observed GFP expressions in ductal organoid cultures. GFP expressions were correlated with Ngn3 expressions and endocrine cell lineage markers. Interestingly, tuft cell markers were also correlated with GFP expressions. Our results demonstrate that in adult mice, Ngn3-positive endocrine precursor cells arise from the pancreatic ducts both in vivo and in vitro experiments indicating that the pancreatic duct could be a potential donor for therapeutic use.

CD82 is a marker to isolate ß cell precursors from human iPS cells and plays a role for the maturation of ß cells.

Generation of pancreatic ß cells from pluripotent stem cells is a key technology to develop cell therapy for insulin-dependent diabetes and considerable efforts have been made to produce ß cells. However, due to multiple and lengthy differentiation steps, production of ß cells is often unstable. It is also desirable to eliminate undifferentiated cells to avoid potential risks of tumorigenesis. To isolate ß cell precursors from late stage pancreatic endocrine progenitor (EP) cells derived from iPS cells, we have identified CD82, a member of the tetraspanin family. CD82+ cells at the EP stage differentiated into endocrine cells more efficiently than CD82- EP stage cells. We also show that CD82+ cells in human islets secreted insulin more efficiently than CD82- cells. Furthermore, knockdown of CD82 expression by siRNA or inhibition of CD82 by monoclonal antibodies in NGN3+ cells suppressed the function of ß cells with glucose-stimulated insulin secretion, suggesting that CD82 plays a role in maturation of EP cells to ß cells.

Bone phenotype in melanocortin 2 receptor-deficient mice.

Considering that stress condition associated with osteoporosis, the hypothalamic-pituitary-adrenal (HPA) axis, which is essential for central stress response system, is implicated in regulating bone mass accrual. Melanocortin 2 receptor (MC2R), the receptor of adrenocorticotropic hormone is expressed in both adrenal gland cells and bone cells. To elucidate the role of HPA axis in bone metabolism, we assessed the skeletal phenotype of MC2R deficient mice (MC2R -/- mice). We first examined bone mineral density and cortical thickness of femur using dual x-ray absorptiometry and micro-computed tomography. We then conducted histomorphometric analysis to calculate the static and dynamic parameters of vertebrae in MC2R -/- mice. The levels of osteoblastic marker genes were examined by quantitative PCR in primary osteoblasts derived from MC2R -/- mice. Based on these observations, bone mineral density of femur in MC2R -/- mice was increasing relative to litter controls. Meanwhile, the thickness of cortical bone of femur in MC2R -/- mice was remarkably elevated. Moreover, serum osteocalcin level was drastically raised in MC2R -/- mice. However, bone histomorphometry revealed that static and dynamic parameters reflecting bone formation and resorption were unchanged in vertebrae of MC2R -/- mice compared to the control, indicating that MC2R function may be specific to appendicular bone than axis bone. Taken together, the HPA axis due to deletion of MC2R is involved in bone metabolism.

Reversible expansion of pancreatic islet progenitors derived from human induced pluripotent stem cells.

Transplantation of pancreatic islets is an effective therapy for severe type 1 diabetes. As donor shortage is a major problem for this therapy, attempts have been made to produce a large number of pancreatic islets from human pluripotent stem cells (hPSCs). However, as the differentiation of hPSCs to pancreatic islets requires multiple and lengthy processes using various expensive cytokines, the process is variable, low efficiency and costly. Therefore, it would be beneficial if islet progenitors could be expanded. Neurogenin3 (NGN3)-expressing pancreatic endocrine progenitor (EP) cells derived from hPSCs exhibited the ability to differentiate into pancreatic islets while their cell cycle was arrested. By using a lentivirus vector, we introduced several growth-promoting genes into NGN3-expressing EP cells. We found that SV40LT expression induced proliferation of the EP cells but reduced the expression of endocrine lineage-commitment factors, NGN3, NEUROD1 and NKX2.2, resulting in the suppression of islet differentiation. By using the Cre-loxP system, we removed SV40LT after the expansion, leading to re-expression of endocrine-lineage commitment genes and differentiation into functional pancreatic islets. Thus, our findings will pave a way to generate a large quantity of functional pancreatic islets through the expansion of EP cells from hPSCs.

Comparative analysis of enteroendocrine cells and their hormones between mouse intestinal organoids and native tissues.

