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.

Assessment of the effects of dentifrice on periodontal disease biomarkers in gingival crevicular fluid.

BACKGROUND: Periodontal disease has been studied primarily from clinical outcomes in lengthy human studies. Comprehensive biochemical profiling (metabolomics) has become a powerful tool for disease characterization and biomarker discovery. In a previous study, we performed a metabolomic analysis of gingival crevicular fluid collected from healthy, gingivitis, and periodontitis sites. Many metabolites associated with inflammation, oxidative stress, tissue degradation, and bacterial metabolism were found to be significantly induced by the diseases. METHODS: A panel of 10 markers was selected from the previous metabolomic study based on their statistical significance. Thirty-nine chronic periodontitis subjects were randomly assigned to a toothpaste regimen: control dentifrice (n = 21) or triclosan-containing dentifrice ([CT] n = 18). Subjects were instructed to use their assigned dentifrice twice daily for 6 weeks. Gingival crevicular fluid samples from six healthy, six gingivitis, and three periodontitis sites were collected from each subject at baseline, 1 week, and 6 weeks. The relative levels of the markers in the samples were determined by mass spectrometry. One-sided matched-paired t tests were performed to compare data from healthy, gingivitis, and periodontitis sites. RESULTS: Statistical analysis indicates that CT significantly decreased the levels of inosine, lysine, putrescine, and xanthine at the gingivitis sites as early as week 1. In contrast, control dentifrice had little effect. CONCLUSIONS: This result provides biochemical confirmation for the therapeutic effects of CT on gingivitis. Biomarkers were significantly altered by CT before clinical changes were observed, suggesting that the markers have predicative value for disease state assessment.

Pre-conditioning activates adenosine utilization in a cost-effective way during myocardial ischaemia.

During pre-conditioning the interstitial concentration of adenosine, in contrast to lactate, presents a die-away curve-pattern for every successive episode of ischaemia. This die-away pattern might not necessarily be attributed to diminished adenosine production. The present study was undertaken to investigate whether pre-conditioning alters the metabolic turnover of adenosine as observed by the lactate production during ischaemia. Interstitial levels of metabolites in pre-conditioned (n=21) and non-preconditioned (n=21) porcine hearts were monitored with microdialysis probes inserted in both ischaemic and non-ischaemic tissue in an open chest heart model. Three subgroups perturbated with either plain microdialysis buffer (control), buffer containing adenosine (375 microM), or buffer containing deoxyadenosine (375 microM) were studied. All animals were subjected to 90 min of equilibrium microdialysis before 40 min of regional myocardial ischaemia and 120 min of reperfusion. Pre-conditioning consisted of four repetitive episodes of 10 min of ischaemia and 20 min of reperfusion. Significantly higher levels of inosine and lactate were found in the ischaemic tissue of the pre-conditioned subgroup receiving adenosine (P < 0.05) compared with the other two subgroups receiving deoxyadenosine and plain buffer, respectively. This difference was only valid for pre-conditioned ischaemic myocardium, and hence equal amounts of inosine and lactate were produced in the non-preconditioned ischaemic myocardium regardless of the presence of adenosine or deoxyadenosine. In the non-ischaemic myocardium baseline levels of metabolites were measured in all subgroups. Pre-conditioning favoured degradation of exogenous adenosine to inosine successively ending up in enhanced lactate production. This was probably because of the involvement of the hexose monophosphate pathway in the pre-conditioned ischaemic myocardium. This route may therefore be supplementary in energy metabolism as a metabolic flow can be started by adenosine ending up in lactate without initial adenosine 5'-triphosphate (ATP) investment. Utilization of adenosine in this way may also explain the successive die-away pattern of adenosine seen in consecutive pre-conditioning cycles.

Noninvasive assessment of myocardial viability by measuring adenosine triphosphate degradation product in coronary sinus effluent of preserved dog heart.

Isolated dog hearts (n = 19) were preserved in cold Collins solution at 4 degrees C for 1 to 26 hours. At the end of preservation the coronary system was flushed out by cold cardioplegic solution, and the hearts were reperfused with supporting dogs. Myocardial biopsies at the end of preservation and coronary sinus effluents at the time of flush-out were analyzed for adenosine 5'-triphosphate and inosine. Both the degree of depletion in adenosine triphosphate content and the accumulation of inosine in the myocardium during preservation correlated significantly to the preservation time (correlation coefficient = -0.85 and 0.86, respectively). There was a significant correlation between the myocardial inosine level and total amount of inosine released into coronary sinus effluent (correlation coefficient = 0.91, p less than 0.001). Also, a significant correlation between the total amount of inosine in coronary sinus effluent and cellular level of adenosine triphosphate was found (correlation coefficient = -0.86, p less than 0.001). Five hearts that failed to resume beating showed significantly higher amounts of released inosine than the other hearts (3.3 +/- 0.9 versus 1.5 +/- 0.5 mumol, p less than 0.005). These results indicate that the inosine level in the coronary sinus effluent reflects the myocardial energy state, and it is suggested that the measurement of inosine level in the effluent may be applicable as a noninvasive method for assessment of myocardial viability in heart preservation.

Urid acid accumulated in isolated kidney grafts: an assessment of agonal ischaemia.

Hypoxanthine-xanthine washed out from kidney tissue during preservation has previously been found to be a reliable measure of the in vitro assessment of the deleterious effect of ischaemia on the functional regenerative ability of the graft. We have now studied the question: can uric acid accumulation in the isolated kidney graft be employed as a retrospective measure of the agonal ischaemia? We have found that uric acid alone accumulates in renal tissue during the agonal phase, in an amount that remains unchanged during the subsequent in vitro ischaemia. The determination of hypoxanthine-xanthine and uric acid in kidney perfusate samples during preservation must thus be presumed to be an optimal clinical-chemical method of assessing the graft ischaemia.