Lineage Tracing of RGS5-CreER-Labeled Cells in Long Bones During Homeostasis and Injury.

Regulator of G protein signaling 5 (RGS5) is a GTPase activator for heterotrimeric G-protein α-subunits, shown to be a marker of pericytes. Bone marrow stromal cell population (BMSCs) is heterogeneous. Populations of mesenchymal progenitors, cells supportive of hematopoiesis, and stromal cells regulating bone remodeling have been recently identified. Periosteal and bone marrow mesenchymal stem cells (MSCs) are participating in fracture healing, but it is difficult to distinguish the source of cells within the callus. Considering that perivascular cells exert osteoprogenitor potential, we generated an RGS5 transgenic mouse model (Rgs5-CreER) which when crossed with Ai9 reporter animals (Rgs5/Tomato), is suitable for lineage tracing during growth and post-injury. Flow cytometry analysis and histology confirmed the presence of Rgs5/Tomato+ cells within CD31+ endothelial, CD45+ hematopoietic, and CD31-CD45- mesenchymal/perivascular cells. A tamoxifen chase showed expansion of Rgs5/Tomato+ cells expressing osterix within the trabeculae positioned between mineralized matrix and vasculature. Long-term chase showed proportion of Rgs5/Tomato+ cells contributes to mature osteoblasts expressing osteocalcin. Following femoral fracture, Rgs5/Tomato+ cells are observed around newly formed bone within the BM cavity and expressed osterix and osteocalcin, while contribution within periosteum was low and limited to fibroblastic callus with very few positive chondrocytes. In addition, BM injury model confirmed that RGS5-Cre labels population of BMSCs expands during injury and participates in osteogenesis. Under homeostatic conditions, lineage-traced RGS5 cells within the trabecular area demonstrate osteoprogenitor capacity that in an injury model contributes to new bone formation primarily within the BM niche.

Endometrial Glucose Transporters in Health and Disease.

Pregnancy loss is a frequent occurrence during the peri-implantation period, when there is high glucose demand for embryonic development and endometrial decidualization. Glucose is among the most essential uterine fluid components required for those processes. Numerous studies associate abnormal glucose metabolism in the endometrium with a higher risk of adverse pregnancy outcomes. The endometrium is incapable of synthesizing glucose, which thus must be delivered into the uterine lumen by glucose transporters (GLUTs) and/or the sodium-dependent glucose transporter 1 (SGLT1). Among the 26 glucose transporters (14 GLUTs and 12 SGLTs) described, 10 (9 GLUTs and SGLT1) are expressed in rodents and 8 (7 GLUTs and SGLT1) in the human uterus. This review summarizes present knowledge on the most studied glucose transporters in the uterine endometrium (GLUT1, GLUT3, GLUT4, and GLUT8), whose data regarding function and regulation are still lacking. We present the recently discovered SGLT1 in the mouse and human endometrium, responsible for controlling glycogen accumulation essential for embryo implantation. Moreover, we describe the epigenetic regulation of endometrial GLUTs, as well as signaling pathways included in uterine GLUT's expression. Further investigation of the GLUTs function in different endometrial cells is of high importance, as numerous glucose transporters are associated with infertility, polycystic ovary syndrome, and gestational diabetes.

Divergent effects of peripheral and global neuropeptide Y deletion.

OBJECTIVES: Neuropeptide Y (NPY) is involved in the coordination of bone mass and adiposity. However, multiple NPY sources exist and their individual contribution to the skeleton and adiposity not known. The objectives of our study were to evaluate the effects of peripheral mesenchymal derived NPY to the skeleton and adiposity and to compare them to the global NPYKO model. METHODS: To study the role of mesenchymal-derived NPY, we crossed conditional NPY (NPYfl/fl) mice with Prx1cre to generate PrxNPYKO mice. The bone phenotype was assessed using micro-CT. The skeletal phenotype of PrxNPYKO mice was subsequently compared to global NPYKO model. We evaluated body weight, adiposity and functionally assessed the feeding response of NPY neurons to determine whether central NPY signaling was altered by Prx1cre. RESULTS: We identified the increase in cortical parameters in PrxNPYKO mice with no changes to cancellous bone. This was the opposite phenotype to global NPYKO mice generated from the same conditional allele. Male NPYKOmice have increased adiposity, while PrxNPYKO mice showed no difference, demonstrating that local mesenchymal-derived NPY does not influence adiposity. CONCLUSION: NPY mediates both positive and negative effects on bone mass via separate regulatory pathways. Deletion of mesenchymal-derived NPY had a positive effect on bone mass.