Endocrine cells in the gastrointestinal tract secrete multiple hormones to maintain homeostasis in the body. In the present study, we generated intestinal organoids from the duodenum, jejunum, and ileum of Neurogenin 3 (Ngn3)-EGFP mice and examined how enteroendocrine cells (EECs) within organoid cultures resemble native epithelial cells in the gut. Transcriptome analysis of EGFP-positive cells from Ngn3-EGFP organoids showed gene expression pattern comparable to EECs in vivo. We also compared mRNAs of five major hormones, namely, ghrelin (Ghrl), cholecystokinin (Cck), Gip, secretin (Sct), and glucagon (Gcg) in organoids and small intestine along the longitudinal axis and found that expression patterns of these hormones in organoids were similar to those in native tissues. These findings suggest that an intestinal organoid culture system can be utilized as a suitable model to study enteroendocrine cell functions in vitro.

Evolutionary History of GLIS Genes Illuminates Their Roles in Cell Reprograming and Ciliogenesis.

The GLIS family transcription factors, GLIS1 and GLIS3, potentiate generation of induced pluripotent stem cells (iPSCs). In contrast, another GLIS family member, GLIS2, suppresses cell reprograming. To understand how these disparate roles arose, we examined evolutionary origins and genomic organization of GLIS genes. Comprehensive phylogenetic analysis shows that GLIS1 and GLIS3 originated during vertebrate whole genome duplication, whereas GLIS2 is a sister group to the GLIS1/3 and GLI families. This result is consistent with their opposing functions in cell reprograming. Glis1 evolved faster than Glis3, losing many protein-interacting motifs. This suggests that Glis1 acquired new functions under weakened evolutionary constraints. In fact, GLIS1 induces induced pluripotent stem cells more strongly. Transcriptomic data from various animal embryos demonstrate that glis1 is maternally expressed in some tetrapods, whereas vertebrate glis3 and invertebrate glis1/3 genes are rarely expressed in oocytes, suggesting that vertebrate (or tetrapod) Glis1 acquired a new expression domain and function as a maternal factor. Furthermore, comparative genomic analysis reveals that glis1/3 is part of a bilaterian-specific gene cluster, together with rfx3, ndc1, hspb11, and lrrc42. Because known functions of these genes are related to cilia formation and function, the last common ancestor of bilaterians may have acquired this cluster by shuffling gene order to establish more sophisticated epithelial tissues involving cilia. This evolutionary study highlights the significance of GLIS1/3 for cell reprograming, development, and diseases in ciliated organs such as lung, kidney, and pancreas.

Mobility and Biomechanical Functions in the Aging Male: Testosterone and the Locomotive Syndrome.

In the current aging society, the occurrence of the locomotive syndrome, a condition in which the locomotive organs are impaired, is increasing. The locomotive system includes support (bones), mobility and impact absorption (joints and intervertebral disks), drive and control (muscles, nerves), and network (blood vessels). The impairment of any of those systems can lead to a major decrease in quality of life. In recent years, several studies on methods to improve and prevent conditions impairing the locomotive syndrome have been conducted. Almost in parallel with the structure supporting mobility and body functions, testosterone levels decrease with age. Testosterone is a hormone-regulating several pathways affecting each aspect of the locomotive syndrome. Testosterone is regulated by the pituitary gland triggering several processes in the body through genomic and non-genomic pathways, affecting muscles, bones, nerves, joints, intervertebral discs, and blood vessels. The purpose of this review is to investigate the role of testosterone in each of the systems involved in the locomotive syndrome.

Anabolic-androgenic steroids: procurement and administration practices of doping athletes.

Performance enhancing substances are becoming increasingly popular amongst bodybuilders and people who want to enhance their physiques. However, due to the rise of the Internet and laws prohibiting sales of these substances without prescription, the route of procurement and administration practices have become more and more dangerous. Prior to the mid-1970's, anabolic steroids were not regulated and easily available from physicians and pharmacies in several countries. In 1990, the United States enacted the Anabolic Steroid Control Act, leading to the proliferation of black markets and underground laboratories. The shift from pharmacy to underground online sites for the procurement of anabolic steroids led to an increase of fake products with low purity and the ability to potentially endanger the health of anabolic steroid users. Underground laboratories emerged both locally and in countries with lax legal regulations. 'Anabolic steroid tourism' and large networks of online resellers emerged, leading to the banalisation of the illegal procurement of anabolic steroids. Furthermore, the increase of anecdotal information spreading on the internet among anabolic steroid user forums nourishes the rampant misinformation and dangerous practices that currently exist. The dosages and ways of administration recommended on these forums can be false and misleading to those who lack a medical background and cannot go to their physician to seek advice because of the fear of repercussions. This review aims to elucidate and describe current practices of the anabolic-androgenic steroids black market and draw attention to potential dangers for users.

