Radioprotective Properties of Riboxin (Inosine) and Indralin under External Irradiation.

A comparative assessment of radioprotective properties of inosine nucleoside (riboxin) and recognized radioprotector indralin was carried out. We analyzed survival of male ICR CD-1 mice weighting 32.2±0.2 g exposed to external X-ray radiation at doses 6.5 and 6.75 Gy and receiving indralin at a dose of 100 or 150 µg/g body weight or riboxin (inosine) at a dose of 100 or 200 µg/g body weight before irradiation. The survival analysis was carried out by the Kaplan-Meier method. The significance was assessed by using the log-rank-test. Inosine showed a significant difference from the irradiated control only at a dose of 100 µg/g body weight at a radiation dose of 6.75 Gy. The survival of animals treated with indralin was significantly higher in comparison with not only the irradiated control group, but also with the groups receiving inosine.

Inosine pretreatment of pregnant rats ameliorates maternal inflammation-mediated hypomyelination in pups via microglia polarization switch.

Periventricular leukomalacia (PVL) is a neurological condition observed in premature infants, characterized by hypomyelination and activation of microglia. Maternal inflammation-induced brain injury in offspring significantly contributes to the development of PVL. Currently, there are no clinical pharmaceutical interventions available for pregnant women to prevent maternal inflammation-mediated brain injury in their offspring. Inosine has been shown to modulate the immune response in diverse stressful circumstances, such as injury, ischemia, and inflammation. The aim of this investigation was to examine the potential prophylactic impact of inosine on offspring PVL induced by maternal inflammation. This was accomplished by administering a 1 mg/ml inosine solution (40 ml daily) to pregnant Sprague-Dawley (SD) rats for 16 consecutive days prior to their intraperitoneal injection of lipopolysaccharide (350 µg/kg, once a day, for two days). The results showed that maternal inosine pretreatment significantly reversed the reduction in MBP and CNPase (myelin-related markers), CC-1 and Olig2 (oligodendrocyte-related markers) in their PVL pups (P7), suggesting that inosine administration during pregnancy could improve hypomyelination and enhance the differentiation of oligodendrocyte precursor cells (OPCs) in their PVL pups. Furthermore, the protective mechanism of inosine against PVL is closely associated with the activation and polarization of microglia. This is evidenced by a notable reduction in the quantity of IBA 1-positive microglia, a decrease in the level of CD86 (a marker for M1 microglia), an increase in the level of Arg 1 (a marker for M2 microglia), as well as a decrease in the level of pro-inflammatory factors TNF-α, IL-1ß, and IL-6, and an increase in the level of anti-inflammatory factors IL-4 and IL-10 in the brain of PVL pups following maternal inosine pretreatment. Taken together, inosine pretreatment of pregnant rats can improve hypomyelination in their PVL offspring by triggering the M1/M2 switch of microglia.

Unlocking CAR T cell potential: Inosine-induced stemness and enhanced potency.

Adenosine (Ado) drives immune suppression in the tumor microenvironment. In this issue of Cancer Cell, Klysz et al. investigate Ado-mediated immunosuppression. Overexpression of Ado deaminase (ADA-OE), metabolizing Ado to inosine (INO), induces stemness and improves CAR T cell functionality. Likewise, exposure to INO enhances CAR T cells' function and induces stemness features.

L-arginine metabolism inhibits arthritis and inflammatory bone loss.

OBJECTIVES: To investigate the effect of the L-arginine metabolism on arthritis and inflammation-mediated bone loss. METHODS: L-arginine was applied to three arthritis models (collagen-induced arthritis, serum-induced arthritis and human TNF transgenic mice). Inflammation was assessed clinically and histologically, while bone changes were quantified by µCT and histomorphometry. In vitro, effects of L-arginine on osteoclast differentiation were analysed by RNA-seq and mass spectrometry (MS). Seahorse, Single Cell ENergetIc metabolism by profilIng Translation inHibition and transmission electron microscopy were used for detecting metabolic changes in osteoclasts. Moreover, arginine-associated metabolites were measured in the serum of rheumatoid arthritis (RA) and pre-RA patients. RESULTS: L-arginine inhibited arthritis and bone loss in all three models and directly blocked TNFα-induced murine and human osteoclastogenesis. RNA-seq and MS analyses indicated that L-arginine switched glycolysis to oxidative phosphorylation in inflammatory osteoclasts leading to increased ATP production, purine metabolism and elevated inosine and hypoxanthine levels. Adenosine deaminase inhibitors blocking inosine and hypoxanthine production abolished the inhibition of L-arginine on osteoclastogenesis in vitro and in vivo. Altered arginine levels were also found in RA and pre-RA patients. CONCLUSION: Our study demonstrated that L-arginine ameliorates arthritis and bone erosion through metabolic reprogramming and perturbation of purine metabolism in osteoclasts.

