Xanthosine, a purine glycoside mediates hepatic glucose homeostasis through inhibition of gluconeogenesis and activation of glycogenesis via regulating the AMPK/ FoxO1/AKT/GSK3ß signaling cascade.

Type 2 Diabetes Mellitus (T2DM) is characterized by hepatic insulin resistance, which results in increased glucose production and reduced glycogen storage in the liver. There is no previous study in the literature that has explored the role of Xanthosine in hepatic insulin resistance. Moreover, mechanistic explanation for the beneficial effects of Xanthosine in lowering glucose production in diabetes is yet to be determined. This study for the first time investigated the beneficial effects of Tribulus terrestris (TT) and its active constituent, Xanthosine on gluconeogenesis and glycogenesis in Free Fatty Acid (FFA)-induced CC1 hepatocytes and streptozotocin (STZ)-induced Wistar rats. Xanthosine enhanced glucose uptake and decreased glucose production through phosphorylation of AMP-activated protein kinase (AMPK) and forkhead box transcription factor O1 (FoxO1), and downregulation of two rate limiting enzymes of gluconeogenesis, phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) expression in FFA-induced CC1 cells. Xanthosine also prevented FFA-induced decreases in the phosphorylation of AKT/Protein kinase B, glycogen synthase kinase-3ß (GSK3ß), and increased glycogen synthase (GS) phosphorylation to increase the glycogen content in the hepatocytes. Moreover, in STZ-induced diabetic rats, oral administration of TT n-butanol fraction (TTBF) enriched with compound Xanthosine (10, 50 & 100 mg/kg body weight) improved insulin sensitivity, reduced fasting blood glucose levels, improved glucose homeostasis by reducing gluconeogenesis via AMPK/FoxO1-mediated PEPCK and G6Pase down-regulation and increasing glycogenesis via AKT/GSK3ß-mediated GS activation. Overall, Xanthosine may be developed further for treating insulin resistance and hyperglycemia in T2DM.

Stem Cells in Mammary Health and Milk Production.

Adult stem cells like mammary and mesenchymal stem cells have received significant attention because these stem cells possess therapeutic potential in treating many animal diseases. These cells can be administered in an autologous or allogenic fashion, either freshly isolated from the donor tissue or previously cultured and expanded in vitro. The expansion of adult stem cells is a prerequisite before therapeutic application because sufficient numbers are required in dosage calculation. Stem cells directly and indirectly (by secreting various growth factors and angiogenic factors called secretome) act to repair and regenerate injured tissues. Recent studies on mammary stem cells showed in vivo and in vitro expansion ability by removing the blockage of asymmetrical cell division. Compounds like purine analogs (xanthosine, xanthine, and inosine) or hormones (progesterone and bST) help increase stem cell population by promoting cell division. Such methodology of enhancing stem cell number, either in vivo or in vitro, may help in preclinical studies for translational research like treating diseases such as mastitis. The application of mesenchymal stem cells has also been shown to benefit mammary gland health due to the 'homing' property of stem cells. In addition to that, the multiple positive effects of stem cell secretome are on mammary tissue; healing and killing bacteria is novel in the production of quality milk. This systematic review discusses some of the studies on stem cells that have been useful in increasing the stem cell population and increasing mammary stem/progenitor cells. Finally, we provide insights into how enhancing mammary stem cell population could potentially increase terminally differentiated cells, ultimately leading to more milk production.

Synthesis of xanthosine 2-phosphate diesters via phosphitylation of the carbonyl group.

O2-Phosphodiesterification of xanthosine has been achieved by a one-pot procedure consisting of the phosphitylation of the 2-carbonyl group of appropriately protected xanthosine derivatives using phosphoramidites and N-(cyanomethyl)dimethylammonium triflate (CMMT), oxidation of the resulting xanthosine 2-phosphite triesters, and deprotection. In addition, a study on the hydrolytic stability of a fully deprotected xanthosine 2-phosphate diester has revealed that it is more stable at higher pH.

Association Between Kidney Clearance of Secretory Solutes and Cardiovascular Events: The Chronic Renal Insufficiency Cohort (CRIC) Study.