Potential application of testosterone replacement therapy as treatment for obesity and type 2 diabetes in men.

Sedentary lifestyle and over-nutrition are the main causes of obesity and type 2 diabetes (T2D). However, the same causes are major triggers of hypogonadism. Many T2D patients show low testosterone levels while hypogonadal men seem to be prone to become diabetic. Testosterone plays a major role in the regulation of muscle mass, adipose tissue, inflammation and insulin sensitivity and is therefore indirectly regulating several metabolic pathways, while T2D is commonly triggered by insulin resistance, increased adipose tissue and inflammation, showing a negative correlation between testosterone levels and T2D. Testosterone replacement therapy (TRT) is widely used in patients with symptoms of hypogonadism, however it is not commonly used as preventive intervention or treatment for T2D patients even though hypogonadal patients share many common symptoms (obesity, insulin insensitivity, increased inflammation, decrease in muscle mass and strength) with T2D patients. Even though TRT is often associated with side effects such as prostatic hypertrophy or cancer, cardiovascular risks due to increase in the number of red blood cells and infertility, several studies have shown that TRT remains a potent intervention improving metabolic functions such as glycated haemoglobin, blood sugar, total cholesterol and visceral fat. The purpose of this review is to discuss the possible benefits and risks of TRT in the prevention and treatment of obesity and T2D and assess the health risks and benefits of common T2D medications and testosterone.

A Method for Identifying Mouse Pancreatic Ducts.

Proper identification of pancreatic ducts is a major challenge for researchers performing partial duct ligation (PDL), because pancreatic ducts, which are covered with acinar cells, are translucent and thin. Although damage to pancreatic ducts may activate quiescent ductal stem cells, which may allow further investigation into ductal stem cells for therapeutic use, there is a lack of effective techniques to visualize pancreatic ducts. In this study, we report a new method for identifying pancreatic ducts. First, we aimed to visualize pancreatic ducts using black, waterproof fountain pen ink. We injected the ink into pancreatic ducts through the bile duct. The flow of ink was observed in pancreatic ducts, revealing their precise architecture. Next, to visualize pancreatic ducts in live animals, we injected fluorescein-labeled bile acid, cholyl-lysyl-fluorescein into the mouse tail vein. The fluorescent probe clearly marked not only the bile duct but also pancreatic ducts when observed with a fluorescent microscope. To confirm whether the pancreatic duct labeling was successful, we performed PDL on Neurogenin3 (Ngn3)-GFP transgenic mice. As a result, acinar tissue is lost. PDL tail pancreas becomes translucent almost completely devoid of acinar cells. Furthermore, strong activation of Ngn3 expression was observed in the ligated part of the adult mouse pancreas at 7 days after PDL.

Expansion of transplanted islets in mice by co-transplantation with adipose tissue-derived mesenchymal stem cells.

The shortage of donor islets is a significant obstacle for widespread clinical application of pancreatic islet transplantation. To investigate whether adipose tissue-derived mesenchymal stem cells (ADSCs) induce expansion of transplanted islets, we performed co-transplantation experiments in a mouse model. Streptozotosin (STZ)-induced diabetic mice transplanted with 50 syngeneic islets remained hyperglycemic. However, hyperglycemia was ameliorated gradually when 50 islets were co-transplanted with ADSCs but not separately grafted into the contralateral kidney. Insulin and proinsulin contents of 120-day grafts containing 50 islets co-transplanted with ADSCs were significantly increased compared with those of 50 isolated islets. The Ki67-positive ratios in islets of the naïve pancreas, at 30 and 120 days grafts were 0.23%, 2.12%, and 1.52%, respectively. Ki67-positive cells were predominantly Pdx1+ and insulin+ cells. These results demonstrate that co-transplantation with ADSCs induces proliferation of transplanted islets in mice, suggesting a potential solution for the low efficiency of islet transplantation.

Identification of novel splicing variants of protein tyrosine phosphatase receptor type Z.