Xie Zhuo Tiao Zhi formula modulates intestinal microbiota and liver purine metabolism to suppress hepatic steatosis and pyroptosis in NAFLD therapy.

BACKGROUND: Current evidence indicates a rising global prevalence of Non-Alcoholic Fatty Liver Disease (NAFLD), which is closely associated to conditions such as obesity, dyslipidemia, insulin resistance, and metabolic syndrome. The relationship between the gut microbiome and metabolites in NAFLD is gaining attention understanding the pathogenesis and progression of dysregulated lipid metabolism and inflammation. The Xie Zhuo Tiao Zhi (XZTZ) decoction has been employed in clinical practice for alleviating hyperlipidemia and symptoms related to metabolic disorders. However, the pharmacological mechanisms underlying the effects of XZTZ remain to be elucidated. PURPOSE: The objective of this study was to examine the pharmacological mechanisms underlying the hypolipidemic and anti-inflammatory effects of XZTZ decoction in a mouse model of NAFLD, as well as the effects of supplementing exogenous metabolites on PO induced cell damage and lipid accumulation in cultured hepatocytes. METHODS: A high-fat diet (HFD) mouse model was established to examine the effects of XZTZ through oral gavage. The general condition of mice and the protective effect of XZTZ on liver injury were evaluated using histological and biochemical methods. Hematoxylin and eosin staining (H&E) staining and oil red O staining were performed to assess inflammatory and lipid accumulation detection, and cytokine levels were quantitatively analyzed. Additionally, the study included full-length 16S rRNA sequencing, liver transcriptome analysis, and non-targeted metabolomics analysis to investigate the relationship among intestinal microbiome, liver metabolic function, and XZTZ decoction. RESULTS: XZTZ had a significant impact on the microbial community structure in NAFLD mice. Notably, the abundance of Ileibacterium valens, which was significantly enriched by XZTZ, exhibited a negative correlation with liver injury biomarkers such as, alanine transaminase (ALT) and aspartate transaminase (AST) activity. Moreover, treatment with XZTZ led to a significant enrichment of the purine metabolism pathway in liver tissue metabolites, with inosine, a purine metabolite, showing a significant positive correlation with the abundance of I. valens. XZTZ and inosine also significantly enhanced fatty acid ß-oxidation, which led to a reduction in the expression of pro-inflammatory cytokines and the inhibition of liver pyroptosis. These effects contributed to the mitigation of liver injury and hepatocyte damage, both in vivo and vitro. Furthermore, the utilization of HPLC fingerprints and UPLC-Q-TOF-MS elucidated the principal constituents within the XZTZ decoction, including naringin, neohesperidin, atractylenolide III, 23-o-Acetylalisol B, pachymic acid, and ursolic acid which are likely responsible for its therapeutic efficacy. Further investigations are imperative to fully uncover and validate the pharmacodynamic mechanisms underlying these observations. CONCLUSION: The administration of XZTZ decoction demonstrates a protective effect on the livers of NAFLD mice by inhibiting lipid accumulation and reducing hepatocyte inflammatory damage. This protective effect is mediated by the upregulation of I.valens abundance in the intestine, highlighting the importance of the gut-liver axis. Furthermore, the presesnce of inosine, adenosine, and their derivatives are important in promoting the protective effects of XZTZ. Furthermore, the in vitro approaching, we provide hitherto undocumented evidence indicating that the inosine significantly improves lipid accumulation, inflammatory damage, and pyroptosis in AML12 cells incubated with free fatty acids.

8-Aminopurines in the Cardiovascular and Renal Systems and Beyond.

