The Journal of physiology
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The Journal of physiology · Feb 2020
Randomized Controlled TrialNicotinamide riboside does not alter mitochondrial respiration, content or morphology in skeletal muscle from obese and insulin-resistant men.
This is the first long-term human clinical trial to report on effects of nicotinamide riboside (NR) on skeletal muscle mitochondrial function, content and morphology. NR supplementation decreases nicotinamide phosphoribosyltransferase (NAMPT) protein abundance in skeletal muscle. NR supplementation does not affect NAD metabolite concentrations in skeletal muscle. Respiration, distribution and quantity of muscle mitochondria are unaffected by NR. NAMPT in skeletal muscle correlates positively with oxidative phosphorylation Complex I, sirtuin 3 and succinate dehydrogenase. ⋯ Preclinical evidence suggests that the nicotinamide adenine dinucleotide (NAD+ ) precursor nicotinamide riboside (NR) boosts NAD+ levels and improves diseases associated with mitochondrial dysfunction. We aimed to determine if dietary NR supplementation in middle-aged, obese, insulin-resistant men affects mitochondrial respiration, content and morphology in skeletal muscle. In a randomized, placebo-controlled clinical trial, 40 participants received 1000 mg NR or placebo twice daily for 12 weeks. Skeletal muscle biopsies were collected before and after the intervention. Mitochondrial respiratory capacity was determined by high-resolution respirometry on single muscle fibres. Protein abundance and mRNA expression were measured by Western blot and quantitative PCR analyses, respectively, and in a subset of the participants (placebo n = 8; NR n = 8) we quantified mitochondrial fractional area and mitochondrial morphology by laser scanning confocal microscopy. Protein levels of nicotinamide phosphoribosyltransferase (NAMPT), an essential NAD+ biosynthetic enzyme in skeletal muscle, decreased by 14% with NR. However, steady-state NAD+ levels as well as gene expression and protein abundance of other NAD+ biosynthetic enzymes remained unchanged. Neither respiratory capacity of skeletal muscle mitochondria nor abundance of mitochondrial associated proteins were affected by NR. Moreover, no changes in mitochondrial fractional area or network morphology were observed. Our data do not support the hypothesis that dietary NR supplementation has significant impact on skeletal muscle mitochondria in obese and insulin-resistant men. Future studies on the effects of NR on human skeletal muscle may include both sexes and potentially provide comparisons between young and older people.
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The Journal of physiology · Feb 2020
Synaptic cleft microenvironment influences potassium permeation and synaptic transmission in hair cells surrounded by calyx afferents in the turtle.
In central regions of vestibular semicircular canal epithelia, the [K+ ] in the synaptic cleft ([K+ ]c ) contributes to setting the hair cell and afferent membrane potentials; the potassium efflux from type I hair cells results from the interdependent gating of three conductances. Elevation of [K+ ]c occurs through a calcium-activated potassium conductance, GBK , and a low-voltage-activating delayed rectifier, GK(LV) , that activates upon elevation of [K+ ]c . Calcium influx that enables quantal transmission also activates IBK , an effect that can be blocked internally by BAPTA, and externally by a CaV 1.3 antagonist or iberiotoxin. Elevation of [K+ ]c or chelation of [Ca2+ ]c linearizes the GK(LV) steady-state I-V curve, suggesting that the outward rectification observed for GK(LV) may result largely from a potassium-sensitive relief of Ca2+ inactivation of the channel pore selectivity filter. Potassium sensitivity of hair cell and afferent conductances allows three modes of transmission: quantal, ion accumulation and resistive coupling to be multiplexed across the synapse. ⋯ In the vertebrate nervous system, ions accumulate in diffusion-limited synaptic clefts during ongoing activity. Such accumulation can be demonstrated at large appositions such as the hair cell-calyx afferent synapses present in central regions of the turtle vestibular semicircular canal epithelia. Type I hair cells influence discharge rates in their calyx afferents by modulating the potassium concentration in the synaptic cleft, [K+ ]c , which regulates potassium-sensitive conductances in both hair cell and afferent. Dual recordings from synaptic pairs have demonstrated that, despite a decreased driving force due to potassium accumulation, hair cell depolarization elicits sustained outward currents in the hair cell, and a maintained inward current in the afferent. We used kinetic and pharmacological dissection of the hair cell conductances to understand the interdependence of channel gating and permeation in the context of such restricted extracellular spaces. Hair cell depolarization leads to calcium influx and activation of a large calcium-activated potassium conductance, GBK , that can be blocked by agents that disrupt calcium influx or buffer the elevation of [Ca2+ ]i , as well as by the specific KCa 1.1 blocker iberiotoxin. Efflux of K+ through GBK can rapidly elevate [K+ ]c , which speeds the activation and slows the inactivation and deactivation of a second potassium conductance, GK(LV) . Elevation of [K+ ]c or chelation of [Ca2+ ]c linearizes the GK(LV) steady-state I-V curve, consistent with a K+ -dependent relief of Ca2+ inactivation of GK(LV) . As a result, this potassium-sensitive hair cell conductance pairs with the potassium-sensitive hyperpolarization-activated cyclic nucleotide-gated channel (HCN) conductance in the afferent and creates resistive coupling at the synaptic cleft.
