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Summary: Using 13C6 glucose labeling coupled to gas chromatography-mass spectrometry and 2D 1H-13C heteronuclear single quantum coherence NMR spectroscopy, we have obtained a comparative high-resolution map of glucose fate underpinning β cell function. In both mouse and human islets, the contribution of glucose to the tricarboxylic acid (TCA) cycle is similar. Pyruvate fueling of the TCA cycle is primarily mediated by the activity of pyruvate dehydrogenase, with lower flux through pyruvate carboxylase. While the conversion of pyruvate to lactate by lactate dehydrogenase (LDH) can be detected in islets of both species, lactate accumulation is 6-fold higher in human islets. Human islets express LDH, with low-moderate LDHA expression and β cell-specific LDHB expression. LDHB inhibition amplifies LDHA-dependent lactate generation in mouse and human β cells and increases basal insulin release. Lastly, cis-instrument Mendelian randomization shows that low LDHB expression levels correlate with elevated fasting insulin in humans. Thus, LDHB limits lactate generation in β cells to maintain appropriate insulin release.

Abstract Sarcopenia is associated with reduced quality of life and increased mortality in patients with end stage liver disease (ESLD). Historically, sarcopenia identification in ESLD utilised L3 skeletal muscle index (SMI). There are few data on muscle quality and function within lower limb muscle groups with high functional relevance. The aim of this prospective case–control study was to evaluate the quadriceps muscle in patients with ESLD. Muscle mass and quality were evaluated using MRI (quadriceps anatomical cross sectional area (ACSA), quadriceps volume index, L3 SMI, quadriceps intermuscular adipose tissue (IMAT)), mid‐arm muscle circumference (MAMC) and ultrasonography (vastus lateralis (VL) thickness and quadriceps ACSA). Muscle strength/function was assessed by handgrip strength, peak quadriceps isokinetic torque and chair rise time. Thirty‐nine patients with ESLD (55 years, 61% male, 48% alcoholic related liver disease (ArLD), 71% Child–Pugh B/C) and 18 age/sex‐matched healthy control participants (HC) were studied. Quadriceps mass was significantly reduced in ESLD versus HC (−17%), but L3 SMI and MAMC were unchanged. Quadriceps IMAT percentage was increased in ESLD (+103%). Handgrip strength (−15%), peak isokinetic torque (−29%), and chair rise time (+56%) were impaired in ESLD. Ultrasound measures of VL thickness (r = 0.56, r = 0.57, r = 0.42) and quadriceps ACSA (r = 0.98, r = 0.86, r = 0.67) correlated to MRI quadriceps ACSA, quadriceps volume and L3 SMI, respectively. Quadriceps muscle mass, quality, and function were impaired in patients with ESLD, whereas conventional assessments of muscle (L3 SMI and MAMC) highlighted no differences between ESLD and HC. Full evaluation of lower limb muscle health is essential in ESLD in order to accurately assess sarcopenia and target future interventions.

Abstract The ADP-ribosyltransferase, PARP1 enzymatically generates and applies the post-translational modification, ADP-Ribose (ADPR). PARP1 roles in genome maintenance are well described, but recent work highlights roles in many fundamental processes including cellular identity and energy homeostasis. Herein, we show in both mouse and human skeletal muscle cells that PARP1-mediated PARylation is a regulator of the myogenic program and the muscle transcriptional response to steroid hormones. Chemical PARP1 modulation impacts the expression of major myocellular proteins, including troponins, key in dictating muscle contractile force. Whilst PARP1 in absence of DNA damage is often assumed to be basally inactive, we show PARylation to be acutely sensitive to extracellular glucose concentrations and the steroid hormone class, glucocorticoids which exert considerable authority over muscle tissue mass. Specifically, we find during myogenesis, a transient and significant rise in PAR. This early-stage differentiation event, if blocked with PARP1 inhibition, reduced the abundance of important muscle proteins in the fully differentiated myotubes. This suggests that PAR targets during early-stage differentiation are central to the proper development of the muscle contractile unit. We also show that reduced PARP1 in myoblasts impacts a variety of metabolic pathways in line with the recorded actions of glucocorticoids. Currently, as both regulators of myogenesis and muscle mass loss, glucocorticoids represent a clinical conundrum. Our work goes on to identify that PARP1 influences transcriptional activation by glucocorticoids of a subset of genes critical to human skeletal muscle pathology. These genes may therefore signify a regulatory battery of targets through which selective glucocorticoid modulation could be achieved. Collectively, our data provide clear links between PARP1-mediated PARylation and skeletal muscle homeostatic mechanisms crucial to tissue mass maintenance and endocrine response.

