Dr Namrata Chowdhury

Background Chondrocytes rely primarily on glycolysis to meet their energy requirements, but can support cell survival and matrix synthesis during periods of nutrient stress by enhancing glycolysis with mitochondrial respiration. Accessing this ‘spare respiratory capacity’ requires optimal mitochondrial function. Impaired mitochondrial function is implicated in osteoarthritis (OA). Metabolic adaptation is evident in early-stage OA, however cartilage from late-stage disease does not seem to have this flexibility. A deeper understanding of these complex metabolic pathways may identify new markers of disease stage, and support therapeutic strategies for treating OA. Objectives Metabolomics has the potential to reveal pathological pathways and identify novel biomarkers. The aim was to identify metabolic processes involved in early stage disease by analysis of metabolites and metabolic function in pro-inflammatory models of cartilage degradation. Methods Macroscopically normal articular cartilage was obtained from equine and bovine metacarpophalangeal joints. Equine cartilage explants (n=6), and primary chondrocytes seeded at 105,000/cm2 (n=4), were cultured for 7 days in serum-free DMEM (Gibco) with or without 10 ng/ml equine interleukin-1β (IL-1β) and 10 ng/ml tumour necrosis factor-α (TNF-α). Secretome metabolite levels were measured using AbsoluteIDQ p180 targeted metabolomics kit (Biocrates), with Waters Xevo TQ-S mass spectrometer coupled to an Acquity UPLC system. PCA and OPLS-DA were performed using SIMCA-P v12.0 software. Metabolic function of primary equine (n=9) and bovine chondrocytes (n=3) was determined using Seahorse XFp and XFe24 analyzers. Cells were treated with species-specific 10 ng/ml IL-1β and/or 10 ng/ml TNF-α for 18 hour, and metabolically challenged with the Mito Stress Test. Metabolite levels, and oxygen consumption rates, were normalised to total cell protein, and values analysed by ANOVA with Tukey's multiple comparison post-tests. Results Cytokine treatment decreased proline, ornithine and alpha-aminoadipic acid (p

Increasing the sampling resolution when examining plasma hormone and metabolite profiles will more accurately describe diurnal/circadian rhythms and expose previously undiscovered ultradian rhythms that underpin metabolic physiology(1). In human research studies this must be tempered by the practical, ethical and safety concerns of repeated cannulation/blood draws. Here we examine the correlation between hormone and metabolite profiles present in interstitial fluid microdialysate(2), and plasma to help solve this dilemma. Hormonal markers of circadian phase (melatonin, cortisol) and metabolites will show similar profiles in plasma and interstitial fluid. Compare time-series hormone and metabolite profiles collected in interstitial fluid using a novel ambulatory microdialysis collection device (U-RHYTHM) with simultaneously drawn plasma samples. All study protocols were reviewed by Health Sciences Faculty Research Ethics Committee, University of Bristol. Fasted healthy male volunteers aged 18-35 (n=3) were fed a standardised breakfast (08:00), lunch (13:00), dinner (19:00) and snack (22:00) (2225kCal [83g protein, 273g carbohydrate, 83g fat, 27g fibre]). Participants remained on the study bed, lights off/sleep occurred between 23:00-07:00 (

Patients with liver cirrhosis can develop hyperammonemia and hepatic encephalopathy (HE), accompanied by pronounced daytime sleepiness. Previous studies with healthy volunteers show that experimental increase in blood ammonium levels increases sleepiness and slows the waking EEG. As ammonium increases adenosine levels in vitro, and adenosine is a known regulator of sleep/wake homeostasis, we hypothesized that the sleepiness-inducing effect of ammonium is mediated by adenosine. Eight adult male Wistar rats were fed with an ammonium-enriched diet for 4 weeks; eight rats on standard diet served as controls. Each animal was implanted with electroencephalography/electromyography (EEG/EMG) electrodes and a microdialysis probe. Sleep EEG recording and cerebral microdialysis were carried out at baseline and after 6 hours of sleep deprivation. Adenosine and metabolite levels were measured by HPLC and targeted LC/MS metabolomics, respectively. Baseline adenosine and metabolite levels (12 of 16 amino acids, taurine, t4-hydroxy-proline and acetylcarnitine) were lower in hyperammonemic animals, while putrescine was higher. After sleep deprivation, hyperammonemic animals exhibited a larger increase in adenosine levels, and a number of metabolites showed a different time-course in the two groups. In both groups the recovery period was characterized by a significant decrease in wakefulness/increase in NREM and REM sleep. However, while control animals exhibited a gradual compensatory effect, hyperammonemic animals showed a significantly shorter recovery phase. In conclusion, the adenosine/metabolite/EEG response to sleep deprivation was modulated by hyperammonemia, suggesting that ammonia affects homeostatic sleep regulation and its metabolic correlates.