Protein tyrosine phosphatase receptor type Z (PTPRZ, also known as PTPζ or RPTPß) is preferentially expressed in the central nervous system (CNS). PTPRZ plays important roles during development and adulthood in CNS myelination, learning and memory. Three splicing isoforms for PTPRZ have been identified to date: two receptor type isoforms, PTPRZ-A and PTPRZ-B, and one secretory isoform, PTPRZ-S. We herein identified novel PTPRZ receptor sub-isoforms without a seven-amino acid sequence encoded by exon 16. This sequence forms a part of the helix-turn-helix segment called the 'wedge' structure, which is located at the N-terminal region in the membrane-proximal protein tyrosine phosphatase domain. In contrast to conventional receptor isoforms with uniform expression, the deleted isoforms were expressed in the brain, but not in the retina, indicating the tissue-specific splicing of exon 16. Biochemical analyses of PTPRZ intracellular regions revealed differences in the characteristics of the deleted form, namely, stronger binding activity to postsynaptic density protein 95 (PSD95) and greater enrichment in the postsynaptic density fraction than the full-length form. Furthermore, the exon 16-deleted form exhibited higher catalytic efficiency in vitro. These results suggest that sub-isoforms of PTPRZ have different functions because of variations in the wedge structure.

Netrin-4 Promotes Differentiation and Migration of Osteoblasts.

BACKGROUND/AIM: While netrin-4 plays a vital role in the vascular system, the role of netrin-1 in osteoblast differentiation is not well understood. In this study we explored whether netrin-4 has functional roles in osteoblasts. MATERIALS AND METHODS: Quantitative reverse-transcriptase polymerase chain reaction (PCR), RNA interference, the generation of plasmids, transfections, measurement of alkaline phosphatase activity, a mineralization assay, a migration assay and a cell proliferation assay were performed. RESULTS: Netrin-4 expression was up-regulated during osteoblast differentiation and an RNA interference experiment showed that small interfering RNA used to silence netrin-4 inhibited osteoblast differentiation. Recombinant mouse netrin-4 promoted alkaline phosphatase (ALP) activity of osteoblasts and enhancement of calcium deposits. Moreover, we constructed a vector containing the netrin-4 gene on the basis of the plasmid pcDNA3.1/V5-His. Overexpression of netrin-4 enhanced differentiation of osteoblasts. Finally, recombinant mouse netrin-4 promoted cell migration of osteoblasts. CONCLUSION: Netrin-4 promotes differentiation and migration of osteoblasts.

Targeting PTPRZ inhibits stem cell-like properties and tumorigenicity in glioblastoma cells.

The R5 subfamily of receptor-type protein tyrosine phosphatases (RPTPs) comprises PTPRZ and PTPRG. A recent study on primary human glioblastomas suggested a close association between PTPRZ1 (human PTPRZ) expression and cancer stemness. However, the functional roles of PTPRZ activity in glioma stem cells have remained unclear. In the present study, we found that sphere-forming cells from the rat C6 and human U251 glioblastoma cell lines showed high expression levels of PTPRZ-B, the short receptor isoform of PTPRZ. Stable PTPRZ knockdown altered the expression levels of stem cell transcription factors such as SOX2, OLIG2, and POU3F2 and decreased the sphere-forming abilities of these cells. Suppressive effects on the cancer stem-like properties of the cells were also observed following the knockdown of PTPRG. Here, we identified NAZ2329, a cell-permeable small molecule that allosterically inhibits both PTPRZ and PTPRG. NAZ2329 reduced the expression of SOX2 in C6 and U251 cells and abrogated the sphere-forming abilities of these cells. Tumor growth in the C6 xenograft mouse model was significantly slower with the co-treatment of NAZ2329 with temozolomide, an alkylating agent, than with the individual treatments. These results indicate that pharmacological inhibition of R5 RPTPs is a promising strategy for the treatment of malignant gliomas.

Functional Roles of Netrin-1 in Osteoblast Differentiation.

AIM: Recent studies have demonstrated that netrin-1 plays a vital role in bone metabolism. Previous studies have shown that osteoblasts produce netrin-1 which affects osteoclast differentiation. However, the role of netrin-1 in osteoblast differentiation is not well understood. In this study, we explored the roles of netrin-1 in osteoblasts. MATERIALS AND METHODS: Quantitative reverse-transcriptase polymerase chain reaction (qPCR), RNA interference for netrin receptors, the generation of netrin-1 plasmid, transfection of plasmids, and cell proliferation assay were performed. RESULTS: During osteoblast differentiation by ascorbic acid, netrin-1 expression was significantly decreased. Gene expression related with osteoblast differentiation was down-regulated by netrin-1 treatment. We also found that osteoblast differentiation by bone morphogenetic protein-4 (BMP-4) was inhibited in the presence of recombinant netrin-1. Forced expression of both BMP-4 and netrin-1 significantly decreased alkaline phosphatase expression. On the other hand, Unc5b, neogenin, and A2b which belong to netrin receptors were expressed by osteoblasts. Moreover, alkaline phosphatase expression was significantly decreased by knockdown for the combination of two receptors among these receptors. CONCLUSION: Netrin-1 is involved in the regulation of osteoblast differentiation.