BackgroundExtra-adrenal glucocorticoid (GC) synthesis at epithelial barriers, such as skin and intestine, has been shown to be important in the local regulation of inflammation. However, the role of local GC synthesis in the lung is less well studied. Based on previous studies and the uncontentious efficacy of corticosteroid therapy in asthma patients, we here investigated the role of 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1/Hsd11b1)-dependent local GC reactivation in the regulation of allergic airway inflammation.MethodsAirway inflammation in Hsd11b1-deficient and C57BL/6 wild type mice was analyzed after injection of lipopolysaccharide (LPS) and anti-CD3 antibody, and in acute and chronic models of airway hypersensitivity induced by house dust mite (HDM) extract. The role of 11β-HSD1 in normal and inflammatory conditions was assessed by high dimensional flow cytometry, histological staining, RT-qPCR analysis, ex vivo tissue cultures, GC-bioassays and protein detection by ELISA and immunoblotting.ResultsHere we show that lung tissue from Hsd11b1-deficient mice synthesized significantly less GC ex vivo compared with wild type animals in response to immune cell stimulation. We further observed a drastically aggravated phenotype in Hsd11b1-deficient mice treated with HDM extract compared to wild type animals. Besides eosinophilic infiltration, Hsd11b1-deficient mice exhibited aggravated neutrophilic infiltration caused by a strong Th17-type immune response.ConclusionWe propose an important role of 11β-HSD1 and local GC in regulating Th17-type rather than Th2-type immune responses in HDM-induced airway hypersensitivity in mice by potentially controlling Toll-like receptor 4 (TLR4) signaling and cytokine/chemokine secretion by airway epithelial cells.

Abstract Background Migraine is a highly prevalent disorder with significant economical and personal burden. Despite the development of effective therapeutics, the causes which precipitate migraine attacks remain elusive. Clinical studies have highlighted altered metabolic flux and mitochondrial function in patients. In vivo animal experiments can allude to the metabolic mechanisms which may underlie migraine susceptibility. Understanding the translational relevance of these studies are important to identifying triggers, biomarkers and therapeutic targets in migraine. Main body Functional imaging studies have suggested that migraineurs feature metabolic syndrome, exhibiting hallmark features including upregulated oxidative phosphorylation yet depleted available free energy. Glucose hypometabolism is also evident in migraine patients and can lead to altered neuronal hyperexcitability such as the incidence of cortical spreading depression (CSD). The association between obesity and increased risk, frequency and worse prognosis of migraine also highlights lipid dysregulation in migraine pathology. Calcitonin gene related peptide (CGRP) has demonstrated an important role in sensitisation and nociception in headache, however its role in metabolic regulation in connection with migraine has not been thoroughly explored. Whether impaired metabolic function leads to increased release of peptides such as CGRP or excessive nociception leads to altered flux is yet unknown. Conclusion Migraine susceptibility may be underpinned by impaired metabolism resulting in depleted energy stores and altered neuronal function. This review discusses both clinical and in vivo studies which provide evidence of altered metabolic flux which contribute toward pathophysiology. It also reviews the translational relevance of animal studies in identifying targets of biomarker or therapeutic development.

Summary: Autophagy is a homeostatic process critical for cellular survival, and its malfunction is implicated in human diseases including neurodegeneration. Loss of autophagy contributes to cytotoxicity and tissue degeneration, but the mechanistic understanding of this phenomenon remains elusive. Here, we generated autophagy-deficient (ATG5−/−) human embryonic stem cells (hESCs), from which we established a human neuronal platform to investigate how loss of autophagy affects neuronal survival. ATG5−/− neurons exhibit basal cytotoxicity accompanied by metabolic defects. Depletion of nicotinamide adenine dinucleotide (NAD) due to hyperactivation of NAD-consuming enzymes is found to trigger cell death via mitochondrial depolarization in ATG5−/− neurons. Boosting intracellular NAD levels improves cell viability by restoring mitochondrial bioenergetics and proteostasis in ATG5−/− neurons. Our findings elucidate a mechanistic link between autophagy deficiency and neuronal cell death that can be targeted for therapeutic interventions in neurodegenerative and lysosomal storage diseases associated with autophagic defect.