Screening of compounds comprising 8-substituted guanine revealed that 8-aminoguanosine and 8-aminoguanine cause diuresis/natriuresis/glucosuria, yet decrease potassium excretion. Subsequent investigations demonstrated that 8-aminoguanosine's effects are mediated by its metabolite 8-aminoguanine. The mechanism by which 8-aminoguanine causes diuresis/natriuresis/glucosuria involves inhibition of PNPase (purine nucleoside phosphorylase), which increases renal interstitial inosine levels. Additional evidence suggests that inosine, via indirect or direct adenosine A2B receptor activation, increases renal medullary blood flow which enhances renal excretory function. Likely, 8-aminoguanine has pleiotropic actions that also alter renal excretory function. Indeed, the antikaliuretic effects of 8-aminoguanine are independent of PNPase inhibition. 8-Aminoguanine is an endogenous molecule; nitrosative stress leads to production of biomolecules containing 8-nitroguanine moieties. Degradation of these biomolecules releases 8-nitroguanosine and 8-nitro-2'-deoxyguanosine which are converted to 8-aminoguanine. Also, guanosine and guanine per se may contribute to 8-aminoguanine formation. 8-Aminoinosine, 8-aminohypoxanthine, and 8-aminoxanthine likewise induce diuresis/natriuresis/glucosuria, yet do not reduce potassium excretion. Thus, there are several pharmacologically active 8-aminopurines with nuanced effects on renal excretory function. Chronic treatment with 8-aminoguanine attenuates hypertension in deoxycorticosterone/salt rats, prevents strokes, and increases lifespan in Dahl salt-sensitive rats on a high salt diet and attenuates the metabolic syndrome in rats; 8-aminoguanosine retards progression of pulmonary hypertension in rats and anemia and organ damage in sickle cell mice. 8-Aminoguanine reverses age-associated lower urinary tract dysfunction and retinal degeneration. 8-Aminopurines represent a new class of agents (and potentially endogenous factors) that have beneficial effects on the cardiovascular system and kidneys and may turn back the clock in age-associated diseases.

Dopaminergic neuroprotective effects of inosine in MPTP-induced parkinsonian mice via brain-derived neurotrophic factor upregulation.

Parkinson's disease (PD) is the second most common neurodegenerative disease. However, no curative or modifying therapy is known. Inosine is a purine nucleoside that increases brain-derived neurotrophic factor (BDNF) expression in the brain through adenosine receptors. Herein, we investigated the neuroprotective effects of inosine and elucidated the mechanisms underlying its pharmacological action. Inosine rescued SH-SY5Y neuroblastoma cells from MPP+ injury in a dose-dependent manner. Inosine protection correlated with BDNF expression and the activation of its downstream signaling cascade, as the TrkB receptor inhibitor, K252a and siRNA against the BDNF gene remarkably reduced the protective effects of inosine. Blocking the A1 or A2A adenosine receptors diminished BDNF induction and the rescuing effect of inosine, indicating a critical role of adenosine A1 and A2A receptors in inosine-related BDNF elevation. We assessed whether the compound could protect dopaminergic neurons from MPTP-induced neuronal injury. Beam-walking and challenge beam tests revealed that inosine pretreatment for 3 weeks reduced the MPTP-induced motor function impairment. Inosine ameliorated dopaminergic neuronal loss and MPTP-mediated astrocytic and microglial activation in the substantia nigra and striatum. Inosine ameliorated the depletion of striatal dopamine and its metabolite following MPTP injection. BDNF upregulation and the activation of its downstream signaling pathway seemingly correlate with the neuroprotective effects of inosine. To our knowledge, this is the first study to demonstrate the neuroprotective effects of inosine against MPTP neurotoxicity via BDNF upregulation. These findings highlight the therapeutic potential of inosine in dopaminergic neurodegeneration in PD brains.

8-Aminoguanine and Its Actions on Renal Excretory Function.