Parkinson’s disease (PD) is a chronic disorder that presents a range of premotor signs, such as sleep disturbances and cognitive decline, which are key non-motor features of the disease. Increasing evidence of a possible association between sleep disruption and the neurodegenerative process suggests that sleep impairment could produce a detectable metabolic signature on the disease. In order to integrate neurocognitive and metabolic parameters, we performed untargeted and targeted metabolic profiling of the rotenone PD model in a chronic sleep restriction (SR) (6 h/day for 21 days) condition. We found that SR combined with PD altered several behavioural (reversal of locomotor activity impairment; cognitive impairment; delay of rest-activity rhythm) and metabolic parameters (branched-chain amino acids, tryptophan pathway, phenylalanine, and lipoproteins, pointing to mitochondrial impairment). If combined, our results bring a plethora of parameters that represents reliable early-phase PD biomarkers which can easily be measured and could be translated to human studies.

Misalignment between internal circadian rhythmicity and externally imposed behavioral schedules, such as occurs in shift workers, has been implicated in elevated risk of metabolic disorders. To determine underlying mechanisms, it is esse ntial to assess whether and how peripheral clocks are disturbed during shift work and to what extent this is linked to the central suprachiasmatic nuclei (SCN) pacemaker and/or misaligned behavioral time cues. Investigating rhythms in circulating metabolites as biomarkers of peripheral clock distur- bances may offer new insight s. We evaluated the impact of misaligned sleep/wake and feeding/fasting cycles on circulating metabolites using a targeted metabolomics approach. Sequential plasma samples obtained during a 24-h constant routine that followed a 3-d simulated night-s hift schedule, compared with a simulated day-shift schedule, we re analyzed for 132 circulating metabolites. Nearly half of these metabolites showed a 24-h rhyth- micity under constant routine following either or both simulated shift schedules. However, while tradition al markers of the circadian clock in the SCN — melatonin, cortisol, and PER3 expression — maintained a stable phase alignment after both schedules, only a few metabo- lites did the same. Many showed reversed rhythms, lost their rhythms, or showed rhythmicity only under constant routine fol- lowing the night-shift schedule. Here, 95% of the metabolites with a 24-h rhythmicity showed rhythms that were driven by behavior- al time cues externally imposed during the preceding simulated shift schedule rather than being driven by the central SCN circa- dian clock. Characterization of these metabolite rhythms will pro- vide insight into the underlying mechanisms linking shift work and metabolic disorders

Bile acids are trans-genomic molecules arising from the concerted metabolism of the human host and the intestinal microbiota and are important for digestion, energy homeostasis and metabolic regulation. While diurnal variation has been demonstrated in the enterohepatic circulation and the gut microbiota, existing human data are poorly resolved, and the influence of the host circadian system has not been determined. Using entrained laboratory protocols, we demonstrate robust daily rhythms in the circulating bile acid pool in healthy male participants. We identify temporal relationships between bile acids and plasma lipids and show that these relationships are lost following sleep deprivation. We also highlight that bile acid rhythmicity is predominantly lost when environmental timing cues are held constant. Here we show that the environment is a stronger determinant of these temporal dynamics than the intrinsic circadian system of the host. This has significance for the intimate relationship between circadian timing and metabolism.Bile acids are important for digestion, energy homeostasis and metabolic regulation. Here the authors show daily rhythms in the circulating bile acid pool which are lost when environmental timing cues are held constant indicating that environment is a stronger determinant of these dynamics than the circadian system.

Metabolic rhythms include rapid, ultradian (hourly) dynamics, but it remains unclear what their relationship to circadian metabolic rhythms is, and what role meal timing plays in coordinating these ultradian rhythms in metabolism. Here, we characterized widespread ultradian rhythms under ad libitum feeding conditions in the plasma metabolome of the vole, the gold standard animal model for behavioral ultradian rhythms, naturally expressing similar to 2-h foraging rhythms throughout the day and night. These ultradian metabolite rhythms co-expressed with diurnal 24-h rhythms in the same metabolites and did not align with food intake patterns. Specifically, under light-dark entrained conditions we showed twice daily entrainment of phase and period of ultradian behavioral rhythms associated with phase adjustment of the ultradian cycle around the light-dark and dark-light transitions. These ultradian activity patterns also drove an ultradian feeding pattern. We used a unique approach to map this behavioral activity/feeding status to high temporal resolution (every 90 min) measures of plasma metabolite profiles across the 24-h light-dark cycle. A total of 148 known metabolites were detected in vole plasma. Supervised, discriminant analysis did not group metabolite concentration by feeding status, instead, unsupervised clustering of metabolite time courses revealed clusters of metabolites that exhibited significant ultradian rhythms with periods different from the feeding cycle. Two clusters with dissimilar ultradian dynamics, one lipid-enriched (period = 3.4 h) and one amino acid-enriched (period = 4.1 h), both showed co-expression with diurnal cycles. A third cluster solely comprised of glycerophospholipids (specifically ether-linked phosphatidylcholines) expressed an 11.9 h ultradian rhythm without co-expressed diurnal rhythmicity. Our findings show coordinated co-expression of diurnal metabolic rhythms with rapid dynamics in feeding and metabolism. These findings reveal that ultradian rhythms are integral to biological timing of metabolic regulation, and will be important in interpreting the impact of circadian desynchrony and meal timing on metabolic rhythms.