Methods of isolating mitochondria commonly utilise mechanical force and shear stress to homogenize tissue followed by purification by multiple rounds of ultracentrifugation. Existing protocols can be time-consuming with some physically impairing integrity of the sensitive mitochondrial double membrane. Here, we describe a method for the recovery of intact, respiring mitochondria from murine skeletal muscle tissue and cell lines using nitrogen cavitation. This protocol results in high-yield, pure and respiring mitochondria without the need for purification gradients or ultracentrifugation. The protocol takes under an hour and requires limited specialised equipment. Our methodology is successful in extracting mitochondria of both cell extracts and skeletal muscle tissue. This represents an improved yield in comparison to many of the existing methods. Western blotting and electron microscopy demonstrate the enrichment of mitochondria with their ultrastructure well-preserved and an absence of contamination from cytoplasmic or nuclear fractions. Using respirometry analysis we show that mitochondria extracted from murine skeletal muscle cell lines (C2C12) and tibialis anterior tissue have an appropriate respiratory control ratio. These measures are indicative of healthy coupled mitochondria. Our method successfully demonstrates the rapid isolation of functional mitochondria and will benefit researchers studying mitochondrial bioenergetics as well as providing greater throughput and application for time-sensitive assays.

Abstract Background Several chronic inflammatory diseases co-exist with and accelerate sarcopenia (reduction in muscle strength, function and mass) and negatively impact on both morbidity and mortality. There is currently limited research on the extent of sarcopenia in such conditions, how to accurately assess it and whether there are generic or disease-specific mechanisms driving sarcopenia. Therefore, this study aims to identify potential mechanisms driving sarcopenia within chronic inflammatory disease via a multi-modal approach; in an attempt to help define potential interventions for future use. Methods This prospective cohort study will consist of a multi-modal assessment of sarcopenia and its underlying mechanisms. Recruitment will target three chronic inflammatory diseases: chronic liver disease (CLD) (n=50), with a subset of NAFLD (n=20), inflammatory bowel disease (IBD) (n=50) and rheumatoid arthritis (RA) (n=50) both before and after therapeutic intervention. In addition, 20 age and sex matched healthy individuals will be recruited for comparison. Participants will undergo 4 assessment visits at weeks 0, 2, 12 and 24. Visits will consist of the following assessments: blood tests, anthropometrics, functional assessment, quadriceps muscle imaging, actigraphy, quality of life questionnaires, food diary collection and muscle biopsy of the vastus lateralis (at weeks 2 and 24 only). In addition, stool and urine samples will be collected for future microbiome and metabolomics analysis. Discussion This is the first study to use a multi-modal assessment model to phenotype sarcopenia in these chronic inflammatory diseases. We hope to identify generic as well as disease-specific mechanisms driving sarcopenia. We appreciate that these cohorts do require separate standards of care treatments which limit comparison between groups. Ethics and dissemination The study is approved by the Health Research Authority - West Midlands Solihull Research Ethics Service Committee Authority (REC reference: 18/WM/0167). Recruitment commenced in January 2019 and will continue until July 2021. The study was halted in March 2020 and again in January 2021 with the COVID-19 pandemic. The findings will be disseminated through peer-reviewed publications and conference presentations. All data will be stored on a secure server. Trial registration ClinicalTrials.gov Identifier: NCT04734496

IntroductionEnd stage liver disease (ESLD) is associated with loss of muscle mass and function, known as sarcopenia, which can increase the risk of complications of ESLD, hospitalization and mortality. Therefore, the accurate assessment of muscle mass is essential to evaluate sarcopenia in ESLD. However, manual segmentation of muscle volume (MV) can be laborious on cross-sectional imaging, due to the number of slices that require analysis. This study aimed to investigate the impact of reducing the number of slices required for MV estimation. Further, we aimed to compare two equations utilized in estimating MV (cylindrical and truncated cone).MethodsThirty eight ESLD patients (23 males; 54.8 ± 10.7 years) were recruited from the Queen Elizabeth University Hospital Birmingham. A 3T MRI scan was completed of the lower limbs. Quadriceps MV was estimated utilizing 1-, 2-, 3-, and 4 cm slice intervals with both cylindrical and truncated cone equations. Absolute and relative error (compared to 1 cm slice interval) was generated for 2-, 3-, and 4 cm slice intervals. L3 skeletal muscle index (SMI) was also calculated in 30 patients.ResultsRelative error increased with slice interval using the cylindrical (0.45 vs. 1.06 vs. 1.72%) and truncated cone equation (0.27 vs. 0.58 vs. 0.74%) for 2, 3, and 4 cm, respectively. Significantly, the cylindrical equation produced approximately twice the error compared to truncated cone, with 3 cm (0.58 vs. 1.06%, P < 0.01) and 4 cm intervals (0.74 vs. 1.72%, P < 0.001). Finally, quadriceps MV was significantly correlated to L3 SMI (r2 = 0.44, P < 0.0001).ConclusionThe use of the truncated equation with a 4 cm slice interval on MRI offers an efficient but accurate estimation of quadricep muscle volume in ESLD patients.