BACKGROUND: The endogenous purine 8-aminoguanine induces diuresis/natriuresis/glucosuria by inhibiting PNPase (purine nucleoside phosphorylase); however, mechanistic details are unknown. METHODS: Here, we further explored in rats 8-aminoguanine's effects on renal excretory function by combining studies using intravenous 8-aminoguanine, intrarenal artery infusions of PNPase substrates (inosine and guanosine), renal microdialysis, mass spectrometry, selective adenosine receptor ligands, adenosine receptor knockout rats, laser doppler blood flow analysis, cultured renal microvascular smooth muscle cells, HEK293 cells expressing A2B receptors and homogeneous time resolved fluorescence assay for adenylyl cyclase activity. RESULTS: Intravenous 8-aminoguanine caused diuresis/natriuresis/glucosuria and increased renal microdialysate levels of inosine and guanosine. Intrarenal inosine, but not guanosine, exerted diuretic/natriuretic/glucosuric effects. In 8-aminoguanine-pretreated rats, intrarenal inosine did not induce additional diuresis/natriuresis/glucosuria. 8-Aminoguanine did not induce diuresis/natriuresis/glucosuria in A2B-receptor knockout rats, yet did so in A1- and A2A-receptor knockout rats. Inosine's effects on renal excretory function were abolished in A2B knockout rats. Intrarenal BAY 60-6583 (A2B agonist) induced diuresis/natriuresis/glucosuria and increased medullary blood flow. 8-Aminoguanine increased medullary blood flow, a response blocked by pharmacological inhibition of A2B, but not A2A, receptors. In HEK293 cells expressing A2B receptors, inosine activated adenylyl cyclase, and this was abolished by MRS 1754 (A2B antagonist). In renal microvascular smooth muscle cells, 8-aminoguanine and forodesine (PNPase inhibitor) increased inosine and 3',5'-cAMP; however, in cells from A2B knockout rats, 8-aminoguanine and forodesine did not augment 3',5'-cAMP yet increased inosine. CONCLUSIONS: 8-Aminoguanine induces diuresis/natriuresis/glucosuria by increasing renal interstitial levels of inosine which, via A2B receptor activation, increases renal excretory function, perhaps in part by increasing medullary blood flow.

Inosine, gut microbiota, and cancer immunometabolism.

This article briefly reviews cancer immunity and the role of gut microbiota in carcinogenesis, followed by an understanding of mechanisms by which inosine is involved in cancer immunometabolism. The immune system plays a paradoxical role in cancer treatment. Antitumor immunity depends on the T-cell priming against tumor antigens, whereas inflammatory mediators trigger the protumor signaling in the tumor microenvironment. Studies link the microbiome with metabolism and immunity-two main factors implicated in carcinogenesis. Gut microbiota has been shown to affect both antitumor immunity and protumor immune signaling. There is mounting evidence that the human microbiome can play a role in the immunotherapeutic effects, both response and resistance. Inosine-5'-monophosphate dehydrogenase (IMPDH) is a highly conservative enzyme widely expressed in mammals. Cell signaling pathways use molecular inosine, a crucial secondary metabolite in purine metabolism and a molecular messenger. Recent research has identified inosine as a critical regulator of immune checkpoint inhibition (ICI) therapeutic response in various tumor types. Some bacterial species were found to produce inosine or its metabolite hypoxanthine and induce T-helper 1 differentiation and effector functions via the inosine-A2AR-cAMP-PKA pathway upon ICI therapy. Also, inosine acts as a substitute carbon source for T-cell metabolism in glucose-restricted environments, i.e., the tumor microenvironment, assisting T-cell proliferation and differentiation while enhancing sensitivity to ICI, reinforcing the notion that inosine metabolism might contribute to antitumor immunity. Also, inosine is a potent agonist of the adenosine receptor, A2AR, and A2AR signaling can affect T-cell responses and antitumor immunity, making the inosine-A2AR pathway blockage a candidate for cancer treatment. Further research is required to investigate inosine as a cancer immunometabolism therapy.

A plastid nucleoside kinase is involved in inosine salvage and control of purine nucleotide biosynthesis.

In nucleotide metabolism, nucleoside kinases recycle nucleosides into nucleotides-a process called nucleoside salvage. Nucleoside kinases for adenosine, uridine, and cytidine have been characterized from many organisms, but kinases for inosine and guanosine salvage are not yet known in eukaryotes and only a few such enzymes have been described from bacteria. Here we identified Arabidopsis thaliana PLASTID NUCLEOSIDE KINASE 1 (PNK1), an enzyme highly conserved in plants and green algae belonging to the Phosphofructokinase B family. We demonstrate that PNK1 from A. thaliana is located in plastids and catalyzes the phosphorylation of inosine, 5-aminoimidazole-4-carboxamide-1-ß-d-ribose (AICA ribonucleoside), and uridine but not guanosine in vitro, and is involved in inosine salvage in vivo. PNK1 mutation leads to increased flux into purine nucleotide catabolism and, especially in the context of defective uridine degradation, to over-accumulation of uridine and UTP as well as growth depression. The data suggest that PNK1 is involved in feedback regulation of purine nucleotide biosynthesis and possibly also pyrimidine nucleotide biosynthesis. We additionally report that cold stress leads to accumulation of purine nucleotides, probably by inducing nucleotide biosynthesis, but that this adjustment of nucleotide homeostasis to environmental conditions is not controlled by PNK1.