RATIONALE & OBJECTIVE: The clearance of protein-bound solutes by the proximal tubules is an innate kidney mechanism for removing putative uremic toxins that could exert cardiovascular toxicity in humans. However, potential associations between impaired kidney clearances of secretory solutes and cardiovascular events among patients with chronic kidney disease (CKD) remains uncertain. STUDY DESIGN: A multicenter, prospective, cohort study. SETTING & PARTICIPANTS: We evaluated 3,407 participants from the Chronic Renal Insufficiency Cohort (CRIC) study. EXPOSURES: Baseline kidney clearances of 8 secretory solutes. We measured concentrations of secretory solutes in plasma and paired 24-hour urine specimens using liquid chromatography-tandem mass spectrometry (LC-MS/MS). OUTCOMES: Incident heart failure, myocardial infarction, and stroke events. ANALYTICAL APPROACH: We used Cox regression to evaluate associations of baseline secretory solute clearances with incident study outcomes adjusting for estimated GFR (eGFR) and other confounders. RESULTS: Participants had a mean age of 56 years; 45% were women; 41% were Black; and the median estimated glomerular filtration rate (eGFR) was 43 mL/min/1.73 m2. Lower 24-hour kidney clearance of secretory solutes were associated with incident heart failure and myocardial infarction but not incident stroke over long-term follow-up after controlling for demographics and traditional risk factors. However, these associations were attenuated and not statistically significant after adjustment for eGFR. LIMITATIONS: Exclusion of patients with severely reduced eGFR at baseline; measurement variability in secretory solutes clearances. CONCLUSIONS: In a national cohort study of CKD, no clinically or statistically relevant associations were observed between the kidney clearances of endogenous secretory solutes and incident heart failure, myocardial infarction, or stroke after adjustment for eGFR. These findings suggest that tubular secretory clearance provides little additional information about the development of cardiovascular disease events beyond glomerular measures of GFR and albuminuria among patients with mild-to-moderate CKD.

Tubular Secretory Clearance Is Associated With Whole-Body Insulin Clearance.

CONTEXT: The kidneys eliminate insulin via glomerular and peritubular mechanisms; consequently, the kidney contribution to insulin clearance may be underestimated by the glomerular filtration rate (GFR) alone. OBJECTIVE: To determine associations of tubular secretory clearance with whole-body insulin clearance and sensitivity in a dedicated study of glucose and insulin metabolism. DESIGN, SETTING, AND PARTICIPANTS: We performed an ancillary, cross-sectional study of tubular secretion in the Study of Glucose and Insulin in Renal Disease (SUGAR). Hyperinsulinemic-euglycemic clamps were performed in 57 nondiabetic persons with chronic kidney disease and 38 persons without kidney disease. INTERVENTION: We measured plasma and 24-hour urine concentrations of endogenous solutes primarily eliminated by tubular secretion. Kidney clearances of secretory solutes were calculated as the amount of blood fully cleared of that solute per minute. MAIN OUTCOME MEASURES: Whole-body insulin clearance, insulin sensitivity. RESULTS: Mean whole-body insulin clearance was 924 ± 228 mL/min. After adjustment for age, sex, Black race, fat and fat-free mass, each 20% lower estimated GFR was associated with a 13 mL/min lower insulin clearance (95% confidence interval [CI], 2-24 mL/min lower). Each 20% lower clearance of isovalerylglycine and xanthosine were associated with a 16 mL/min lower (95% CI, 5-26 mL/min lower) and 19 mL/min lower (95% CI, 7-31 mL/min lower) insulin clearance, respectively. Neither estimated GFR nor secretory solute clearances were associated with insulin sensitivity after adjustment. CONCLUSIONS: These results highlight the importance of tubular secretory pathways to insulin elimination but suggest that kidney functions in aggregate contribute only modestly to systemic insulin clearance.

Purification and characterization of theobromine synthase in a Theobromine-Enriched wild tea plant (Camellia gymnogyna Chang) from Dayao Mountain, China.

Camellia gymnogyna Chang (CgC), a wild tea plant, was discovered on Dayao Mountain, China. However, research regarding this tea plant is limited. Our study found that CgC contains theobromine, caffeine, and theacrine, among which theobromine content was the highest (14.37-39.72 mg/g). In addition, theobromine synthase (TS) was partially purified from CgC leaves, up to 35.87-fold, with consecutive chromatography, and its molecular weight was found to be approximately 62 kDa. The optimum reaction time, pH, and temperature for theobromine synthase from 7-methylxanthine was found to be 6 h, 4, and 45 °C, respectively. TS expression at both mRNA and protein stages was higher in the first than in the fourth leaf (P < 0.05). Subcellular localization of TS indicated that it was localized in the nucleus. These results indicate that CgC can be of scientific value and could lead to efficient utilization of this rare wild tea germplasm.