Abstract Background Headache is the dominant factor for quality of life related disability in idiopathic intracranial hypertension (IIH) and typically has migraine-like characteristics. There are currently no evidence-based therapeutics for headache in IIH, and consequently this is an important unmet clinical need. Case series We report a series of seven patients in whom headaches were the presenting feature of IIH and the headaches had migraine-like characteristics, as is typical in many IIH patients. Papilloedema settled (ocular remission) but headaches continued. These headaches responded markedly to erenumab, a monoclonal antibody targeted against the calcitonin gene related peptide (CGRP) receptor. Of note, there was a recurrence of raised ICP, as evidenced by a return of the papilloedema, however the headaches did not recur whilst treated with erenumab. Conclusions Those with prior IIH who have their headaches successfully treated with CGRP therapy, should remain under close ocular surveillance (particularly when weight gain is evident) as papilloedema can re-occur in the absence of headache. These cases may suggest that CGRP could be a mechanistic driver for headache in patients with active IIH.

Abstract Background Hexose-6-Phosphate Dehydrogenase (H6PD) is a generator of NADPH in the Endoplasmic/Sarcoplasmic Reticulum (ER/SR). Interaction of H6PD with 11β-hydroxysteroid dehydrogenase type 1 provides NADPH to support oxo-reduction of inactive to active glucocorticoids, but the wider understanding of H6PD in ER/SR NAD(P)(H) homeostasis is incomplete. Lack of H6PD results in a deteriorating skeletal myopathy, altered glucose homeostasis, ER stress and activation of the unfolded protein response. Here we further assess muscle responses to H6PD deficiency to delineate pathways that may underpin myopathy and link SR redox status to muscle wide metabolic adaptation. Methods We analysed skeletal muscle from H6PD knockout (H6PDKO), H6PD and NRK2 double knockout (DKO) and wild-type (WT) mice. H6PDKO mice were supplemented with the NAD+ precursor nicotinamide riboside. Skeletal muscle samples were subjected to biochemical analysis including NAD(H) measurement, LC-MS based metabolomics, Western blotting, and high resolution mitochondrial respirometry. Genetic and supplement models were assessed for degree of myopathy compared to H6PDKO. Results H6PDKO skeletal muscle showed adaptations in the routes regulating nicotinamide and NAD+ biosynthesis, with significant activation of the Nicotinamide Riboside Kinase 2 (NRK2) pathway. Associated with changes in NAD+ biosynthesis, H6PDKO muscle had impaired mitochondrial respiratory capacity with altered mitochondrial acylcarnitine and acetyl-CoA metabolism. Boosting NAD+ levels through the NRK2 pathway using the precursor nicotinamide riboside elevated NAD+/NADH but had no effect to mitigate ER stress and dysfunctional mitochondrial respiratory capacity or acetyl-CoA metabolism. Similarly, H6PDKO/NRK2 double KO mice did not display an exaggerated timing or severity of myopathy or overt change in mitochondrial metabolism despite depression of NAD+ availability. Conclusions These findings suggest a complex metabolic response to changes in muscle SR NADP(H) redox status that result in impaired mitochondrial energy metabolism and activation of cellular NAD+ salvage pathways. It is possible that SR can sense and signal perturbation in NAD(P)(H) that cannot be rectified in the absence of H6PD. Whether NRK2 pathway activation is a direct response to changes in SR NAD(P)(H) availability or adaptation to deficits in metabolic energy availability remains to be resolved.