Effects of different levels of inosine-5'-monophosphate (5'-IMP) supplementation on the growth performance and meat quality of finishing pigs (75 to 100 kg).

This study aimed to assess the effects of dietary supplementation of inosine-5'-monophosphate (5'-IMP) on energy efficiency, growth performance, carcass characteristics, meat quality, oxidative status, and biochemical profile of blood plasma in finishing pigs. Fifty-four crossbred castrated male pigs were distributed in a randomized block design consisting of nine blocks, with six treatments per block and one animal per treatment per block. Experimental diets were as follows: positive control diet (PC, 3300 kcal ME/kg), negative control diet (NC, 3200 kcal ME/kg), and four diets prepared by supplementing the NC diet with 0.050%, 0.100%, 0.150%, or 0.200% 5'-IMP. Based on regression analysis, supplementation with 0.129% 5'-IMP increased average daily weight gain (1.30 kg). Backfat thickness, pH45minutes and redness of m. Longissimus Lumborum (LL) increased linearly with 5'-IMP supplementation level. Drip loss and pH at 24 h post-slaughter had a quadratic response to 5'-IMP supplementation. It is concluded that 5'-IMP supplementation positively influenced growth performance, carcass characteristics and LL meat quality in finishing barrows.

The impact of inosine on hippocampal synaptic transmission and plasticity involves the release of adenosine through equilibrative nucleoside transporters rather than the direct activation of adenosine receptors.

Inosine has robust neuroprotective effects, but it is unclear if inosine acts as direct ligand of adenosine receptors or if it triggers metabolic effects indirectly modifying the activity of adenosine receptors. We now combined radioligand binding studies with electrophysiological recordings in hippocampal slices to test how inosine controls synaptic transmission and plasticity. Inosine was without effect at 30 µM and decreased field excitatory post-synaptic potentials by 14% and 33% at 100 and 300 µM, respectively. These effects were prevented by the adenosine A1 receptor antagonist DPCPX. Inosine at 300 (but not 100) µM also decreased the magnitude of long-term potentiation (LTP), an effect prevented by DPCPX and by the adenosine A2A receptor antagonist SCH58261. Inosine showed low affinity towards human and rat adenosine receptor subtypes with Ki values of > 300 µM; only at the human and rat A1 receptor slightly higher affinities with Ki values of around 100 µM were observed. Affinity of inosine at the rat A3 receptor was higher (Ki of 1.37 µM), while it showed no interaction with the human orthologue. Notably, the effects of inosine on synaptic transmission and plasticity were abrogated by adenosine deaminase and by inhibiting equilibrative nucleoside transporters (ENT) with dipyridamole and NBTI. This shows that the impact of inosine on hippocampal synaptic transmission and plasticity is not due to a direct activation of adenosine receptors but is instead due to an indirect modification of the tonic activation of these adenosine receptors through an ENT-mediated modification of the extracellular levels of adenosine.

Inhibition of UBA6 by inosine augments tumour immunogenicity and responses.

Anti-cancer immunity and response to immune therapy is influenced by the metabolic states of the tumours. Immune checkpoint blockade therapy (ICB) is known to involve metabolic adaptation, however, the mechanism is not fully known. Here we show, by metabolic profiling of plasma samples from melanoma-bearing mice undergoing anti-PD1 and anti-CTLA4 combination therapy, that higher levels of purine metabolites, including inosine, mark ICB sensitivity. Metabolic profiles of ICB-treated human cancers confirm the association between inosine levels and ICB sensitivity. In mouse models, inosine supplementation sensitizes tumours to ICB, even if they are intrinsically ICB resistant, by enhancing T cell-mediated cytotoxicity and hence generating an immunologically hotter microenvironment. We find that inosine directly inhibits UBA6 in tumour cells, and lower level of UBA6 makes the tumour more immunogenic and this is reflected in favourable outcome following ICB therapy in human melanomas. Transplanted mouse melanoma and breast cancer cells with genetic ablation of Uba6 show higher sensitivity to ICB than wild type tumours. Thus, we provide evidence of an inosine-regulated UBA6-dependent pathway governing tumour-intrinsic immunogenicity and hence sensitivity to immune checkpoint inhibition, which might provide targets to overcome ICB resistance.

miR-351 promotes atherosclerosis in diabetes by inhibiting the ITGB3/PIK3R1/Akt pathway and induces endothelial cell injury and lipid accumulation.