Alterations of Proximal Tubular Secretion in Autosomal Dominant Polycystic Kidney Disease.

BACKGROUND AND OBJECTIVES: In autosomal dominant polycystic kidney disease (ADPKD), the GFR often remains normal despite significant nephron loss. Proximal tubular secretory clearance may be reduced in ADPKD before detectable changes in GFR. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: We used targeted mass spectrometry to quantify secretory solutes from blood and urine samples from 31 patients with ADPKD and preserved GFR (mean eGFR =111±11 ml/min per 1.73 m2) and 25 healthy control individuals as well as from 95 patients with ADPKD and reduced GFR (mean eGFR =53±21 ml/min per 1.73 m2) and 92 individuals with non-ADPKD CKD. We used linear regression to compare the fractional excretion of each solute between ADPKD and control groups. Among 112 patients with ADPKD, we used linear regression to determine associations of solute fractional excretion with height-adjusted total kidney volume. RESULTS: After adjusting for demographics, clinical characteristics, and kidney function measures, the fractional excretions of three secretory solutes were lower in patients with ADPKD and preserved GFR compared with healthy individuals: 52% lower cinnamoylglycine excretion (95% confidence interval, 24% to 70%), 53% lower tiglylglycine excretion (95% confidence interval, 23% to 71%), and 91% lower xanthosine excretion (95% confidence interval, 83% to 95%). In addition to lower excretions of tiglylglycine and xanthosine, patients with ADPKD and reduced GFR also demonstrated 37% lower dimethyluric acid excretion (95% confidence interval, 21% to 50%), 58% lower hippurate excretion (95% confidence interval, 48% to 66%), 48% lower isovalerylglycine excretion (95% confidence interval, 37% to 56%), and 31% lower pyridoxic acid excretion (95% confidence interval, 16% to 42%) compared with patients with non-ADPKD CKD and comparable eGFR. Among patients with ADPKD, solute fractional excretions were not associated with differences in kidney volume. CONCLUSIONS: Patients with ADPKD and preserved and reduced GFR demonstrate lower tubular secretory solute excretion compared with healthy controls and patients with non-ADPKD CKD. Our results suggest that tubular secretion is impaired in ADPKD independent of GFR.

Evaluation of xanthosine treatment on gene expression of mammary glands in early lactating goats.

This study examined the hypothesis that xanthosine (XS) treatment would promote mammary-specific gene expression and stem cell transcripts and have a positive influence on milk yield of dairy goats. Seven primiparous Beetal goats were assigned to the study. Five days after kidding, one gland (either left or right) was infused with XS (TRT) twice daily for 3 d and the other gland with no XS infusion served as a control (CON). Mammary biopsies were collected at 10 d and RNA was isolated. Gene expression analysis of milk synthesis genes, mammary stem/progenitor cell markers, cell proliferation and differentiation markers were performed using real time quantitative PCR (RT-qPCR). Results showed that the transcripts of milk synthesis genes (BLG4, CSN2, LALBA, FABP3, CD36) and mammary stem/progenitor cell markers (ALDH1 and NR5A2) were increased in as a result of XS treatment. Average milk yield in TRT glands was increased marginally (approximately ~2% P = 0·05, paired t-test) per gland relative to CON gland until 7 wk. After 7 wk, milk yield of TRT and CON glands did not differ. Analysis of milk composition revealed that protein, lactose, fat and solids-not-fat percentages remained the same in TRT and CON glands. These results suggest that XS increases expression of milk synthesis genes, mammary stem/progenitor cells and has a small effect on milk yield.

Examination of the xanthosine response on gene expression of mammary epithelial cells using RNA-seq technology.