Antidiabetic Effects of a Tripeptide That Decreases Abundance of Na+-d-glucose Cotransporter SGLT1 in the Brush-Border Membrane of the Small Intestine.

In enterocytes, protein RS1 (RSC1A1) mediates an increase of glucose absorption after ingestion of glucose-rich food via upregulation of Na+-d-glucose cotransporter SGLT1 in the brush-border membrane (BBM). Whereas RS1 decelerates the exocytotic pathway of vesicles containing SGLT1 at low glucose levels between meals, RS1-mediated deceleration is relieved after ingestion of glucose-rich food. Regulation of SGLT1 is mediated by RS1 domain RS1-Reg, in which Gln-Ser-Pro (QSP) is effective. In contrast to QSP and RS1-Reg, Gln-Glu-Pro (QEP) and RS1-Reg with a serine to glutamate exchange in the QSP motif downregulate the abundance of SGLT1 in the BBM at high intracellular glucose concentrations by about 50%. We investigated whether oral application of QEP improves diabetes in db/db mice and affects the induction of diabetes in New Zealand obese (NZO) mice under glucolipotoxic conditions. After 6-day administration of drinking water containing 5 mM QEP to db/db mice, fasting glucose was decreased, increase of blood glucose in the oral glucose tolerance test was blunted, and insulin sensitivity was increased. When QEP was added for several days to a high fat/high carbohydrate diet that induced diabetes in NZO mice, the increase of random plasma glucose was prevented, accompanied by lower plasma insulin levels. QEP is considered a lead compound for development of new antidiabetic drugs with more rapid cellular uptake. In contrast to SGLT1 inhibitors, QEP-based drugs may be applied in combination with insulin for the treatment of type 1 and type 2 diabetes, decreasing the required insulin amount, and thereby may reduce the risk of hypoglycemia.

Apigenin, a dietary flavonoid, induces apoptosis, DNA damage, and oxidative stress in human breast cancer MCF-7 and MDA MB-231 cells.

Apigenin is found in several dietary plant foods such as vegetables and fruits. To investigate potential anticancer properties of apigenin on human breast cancer, ER-positive MCF-7 and triple-negative MDA MB-231 cells were used. Moreover, toxicological safety of apigenin towards normal cells was evaluated in human lymphocytes. Cytotoxicity of apigenin towards cancer cells was evaluated by MTT assay whereas further genotoxic and oxidative stress parameters were measured by comet and lipid peroxidation assays, respectively. In order to examine the type of cell death induced by apigenin, several biomarkers were used. Toxicological safety towards normal cells was evaluated by cell viability and comet assays. After the treatment with apigenin, we observed changes in cell morphology in a dose- (10 to 100 µM) and time-dependent manner. Moreover, apigenin caused cell death in both cell lines leading to significant toxicity and dominantly to apoptosis. Furthermore, apigenin proved to be genotoxic towards the selected cancer cells with a potential to induce oxidative damage to lipids. Of great importance is that no significant cytogenotoxic effects were detected in normal cells. The observed cytogenotoxic and pro-cell death activities of apigenin coupled with its low toxicity towards normal cells indicate that this natural product could be used as a future anticancer modality. Therefore, further analysis to determine the exact mechanism of action and in vivo studies on animal models are warranted.

Sex-independent expression of chloride/formate exchanger Cfex (Slc26a6) in rat pancreas, small intestine, and liver, and male-dominant expression in kidneys.