BACKGROUND: The miR-351 gene is significantly upregulated in diabetic mice with atherosclerosis. However, the mechanism by which its presence is important for the overall disease has not been elucidated. Therefore, this study will investigate the mechanism of miR-351 in the process of diabetes mellitus with atherosclerosis through miR-351 gene knockout mice. METHODS: In this study, miR-351-/- C57BL/6 mice were first induced to form a type 2 diabetes mellitus model with atherosclerosis by STZ injection and a high-fat diet. Pathological tests (oil red O, HE, and Masson staining) combined with biochemical indices (TC, TG, LDL-C, HDL-C, TNF-α, hs-CRP, NO, SOD, MDA, CAT, and GSH-Px) were performed to evaluate the pathological degree of atherosclerosis in each group. Mouse aortic endothelial cells were treated with oxidized low-density lipoprotein (ox-LDL) and 30 mM glucose to establish a diabetic atherosclerosis cell model. Combined with cell oil red O staining and flow cytometry, the effects of silencing miR-351 on lipid accumulation and cell apoptosis in the diabetic atherosclerosis cell model were determined. Fluorescence in situ hybridization was used to detect the localization and transcription levels of miR-351 in cells. The target genes of miR-351 were predicted by bioinformatics and verified by dual-luciferase activity reporting. Western blotting was used to detect the expression levels of phosphorylated inosine 3-kinase regulatory subunit 1 (PIK3R1)/serine/threonine kinase 1 (Akt) and apoptosis-related proteins after transfection with integrin subunit ß3 (ITGB3) small interfering ribonucleic acid (siRNA). RESULTS: The expression of the miR-351 gene was significantly increased in the high-fat wild-type (HWT) group, and its expression was significantly decreased in the knockout mice. Silencing miR-351 effectively alleviated atherosclerosis in mice. The levels of miR-351 expression, apoptosis, lipid accumulation, and oxidative stress in ox-LDL + high glucose-induced endothelial cells were significantly increased. These phenomena were effectively inhibited in lentivirus-infected miR-351-silenced cell lines. Bioinformatics predicted that miR-351-5p could directly target the ITGB3 gene. Transfection of ITGB3 siRNA reversed the downregulation of apoptosis, decreased oil accumulation, and decreased oxidative stress levels induced by miR-351 silencing. In addition, it inhibited the activation of the PIK3R1/Akt pathway. CONCLUSION: Silencing miR-351 upregulates ITGB3 and activates the PIK3R1/Akt pathway, thereby exerting anti-apoptosis and protective effects on endothelial cells.

Succinate and inosine coordinate innate immune response to bacterial infection.

Macrophages restrict bacterial infection partly by stimulating phagocytosis and partly by stimulating release of cytokines and complement components. Here, we treat macrophages with LPS and a bacterial pathogen, and demonstrate that expression of cytokine IL-1ß and bacterial phagocytosis increase to a transient peak 8 to 12 h post-treatment, while expression of complement component 3 (C3) continues to rise for 24 h post-treatment. Metabolomic analysis suggests a correlation between the cellular concentrations of succinate and IL-1ß and of inosine and C3. This may involve a regulatory feedback mechanism, whereby succinate stimulates and inosine inhibits HIF-1α through their competitive interactions with prolyl hydroxylase. Furthermore, increased level of inosine in LPS-stimulated macrophages is linked to accumulation of adenosine monophosphate and that exogenous inosine improves the survival of bacterial pathogen-infected mice and tilapia. The implications of these data suggests potential therapeutic tools to prevent, manage or treat bacterial infections.

Apoptotic brown adipocytes enhance energy expenditure via extracellular inosine.