BACKGROUND: Xanthosine treatment has been previously reported to increase mammary stem cell population and milk production in cattle and goats. However, the underlying molecular mechanisms associated with the increase in stem cell population and milk production remain unclear. METHODS: Primiparous Beetal goats were assigned to the study. Five days post-partum, one mammary gland of each goat was infused with xanthosine (TRT) twice daily (2×) for 3 days consecutively, and the other gland served as a control (CON). Milk samples from the TRT and CON glands were collected on the 10th day after the last xanthosine infusion and the total RNA was isolated from milk fat globules (MEGs). Total RNA in MFGs was mainly derived from the milk epithelial cells (MECs) as evidenced by expression of milk synthesis genes. Significant differentially expressed genes (DEGs) were subjected to Gene Ontology (GO) terms using PANTHER and gene networks were generated using STRING db. RESULTS: Preliminary analysis indicated that each individual goat responded to xanthosine treatment differently, with this trend being correlated with specific DEGs within the same animal's mammary gland. Several pathways are impacted by these DEGs, including cell communication, cell proliferation and anti-microbials. CONCLUSIONS: This study provides valuable insights into transcriptomic changes in milk producing epithelial cells in response to xanthosine treatment. Further characterization of DEGs identified in this study is likely to delineate the molecular mechanisms of increased milk production and stem or progenitor cell population by the xanthosine treatment.

In vivo response of xanthosine on mammary gene expression of lactating Beetal goat.

Xanthosine is hypothesized to increase stem cell number by promoting symmetrical cell division. Stem cells, in particular mammary stem/progenitor cells are important for gland growth and tissue repair. Molecular mechanism of xanthosine effects on mammary tissue is very limited therefore, a detailed study is warranted. The objective of this study was to evaluate transcriptomic changes in mammary gland infused/not infused with xanthosine of lactating goat. Seven primiparous Beetal goats on day 5 after kidding, were selected for the study. One gland of each goat was infused with xanthosine (TRT gland) twice daily for 3 days while the other gland did not receive any xanthosine and served as control (CON gland). Biopsy of mammary tissues was taken from TRT and CON glands, 2 days after the last day of treatment that is on day 10 after kidding. Illumina RNA-sequencing (RNA-seq) was performed for global gene expression analysis of contralateral glands. Of 382 differentially expressed genes (DEGs), 372 genes were annotated to the goat genome. Gene ontology analyses revealed majority of the DEGs to be associated with metabolic pathways (glycan and lipid metabolism), biosynthesis of antibiotics and peroxisome proliferator-activated receptor signalling pathways. These molecular pathways are either directly or indirectly involved with lipid metabolism in mammary tissue and host adaptive immune response. Expression of stem cell marker namely aldehyde dehydrogenase enzymes (ALDH1A1, ALDH3B1) were upregulated in the treatment gland. Real-time quantitative PCR (RT-qPCR) analyses of selected DEGs showed their expression profiles to be in agreement with results of RNA-seq. To our knowledge, this is the first study that describes effects of xanthosine on transcriptomic changes of mammary tissue. This information can be used further to dissect the molecular mechanisms underlying effects of xanthosine to improve production potential and udder health.

Ultrafast Electronic Deactivation Dynamics of Xanthosine Monophosphate.

Ultrafast energy dissipation is a crucial factor for the photostability of DNA and RNA, but even some of the key electronic deactivation pathways in monomeric nucleic acid building stones are still controversial. Here, we report on the excited-state dynamics of the rare nucleotide xanthosine monophosphate as a function of deprotonation state (XMP vs. XMP - ) and excitation wavelength ( λ pump = 278-243 nm) by femtosecond time-resolved fluorescence and absorption spectroscopy. We show that the predominating relaxation channel leads to a return of the photo-excited molecules to the electronic ground state in τ∼1 ps. The mechanism likely involves an out-of-plane deformation of the five-membered ring, different from the main electronic deactivation pathways in the canonical purine bases adenine and guanine. The results are discussed in terms of the structural and electronic differences of XMP compared to the canonical nucleotides.

Mammary stem cells: expansion and animal productivity.

Identification and characterization of mammary stem cells and progenitor cells from dairy animals is important in the understanding of mammogenesis, tissue turnover, lactation persistency and regenerative therapy. It has been realized by many investigators that altered lactation, long dry periods (non-milking period between two consecutive lactation cycles), abrupt cessation of lactation (common in water buffaloes) and disease conditions like mastitis, greatly reduce milk yield thus render huge financial losses within the dairy sector. Cellular manipulation of specialized cell types within the mammary gland, called mammary stem cells (MaSCs)/progenitor cells, might provide potential solutions to these problems and may improve milk production. In addition, MaSCs/progenitor cells could be used in regenerative therapy against tissue damage caused by mastitis. This review discusses methods of MaSC/progenitor cell manipulation and their mechanisms in bovine and caprine animals. Author believes that intervention of MaSCs/progenitor cells could lessen the huge financial losses to the dairy industry globally.