The α-ketoglutarate–dependent dioxygenase, prolyl-4-hydroxylase 3 (PHD3), is an HIF target that uses molecular oxygen to hydroxylate peptidyl prolyl residues. Although PHD3 has been reported to influence cancer cell metabolism and liver insulin sensitivity, relatively little is known about the effects of this highly conserved enzyme in insulin-secreting β cells in vivo. Here, we show that the deletion of PHD3 specifically in β cells (βPHD3KO) was associated with impaired glucose homeostasis in mice fed a high-fat diet. In the early stages of dietary fat excess, βPHD3KO islets energetically rewired, leading to defects in the management of pyruvate fate and a shift from glycolysis to increased fatty acid oxidation (FAO). However, under more prolonged metabolic stress, this switch to preferential FAO in βPHD3KO islets was associated with impaired glucose-stimulated ATP/ADP rises, Ca2+ fluxes, and insulin secretion. Thus, PHD3 might be a pivotal component of the β cell glucose metabolism machinery in mice by suppressing the use of fatty acids as a primary fuel source during the early phases of metabolic stress.

BACKGROUND Idiopathic intracranial hypertension (IIH) is a condition predominantly affecting obese women of reproductive age. Recent evidence suggests that IIH is a disease of metabolic dysregulation, androgen excess, and an increased risk of cardiovascular morbidity. Here we evaluate systemic and adipose specific metabolic determinants of the IIH phenotype.METHODS In fasted, matched IIH (n = 97) and control (n = 43) patients, we assessed glucose and insulin homeostasis and leptin levels. Body composition was assessed along with an interrogation of adipose tissue function via nuclear magnetic resonance metabolomics and RNA sequencing in paired omental and subcutaneous biopsies in a case-control study.RESULTS We demonstrate an insulin- and leptin-resistant phenotype in IIH in excess of that driven by obesity. Adiposity in IIH is preferentially centripetal and is associated with increased disease activity and insulin resistance. IIH adipocytes appear transcriptionally and metabolically primed toward depot-specific lipogenesis.CONCLUSION These data show that IIH is a metabolic disorder in which adipose tissue dysfunction is a feature of the disease. Managing IIH as a metabolic disease could reduce disease morbidity and improve cardiovascular outcomes.FUNDING This study was supported by the UK NIHR (NIHR-CS-011-028), the UK Medical Research Council (MR/K015184/1), Diabetes UK, Wellcome Trust (104612/Z/14/Z), the Sir Jules Thorn Award, and the Midlands Neuroscience Teaching and Research Fund.

Summary: Nonalcoholic fatty liver disease (NAFLD) is currently the most prevalent form of liver disease worldwide. This term encompasses a spectrum of pathologies, from benign hepatic steatosis to non-alcoholic steatohepatitis, which have, to date, been challenging to model in the laboratory setting. Here, we present a human pluripotent stem cell (hPSC)-derived model of hepatic steatosis, which overcomes inherent challenges of current models and provides insights into the metabolic rewiring associated with steatosis. Following induction of macrovesicular steatosis in hepatocyte-like cells using lactate, pyruvate, and octanoate (LPO), respirometry and transcriptomic analyses revealed compromised electron transport chain activity. 13C isotopic tracing studies revealed enhanced TCA cycle anaplerosis, with concomitant development of a compensatory purine nucleotide cycle shunt leading to excess generation of fumarate. This model of hepatic steatosis is reproducible, scalable, and overcomes the challenges of studying mitochondrial metabolism in currently available models.

Sarcopenia is a common complication affecting liver disease patients, yet the underlying mechanisms remain unclear. We aimed to elucidate the cellular mechanisms that drive sarcopenia progression using an in vitro model of liver disease. C2C12 myotubes were serum and amino acid starved for 1-h and subsequently conditioned with fasted ex vivo serum from four non-cirrhotic non-alcoholic fatty liver disease patients (NAFLD), four decompensated end-stage liver disease patients (ESLD) and four age-matched healthy controls (CON) for 4- or 24-h. After 4-h C2C12 myotubes were treated with an anabolic stimulus (5 mM leucine) for 30-min. Myotube diameter was reduced following treatment with serum from ESLD compared with CON (−45%) and NAFLD (−35%; p < 0.001 for both). A reduction in maximal mitochondrial respiration (24% and 29%, respectively), coupling efficiency (~12%) and mitophagy (~13%) was identified in myotubes conditioned with NAFLD and ESLD serum compared with CON (p < 0.05 for both). Myostatin (43%, p = 0.04) and MuRF-1 (41%, p = 0.03) protein content was elevated in myotubes treated with ESLD serum compared with CON. Here we highlight a novel, experimental platform to further probe changes in circulating markers associated with liver disease that may drive sarcopenia and develop targeted therapeutic interventions.