Brown adipose tissue (BAT) dissipates energy1,2 and promotes cardiometabolic health3. Loss of BAT during obesity and ageing is a principal hurdle for BAT-centred obesity therapies, but not much is known about BAT apoptosis. Here, untargeted metabolomics demonstrated that apoptotic brown adipocytes release a specific pattern of metabolites with purine metabolites being highly enriched. This apoptotic secretome enhances expression of the thermogenic programme in healthy adipocytes. This effect is mediated by the purine inosine that stimulates energy expenditure in brown adipocytes by the cyclic adenosine monophosphate-protein kinase A signalling pathway. Treatment of mice with inosine increased BAT-dependent energy expenditure and induced 'browning' of white adipose tissue. Mechanistically, the equilibrative nucleoside transporter 1 (ENT1, SLC29A1) regulates inosine levels in BAT: ENT1-deficiency increases extracellular inosine levels and consequently enhances thermogenic adipocyte differentiation. In mice, pharmacological inhibition of ENT1 as well as global and adipose-specific ablation enhanced BAT activity and counteracted diet-induced obesity, respectively. In human brown adipocytes, knockdown or blockade of ENT1 increased extracellular inosine, which enhanced thermogenic capacity. Conversely, high ENT1 levels correlated with lower expression of the thermogenic marker UCP1 in human adipose tissues. Finally, the Ile216Thr loss of function mutation in human ENT1 was associated with significantly lower body mass index and 59% lower odds of obesity for individuals carrying the Thr variant. Our data identify inosine as a metabolite released during apoptosis with a 'replace me' signalling function that regulates thermogenic fat and counteracts obesity.

SAXS Analysis and Characterization of Anticancer Activity of PNP-UDP Family Protein from Putranjiva roxburghii.

A class of plant defense and storage proteins, including Putranjiva roxburghii PNP protein (PRpnp), belongs to PNP-UDP family. The PRpnp and related plant proteins contain a disrupted PNP-UDP domain as revealed in previous studies. In PRpnp, the insert disrupting the domain contains the trypsin inhibitory site. In the present work, we analyzed native PRpnp (nPRpnp) complex formation with trypsin and inosine using SAXS experiments and established its dual functionality. Results indicated a relatively compact nPRpnp:Inosine structure, whereas trypsin complex showed conformational changes/flexibility. nPRpnp also exhibited a strong anti-cancer activity toward breast cancer (MCF-7), prostate cancer (DU-145) and hepatocellular carcinoma (HepG2) cell lines. MCF-7 and DU-145 were more sensitive to nPRpnp treatment as compared to HepG2. However, nPRpnp treatment showed no effect on the viability of HEK293 cells indicating that nPRpnp is specific for targeting the viability of only cancer cells. Further, acridine orange, DAPI and DNA fragmentation studies showed that cytotoxic effect of nPRpnp is mediated through induction of apoptosis as evident from the apoptosis-associated morphological changes and nuclear fragmentation observed after PRpnp treatment of cancer cells. These results suggest that PRpnp has the potential to be used as an anticancer agent. This is first report of anticancer activity as well as SAXS-based analysis for a PNP enzyme with trypsin inhibitory activity.

Inosine attenuates 3-nitropropionic acid-induced Huntington's disease-like symptoms in rats via the activation of the A2AR/BDNF/TrKB/ERK/CREB signaling pathway.

Huntington's disease (HD) is an autosomal dominant inherited neurodegenerative disease characterized by involuntary bizarre movements, psychiatric symptoms, dementia, and early death. Several studies suggested neuroprotective activities of inosine; however its role in HD is yet to be elucidated. The current study aimed to demonstrate the neuroprotective effect of inosine in 3-nitropropionic acid (3-NP)-induced neurotoxicity in rats while investigating possible underlying mechanisms. Rats were randomly divided into five groups; group 1 received i.p. injections of 1% DMSO, whereas groups 2, 3, 4, and 5 received 3-NP (10 mg/kg, i.p.) for 14 days, concomitantly with inosine (200 mg/kg., i.p.) in groups 3, 4, and 5, SCH58261, a selective adenosine 2A receptor (A2AR) antagonist, (0.05 mg/kg, i.p.) in group 4, and PD98059, an extracellular signal-regulated kinase (ERK) inhibitor, (0.3 mg/kg, i.p.) in group 5. Treatment with inosine mitigated 3-NP-induced motor abnormalities and body weight loss. Moreover, inosine boosted the striatal brain-derived neurotrophic factor (BDNF) level, p-tropomyosin receptor kinase B (TrKB), p-ERK, and p-cAMP response element-binding protein (CREB) expression, which subsequently suppressed oxidative stress biomarkers (malondialdehyde and nitric oxide) and pro-inflammatory cytokines (tumor necrosis factor alpha and interleukin-1ß) and replenished the glutathione content. Similarly, histopathological analyses revealed decreased striatal injury score, the expression of the glial fibrillary acidic protein, and neuronal loss after inosine treatment. These effects were attenuated by the pre-administration of SCH58261 or PD98059. In conclusion, inosine attenuated 3-NP-induced HD-like symptoms in rats, at least in part, via the activation of the A2AR/BDNF/TrKB/ERK/CREB signaling pathway.