Chloride/formate exchanger (CFEX; SLC26A6) mediates oxalate transport in various mammalian organs. Studies in Cfex knockout mice indicated its possible role in development of male-dominant hyperoxaluria and oxalate urolithiasis. Rats provide an important model for studying this pathophysiological condition, but data on Cfex (rCfex) localisation and regulation in their organs are limited. Here we applied the RT-PCR and immunochemical methods to investigate rCfex mRNA and protein expression and regulation by sex hormones in the pancreas, small intestine, liver, and kidneys from intact prepubertal and adult as well as gonadectomised adult rats treated with sex hormones. rCfex cDNA-transfected HEK293 cells were used to confirm the specificity of the commercial anti-CFEX antibody. Various biochemical parameters were measured in 24-h urine collected in metabolic cages. rCfex mRNA and related protein expression varied in all tested organs. Sex-independent expression of the rCfex protein was detected in pancreatic intercalated ducts (apical domain), small intestinal enterocytes (brush-border membrane; duodenum > jejunum > ileum), and hepatocytes (canalicular membrane). In kidneys, the rCfex protein was immunolocalised to the proximal tubule brush-border with segment-specific pattern (S1=S2

Loss of Endometrial Sodium Glucose Cotransporter SGLT1 is Detrimental to Embryo Survival and Fetal Growth in Pregnancy.

Embryo implantation requires a hospitable uterine environment. A key metabolic change that occurs during the peri-implantation period, and throughout early pregnancy, is the rise in endometrial glycogen content. Glycogen accumulation requires prior cellular uptake of glucose. Here we show that both human and murine endometrial epithelial cells express the high affinity Na+-coupled glucose carrier SGLT1. Ussing chamber experiments revealed electrogenic glucose transport across the endometrium in wild type (Slc5a1 +/+) but not in SGLT1 deficient (Slc5a1 -/-) mice. Endometrial glycogen content, litter size and weight of offspring at birth were significantly lower in Slc5a1 -/- mice. In humans, SLC5A1 expression was upregulated upon decidualization of primary endometrial stromal cells. Endometrial SLC5A1 expression during the implantation window was attenuated in patients with recurrent pregnancy loss when compared with control subjects. Our findings reveal a novel mechanism establishing adequate endometrial glycogen stores for pregnancy. Disruption of this histiotrophic pathway leads to adverse pregnancy outcome.

Distribution of organic anion transporters NaDC3 and OAT1-3 along the human nephron.

The initial step in renal secretion of organic anions (OAs) is mediated by transporters in the basolateral membrane (BLM). Contributors to this process are primary active Na(+)-K(+)-ATPase (EC 3.6.3.9), secondary active Na(+)-dicarboxylate cotransporter 3 (NaDC3/SLC13A3), and tertiary active OA transporters (OATs) OAT1/SLC22A6, OAT2/SLC22A7, and OAT3/SLC22A8. In human kidneys, we analyzed the localization of these transporters by immunochemical methods in tissue cryosections and isolated membranes. The specificity of antibodies was validated with human embryonic kidney-293 cells stably transfected with functional OATs. Na(+)-K(+)-ATPase was immunolocalized to the BLM along the entire human nephron. NaDC3-related immunostaining was detected in the BLM of proximal tubules and in the BLM and/or luminal membrane of principal cells in connecting segments and collecting ducts. The thin and thick ascending limbs, macula densa, and distal tubules exhibited no reactivity with the anti-NaDC3 antibody. OAT1-OAT3-related immunostaining in human kidneys was detected only in the BLM of cortical proximal tubules; all three OATs were stained more intensely in S1/S2 segments compared with S3 segment in medullary rays, whereas the S3 segment in the outer stripe remained unstained. Expression of NaDC3, OAT1, OAT2, and OAT3 proteins exhibited considerable interindividual variability in both male and female kidneys, and sex differences in their expression could not be detected. Our experiments provide a side-by-side comparison of basolateral transporters cooperating in renal OA secretion in the human kidney.