Summary: Vitamin-D-binding protein (DBP) or group-specific component of serum (GC-globulin) carries vitamin D metabolites from the circulation to target tissues. DBP is highly localized to the liver and pancreatic α cells. Although DBP serum levels, gene polymorphisms, and autoantigens have all been associated with diabetes risk, the underlying mechanisms remain unknown. Here, we show that DBP regulates α cell morphology, α cell function, and glucagon secretion. Deletion of DBP leads to smaller and hyperplastic α cells, altered Na+ channel conductance, impaired α cell activation by low glucose, and reduced rates of glucagon secretion both in vivo and in vitro. Mechanistically, this involves reversible changes in islet microfilament abundance and density, as well as changes in glucagon granule distribution. Defects are also seen in β cell and δ cell function. Immunostaining of human pancreata reveals generalized loss of DBP expression as a feature of late-onset and long-standing, but not early-onset, type 1 diabetes. Thus, DBP regulates α cell phenotype, with implications for diabetes pathogenesis.

Glucocorticoids provide indispensable anti-inflammatory therapies. However, metabolic adverse effects including muscle wasting restrict their use. The enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11β-HSD1) modulates peripheral glucocorticoid responses through pre-receptor metabolism. This study investigates how 11β-HSD1 influences skeletal muscle responses to glucocorticoid therapy for chronic inflammation. We assessed human skeletal muscle biopsies from patients with rheumatoid arthritis and osteoarthritis for 11β-HSD1 activity ex vivo. Using the TNF-α-transgenic mouse model (TNF-tg) of chronic inflammation, we examined the effects of corticosterone treatment and 11β-HSD1 global knock-out (11βKO) on skeletal muscle, measuring anti-inflammatory gene expression, muscle weights, fiber size distribution, and catabolic pathways. Muscle 11β-HSD1 activity was elevated in patients with rheumatoid arthritis and correlated with inflammation markers. In murine skeletal muscle, glucocorticoid administration suppressed IL6 expression in TNF-tg mice but not in TNF-tg11βKO mice. TNF-tg mice exhibited reductions in muscle weight and fiber size with glucocorticoid therapy. In contrast, TNF-tg11βKO mice were protected against glucocorticoid-induced muscle atrophy. Glucocorticoid-mediated activation of catabolic mediators (FoxO1, Trim63) was also diminished in TNF-tg11βKO compared to TNF-tg mice. In summary, 11β-HSD1 knock-out prevents muscle atrophy associated with glucocorticoid therapy in a model of chronic inflammation. Targeting 11β-HSD1 may offer a strategy to refine the safety of glucocorticoids.

Inactive cortisone is converted into active cortisol by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). Excessive levels of active glucocorticoids could deteriorate skin barrier function; barrier impairment is also observed in aged skin. In this study, we aimed to determine whether permeability barrier impairment in the aged skin could be related to increased 11β-HSD1 expression. Aged humans (n = 10) showed increased cortisol in the stratum corneum (SC) and oral epithelium, compared to young subjects (n = 10). 11β-HSD1 expression (as assessed via immunohistochemical staining) was higher in the aged murine skin. Aged hairless mice (56-week-old, n = 5) manifested greater transepidermal water loss, lower SC hydration, and higher levels of serum inflammatory cytokines than the young mice (8-week-old, n = 5). Aged 11β-HSD1 knockout mice (n = 11), 11β-HSD1 inhibitor (INHI)-treated aged wild type (WT) mice (n = 5) and young WT mice (n = 10) exhibited reduced SC corticosterone level. Corneodesmosome density was low in WT aged mice (n = 5), but high in aged 11β-HSD1 knockout and aged INHI-treated WT mice. Aged mice exhibited lower SC lipid levels; this effect was reversed by INHI treatment. Therefore, upregulation of 11β-HSD1 in the aged skin increases the active-glucocorticoid levels; this suppresses SC lipid biosynthesis, leading to impaired epidermal permeability barrier.