Differential Effects of Toll-Like Receptor Activation and Differential Mediation by MAP Kinases of Immune Responses in Microglial Cells.

Microglial activation is believed to play a role in many psychiatric and neurodegenerative diseases. Based largely on evidence from other cell types, it is widely thought that MAP kinase (ERK, JNK and p38) signalling pathways contribute strongly to microglial activation following immune stimuli acting on toll-like receptor (TLR) 3 or TLR4. We report here that exposure of SimA9 mouse microglial cell line to immune mimetics stimulating TLR4 (lipopolysaccharide-LPS) or TLR7/8 (resiquimod/R848), results in marked MAP kinase activation, followed by induction of nitric oxide synthase, and various cytokines/chemokines. However, in contrast to TLR4 or TLR7/8 stimulation, very few effects of TLR3 stimulation by poly-inosine/cytidine (polyI:C) were detected. Induction of chemokines/cytokines at the mRNA level by LPS and resiquimod were, in general, only marginally affected by MAP kinase inhibition, and expression of TNF, Ccl2 and Ccl5 mRNAs, along with nitrite production, were enhanced by p38 inhibition in a stimulus-specific manner. Selective JNK inhibition enhanced Ccl2 and Ccl5 release. Many distinct responses to stimulation of TLR4 and TLR7 were observed, with JNK mediating TNF protein induction by the latter but not the former, and suppressing Ccl5 release by the former but not the latter. These data reveal complex modulation by MAP kinases of microglial responses to immune challenge, including a dampening of some responses. They demonstrate that abnormal levels of JNK or p38 signalling in microglial cells will perturb their profile of cytokine and chemokine release, potentially contributing to abnormal inflammatory patterns in CNS disease states.

Inosine mitigated diabetic peripheral neuropathy via modulating GLO1/AGEs/RAGE/NF-κB/Nrf2 and TGF-ß/PKC/TRPV1 signaling pathways.

Inosine is a dietary supplement that is widely used for managing numerous central neurological disorders. Interestingly, recent experimental investigation of inosine revealed its potential to promote peripheral neuroprotection after sciatic nerve injury. Such investigation has guided the focus of the current study to expose the potential of inosine in mitigating diabetic peripheral neuropathy (DPN) in rats and to study the possible underlying signaling pathways. Adult male Wistar rats were arbitrarily distributed into four groups. In the first group, animals received saline daily for 15 days whereas rats of the remaining groups received a single injection of both nicotinamide (50 mg/Kg/i.p.) and streptozotocin (52.5 mg/Kg/i.p.) for DPN induction. Afterward, inosine (10 mg/Kg/p.o.) was administered to two groups, either alone or in combination with caffeine (3.75 mg/Kg/p.o.), an adenosine receptor antagonist. As a result, inosine showed a hypoglycemic effect, restored the sciatic nerve histological structure, enhanced myelination, modulated conduction velocities and maintained behavioral responses. Furthermore, inosine increased GLO1, reduced AGE/RAGE axis and oxidative stress which in turn, downregulated NF-κB p65 and its phosphorylated form in the sciatic nerves. Inosine enhanced Nrf2 expression and its downstream molecule HO-1, resulting in increased CAT and SOD along with lowered MDA. Moreover, pain was relieved due to suppression of PKC and TRPV1 expression, which ultimately lead to reduced SP and TGF-ß. The potential effects of inosine were nearly blocked by caffeine administration; this emphasizes the role of adenosine receptors in inosine-mediated neuroprotective effects. In conclusion, inosine alleviated hyperglycemia-induced DPN via modulating GLO1/AGE/RAGE/NF-κB p65/Nrf2 and TGF-ß/PKC/TRPV1/SP pathways.