Xanthosine administration does not affect the proportion of epithelial stem cells in bovine mammary tissue, but has a latent negative effect on cell proliferation.

The challenge in manipulating the proportion of somatic stem cells lies in having to override tissue homeostasis. Xanthosine infusion via the teat canal has been reported to augment the number of label-retaining cells in the mammary gland of 3-month-old bovine calves. To further delineate xanthosine׳s effect on defined stem cells in the mammary gland of heifers-which are candidates for increased prospective milk production following such manipulation-bovine mammary parenchymal tissue was transplanted and integrated into the cleared mammary fat pad of immunodeficient mice. Xanthosine administration for 14 days did not affect the number of label-retaining cells after 10- and 11-week chases. No change in stem cell proportion, analyzed according to CD49f and CD24 expression, was noted. Clone formation and propagation rate of cultured cells, as well as expression of stem cell markers, were also unaffected. In contrast, a latent 50% decrease in bovine mammary cell proliferation rate was observed 11 weeks after xanthosine administration. Tumor development in mice was also limited by xanthosine administration. These effects may have resulted from an initial decrease in expression of the rate-limiting enzyme in guanine synthesis, IMPDH. The data indicate that caution should be exerted when considering xanthosine for stem cell manipulation.

ITPA (inosine triphosphate pyrophosphatase): from surveillance of nucleotide pools to human disease and pharmacogenetics.

Cellular nucleotide pools are often contaminated by base analog nucleotides which interfere with a plethora of biological reactions, from DNA and RNA synthesis to cellular signaling. An evolutionarily conserved inosine triphosphate pyrophosphatase (ITPA) removes the non-canonical purine (d)NTPs inosine triphosphate and xanthosine triphosphate by hydrolyzing them into their monophosphate form and pyrophosphate. Mutations in the ITPA orthologs in model organisms lead to genetic instability and, in mice, to severe developmental anomalies. In humans there is genetic polymorphism in ITPA. One allele leads to a proline to threonine substitution at amino acid 32 and causes varying degrees of ITPA deficiency in tissues and plays a role in patients' response to drugs. Structural analysis of this mutant protein reveals that the protein is destabilized by the formation of a cavity in its hydrophobic core. The Pro32Thr allele is thought to cause the observed dominant negative effect because the resulting active enzyme monomer targets both homo- and heterodimers to degradation.

Ex vivo Expansion of Human Adult Pancreatic Cells with Properties of Distributed Stem Cells by Suppression of Asymmetric Cell Kinetics.

Transplantation therapy for type I diabetes (T1D) might be improved if pancreatic stem cells were readily available for investigation. Unlike macroscopic islets, pancreatic tissue stem cells could more easily access the retroperitoneal pancreatic environment and thereby might achieve more effective pancreatic regeneration. Unfortunately, whether the adult pancreas actually contains renewing stem cells continues as a controversial issue in diabetes research. We evaluated a new method developed in our lab for expanding renewing distributed stem cells (DSCs) from adult tissues as a means to provide more evidence for adult pancreatic stem cells, and potentially advance their availability for future clinical investigation. The new method was designed to switch DSCs from asymmetric self-renewal to symmetric self-renewal, which promotes their exponential expansion in culture with reduced production of differentiated cells. Called suppression of asymmetric cell kinetics (SACK), the method uses natural purine metabolites to accomplish the self-renewal pattern shift. The SACK purine metabolites xanthine, xanthosine, and hypoxanthine were evaluated for promoting expansion of DSCs from the pancreas of adult human postmortem donors. Xanthine and xanthosine were effective for deriving both pooled and clonal populations of cells with properties indicative of human pancreatic DSCs. The expanded human cell strains had signature SACK agent-suppressible asymmetric cell kinetics, produced Ngn3+ bipotent precursors for α-cells and ß-cells, and were non-tumorigenic in immunodeficient mice. Our findings support the existence of pancreatic DSCs in the adult human pancreas and indicate a potential path to increasing their availability for future clinical evaluation.