Migraine is a highly prevalent and disabling primary headache disorder, however its pathophysiology remains unclear, hindering successful treatment. A number of key secondary headache disorders have headaches that mimic migraine. Evidence has suggested a role of mitochondrial dysfunction and an imbalance between energetic supply and demand that may contribute towards migraine susceptibility. Targeting these deficits with nutraceutical supplementation may provide an additional adjunctive therapy. Neuroimaging techniques have demonstrated a metabolic phenotype in migraine similar to mitochondrial cytopathies, featuring reduced free energy availability and increased metabolic rate. This is reciprocated in vivo when modelling a fundamental mechanism of migraine aura, cortical spreading depression. Trials assessing nutraceuticals successful in the treatment of mitochondrial cytopathies including magnesium, coenzyme q10 and riboflavin have also been conducted in migraine. Although promising results have emerged from nutraceutical trials in patients with levels of minerals or vitamins below a critical threshold, they are confounded by lacking control groups or cohorts that are not large enough to be representative. Energetic imbalance in migraine may be relevant in driving the tissue towards maximum metabolic capacity, leaving the brain lacking in free energy. Personalised medicine considering an individual’s deficiencies may provide an approach to ameliorate migraine.

Objective: Augmenting nicotinamide adenine dinucleotide (NAD+) availability may protect skeletal muscle from age-related metabolic decline. Dietary supplementation of NAD+ precursors nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) appear efficacious in elevating muscle NAD+. Here we sought to identify the pathways skeletal muscle cells utilize to synthesize NAD+ from NMN and NR and provide insight into mechanisms of muscle metabolic homeostasis. Methods: We exploited expression profiling of muscle NAD+ biosynthetic pathways, single and double nicotinamide riboside kinase 1/2 (NRK1/2) loss-of-function mice, and pharmacological inhibition of muscle NAD+ recycling to evaluate NMN and NR utilization. Results: Skeletal muscle cells primarily rely on nicotinamide phosphoribosyltransferase (NAMPT), NRK1, and NRK2 for salvage biosynthesis of NAD+. NAMPT inhibition depletes muscle NAD+ availability and can be rescued by NR and NMN as the preferred precursors for elevating muscle cell NAD+ in a pathway that depends on NRK1 and NRK2. Nrk2 knockout mice develop normally and show subtle alterations to their NAD+ metabolome and expression of related genes. NRK1, NRK2, and double KO myotubes revealed redundancy in the NRK dependent metabolism of NR to NAD+. Significantly, these models revealed that NMN supplementation is also dependent upon NRK activity to enhance NAD+ availability. Conclusions: These results identify skeletal muscle cells as requiring NAMPT to maintain NAD+ availability and reveal that NRK1 and 2 display overlapping function in salvage of exogenous NR and NMN to augment intracellular NAD+ availability.

Background: Skeletal muscle is central to whole body metabolic homeostasis, with age and disease impairing its ability to function appropriately to maintain health. Inadequate NAD+ availability is proposed to contribute to pathophysiology by impairing metabolic energy pathway use. Despite the importance of NAD+ as a vital redox cofactor in energy production pathways being well-established, the wider impact of disrupted NAD+ homeostasis on these pathways is unknown. Methods: We utilised skeletal muscle myotube models to induce NAD+ depletion, repletion and excess and conducted metabolic tracing to provide comprehensive and detailed analysis of the consequences of altered NAD+ metabolism on central carbon metabolic pathways. We used stable isotope tracers, [1,2-13C] D-glucose and [U-13C] glutamine, and conducted combined 2D-1H,13C-heteronuclear single quantum coherence (HSQC) NMR spectroscopy and GC-MS analysis. Results: NAD+ excess driven by nicotinamide riboside (NR) supplementation within skeletal muscle cells resulted in enhanced nicotinamide clearance, but had no effect on energy homeostasis or central carbon metabolism. Nicotinamide phosphoribosyltransferase (NAMPT) inhibition induced NAD+ depletion and resulted in equilibration of metabolites upstream of glyceraldehyde phosphate dehydrogenase (GAPDH). Aspartate production through glycolysis and TCA cycle activity was increased in response to low NAD+, which was rapidly reversed with repletion of the NAD+ pool using NR. NAD+ depletion reversibly inhibits cytosolic GAPDH activity, but retains mitochondrial oxidative metabolism, suggesting differential effects of this treatment on sub-cellular pyridine pools. When supplemented, NR efficiently reversed these metabolic consequences. However, the functional relevance of increased aspartate levels after NAD+ depletion remains unclear, and requires further investigation. Conclusions: These data highlight the need to consider carbon metabolism and clearance pathways when investigating NAD+ precursor usage in models of skeletal muscle physiology.