Differentiation of Islet Progenitors Regulated by Nicotinamide into Transcriptome-Verified ß Cells That Ameliorate Diabetes.

Developmental stage-specific differentiation of stem or progenitor cells into safe and functional cells is of fundamental importance in regenerative medicine, including ß-cell replacement. However, the differentiation of islet progenitor cells (IPCs) into insulin-secreting ß cells remains elusive. Here, we report that the multifunctional molecule nicotinamide (NIC) is a specific differentiation regulator of mouse IPCs. The differentiated cells regulated by NIC exhibited many characteristics of adult ß cells, including ameliorating preclinical diabetes and a highly comparable transcriptome profile. Gene set enrichment analysis showed that during differentiation, numerous IPC transcription factor genes, including Ngn3, Pax4, Fev, and Mycl1, were all down regulated. Pharmacological, biochemical, and gene knockdown analyses collectively demonstrated that NIC regulated the differentiation via inhibiting Sirt1 (silent information regulator transcript 1). Finally, NIC also regulates human IPC differentiation. Thus, our study advances islet developmental biology and impacts on translational research and regenerative therapies to diabetes and other diseases. Stem Cells 2017;35:1341-1354.

Adipsic hypernatremia without hypothalamic lesions accompanied by autoantibodies to subfornical organ.

Adipsic (or essential) hypernatremia is a rare hypernatremia caused by a deficiency in thirst regulation and vasopressin release. In 2010, we reported a case in which autoantibodies targeting the sensory circumventricular organs (sCVOs) caused adipsic hypernatremia without hypothalamic structural lesions demonstrable by magnetic resonance imaging (MRI); sCVOs include the subfornical organ (SFO) and organum vasculosum of the lamina terminalis (OVLT), which are centers for the monitoring of body-fluid conditions and the control of water and salt intakes, and harbor neurons innervating hypothalamic nuclei for vasopressin release. We herein report three newly identified patients (3- to 8-year-old girls on the first visit) with similar symptoms. The common features of the patients were extensive hypernatremia without any sensation of thirst and defects in vasopressin response to serum hypertonicity. Despite these features, we could not detect any hypothalamic structural lesions by MRI. Immunohistochemical analyses using the sera of the three patients revealed that antibodies specifically reactive to the mouse SFO were present in the sera of all cases; in one case, the antibodies also reacted with the mouse OVLT. The immunoglobulin (Ig) fraction of serum obtained from one patient was intravenously injected into wild-type mice to determine whether the mice developed similar symptoms. Mice injected with a patient's Ig showed abnormalities in water/salt intake, vasopressin release, and diuresis, which resultantly developed hypernatremia. Prominent cell death and infiltration of reactive microglia was observed in the SFO of these mice. Thus, autoimmune destruction of the SFO may be the cause of the adipsic hypernatremia. This study provides a possible explanation for the pathogenesis of adipsic hypernatremia without demonstrable hypothalamus-pituitary lesions.

An inhibitor of fibroblast growth factor receptor-1 (FGFR1) promotes late-stage terminal differentiation from NGN3+ pancreatic endocrine progenitors.

Human induced pluripotent stem cells (hiPSCs) provide a potential resource for regenerative medicine. To identify the signalling pathway(s) contributing to the development of functional ß cells, we established a tracing model consisting of dual knock-in hiPSCs (INS-Venus/NGN3-mCherry) (hIveNry) expressing the fluorescent proteins Venus and mCherry under the control of intrinsic insulin (INS) and neurogenin 3 (NGN3) promoters, respectively. hIveNry iPSCs differentiated into NGN3- and mCherry-positive endocrine progenitors and then into Venus-positive ß cells expressing INS, PDX1, NKX6.1, and glucokinase (GCK). Using these cells, we conducted high-throughput screening of chemicals and identified a specific kinase inhibitor of fibroblast growth factor receptor 1 (FGFR1) that acted in a stage-dependent manner to promote the terminal differentiation of pancreatic endocrine cells, including ß cells, from the intermediate stage of pancreatic endocrine progenitors while blocking the early development of pancreatic progenitors. This FGFR1 inhibitor augmented the expression of functional ß cell markers (SLC30A8 and ABCC8) and improved glucose-stimulated INS secretion. Our findings indicate that the hIveNry model could provide further insights into the mechanisms of hiPS-derived ß cell differentiation controlled by FGFR1-mediated regulatory pathways in a temporal-dependent fashion.