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The Journal of physiology · Jan 2020
Active cooling temperature required to achieve therapeutic hypothermia correlates with short-term outcome in neonatal hypoxic-ischaemic encephalopathy.
Hypoxic-ischaemic encephalopathy (HIE) affects 2-4/1000 live term births. Treatment with therapeutic hypothermia (TH) improves the long-term neurodevelopmental outcome of neonates with moderate to severe HIE. However, early prediction of outcome still remains challenging, and no reliable and easily obtainable biomarker has been identified to date. Neonates with HIE display impaired thermoregulation, resulting in spontaneous hypothermia. The degree of cooling required to achieve TH may therefore act as a biomarker of injury severity. The present study demonstrates a correlation between servo-controlled mattress temperature during TH and short-term outcome. Neonates with an unfavourable outcome require less cooling to maintain a core temperature between 33 and 34°C during TH compared to neonates with a favourable outcome. The degree of impaired temperature regulation was strongly associated with a high magnetic resonance imaging injury score and death. Cooling device output temperature is a potential and easily obtainable early physiological biomarker of outcome in infants with HIE undergoing TH. ⋯ Neonatal hypoxic-ischaemic encephalopathy (HIE) is a leading cause of death and disability in children. Therapeutic hypothermia (TH) at 33.5°C for 72 h is the only therapy to date shown to improve outcome in moderate to severe HIE; however, assessment of severity and prediction of outcome remains challenging. Infants with HIE display significant physiological perturbations, including spontaneous hypothermia. We hypothesized that neonates with more severe brain injury on magnetic resonance imaging (MRI) would exhibit a greater degree of spontaneous hypothermia, and thus require less active cooling to attain TH. Twenty-eight neonates with moderate or severe HIE treated with TH were included in the present study. MRI images obtained on day of life 4-7 were scored according to standardized injury criteria. Unfavourable outcome was defined as death or significant grey matter injury on MRI according to a previously validated scoring system. A significantly higher cooling device output temperature was seen in infants with an unfavourable outcome. All neonates who required the mattress to provide a temperature ≥32°C to maintain their core body temperature at 33.5°C had a high likelihood of unfavourable outcome (likelihood ratio = 14.4). By contrast, infants who never required a device output temperature ≥32°C had a low likelihood of an unfavourable outcome (likelihood ratio = 0.07, P < 0.001). Infants with significant grey matter injury on MRI require less active cooling to maintain target temperature during TH. The cooling device output temperature has the potential to be an easily accessible physiological biomarker and predictor of injury and mortality in neonates with moderate or severe HIE.
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The Journal of physiology · Jan 2020
Differential impact of two critical respiratory centres in opioid-induced respiratory depression in awake mice.
The main cause of death from opioid overdose is respiratory depression due to the activation of µ-opioid receptors (MORs). We conditionally deleted MORs from neurons in two key areas of the brainstem respiratory circuitry (the Kölliker-Fuse nucleus (KF) and pre-Bötzinger complex (preBötC)) to determine their role in opioid-induced respiratory disturbances in adult, awake mice. Deletion of MORs from KF neurons attenuated respiratory rate depression at all doses of morphine. Deletion of MORs from preBötC neurons attenuated rate depression at the low dose, but had no effect on rate following high doses of morphine. Instead, high doses of morphine increased the occurrence of apnoeas. The results indicate that opioids affect distributed key areas of the respiratory network in a dose-dependent manner and countering the respiratory effects of high dose opioids via the KF may be an effective approach to combat overdose. ⋯ The primary cause of death from opioid overdose is respiratory failure. High doses of opioids cause severe rate depression and increased risk of fatal apnoea, which correlate with increasing irregularities in breathing pattern. µ-Opioid receptors (MORs) are widely distributed throughout the brainstem respiratory network, but the mechanisms underlying respiratory depression are poorly understood. The medullary pre-Bötzinger complex (preBötC) and the pontine Kölliker-Fuse nucleus (KF) are considered critical for inducing opioid-related respiratory disturbances. We used a conditional knockout approach to investigate the roles and relative contribution of MORs in KF and preBötC neurons in opioid-induced respiratory depression in awake adult mice. The results revealed dose-dependent and region-specific opioid effects on the control of both respiratory rate and pattern. Respiratory depression induced by an anti-nociceptive dose of morphine was significantly attenuated following deletion of MORs from either the KF or the preBötC, suggesting cumulative network effects on respiratory rate control at low opioid doses. Deletion of MORs from KF neurons also relieved rate depression at near-maximal respiratory depressant doses of morphine. Meanwhile, deletion of MORs from the preBötC had no effect on rate following administration of high doses of morphine. Instead, a severe ataxic breathing pattern emerged with many apnoeas. We conclude that opioids affect distributed areas of the respiratory network and opioid-induced respiratory depression cannot be attributed to only one area in isolation. However, countering the effects of near maximal respiratory depressant doses of opioids in the KF may be a powerful approach to combat opioid overdose.