Summary: Nicotinamide adenine dinucleotide (NAD+) is modulated by conditions of metabolic stress and has been reported to decline with aging in preclinical models, but human data are sparse. Nicotinamide riboside (NR) supplementation ameliorates metabolic dysfunction in rodents. We aimed to establish whether oral NR supplementation in aged participants can increase the skeletal muscle NAD+ metabolome and if it can alter muscle mitochondrial bioenergetics. We supplemented 12 aged men with 1 g NR per day for 21 days in a placebo-controlled, randomized, double-blind, crossover trial. Targeted metabolomics showed that NR elevated the muscle NAD+ metabolome, evident by increased nicotinic acid adenine dinucleotide and nicotinamide clearance products. Muscle RNA sequencing revealed NR-mediated downregulation of energy metabolism and mitochondria pathways, without altering mitochondrial bioenergetics. NR also depressed levels of circulating inflammatory cytokines. Our data establish that oral NR is available to aged human muscle and identify anti-inflammatory effects of NR. : Elhassan et al. show that oral nicotinamide riboside increases the NAD+ metabolome in aged human skeletal muscle, without apparently altering mitochondrial bioenergetics. Measures of muscle and whole-body metabolism are also unchanged. Nicotinamide riboside reduces the levels of circulating inflammatory cytokines. Studies in relevant human disease models are warranted. Keywords: nicotinamide adenine dinucleotide, metabolism, aging, inflammation, cell adhesion

Sorafenib is a multi-kinase inhibitor and one of the few systemic treatment options for patients with advanced hepatocellular carcinomas (HCCs). Resistance to sorafenib develops frequently and could be mediated by the nicotinamide adenine dinucleotide (NAD)-dependent deacetylase sirtuin (SIRT)1. We aimed to test whether sorafenib efficacy is influenced by cellular NAD levels and NAD-dependent SIRT1 function. We analyzed sorafenib effects on apoptosis induction, NAD salvage, mitochondrial function, and related signaling pathways in HCC cell lines (HepG2, Hep3B, und HUH7) overexpressing SIRT1 or supplemented with the NAD metabolite nicotinamide mononucleotide (NMN) compared to controls. Treatment of HCC cell lines with sorafenib dose-dependently induced apoptosis and a significant decrease in cellular NAD concentrations. The SIRT1 protein was downregulated in HUH7 cells but not in Hep3B cells. After sorafenib treatment, mitochondrial respiration in permeabilized cells was lower, citrate synthase activity was attenuated, and cellular adenosine triphosphate (ATP) levels were decreased. Concomitant to increased phosphorylation of adenosine monophosphate (AMP)-activated protein kinase (AMPK), sorafenib treatment led to decreased activity of the mechanistic target of rapamycin (mTOR), indicative of energy deprivation. Transient overexpression of SIRT1, as well as NAD repletion by NMN, decreased sorafenib-induced apoptosis. We can, therefore, conclude that sorafenib influences the NAD/SIRT1/AMPK axis. Overexpression of SIRT1 could be an underlying mechanism of resistance to sorafenib treatment in HCC.

11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) mediates glucocorticoid activation and is currently considered as therapeutic target to treat metabolic diseases; however, biomarkers to assess its activity in vivo are still lacking. Recent in vitro experiments suggested that human 11β-HSD1 metabolizes the secondary bile acid 7-oxolithocholic acid (7-oxoLCA) to chenodeoxycholic acid (CDCA) and minor amounts of ursodeoxycholic acid (UDCA). Here, we provide evidence from in vitro and in vivo studies for a major role of 11β-HSD1 in the oxidoreduction of 7-oxoLCA and compare its level and metabolism in several species. Hepatic microsomes from liver-specific 11β-HSD1-deficient mice were devoid of 7-oxoLCA oxidoreductase activity. Importantly, circulating and intrahepatic levels of 7-oxoLCA and its taurine conjugate were significantly elevated in mouse models of 11β-HSD1 deficiency. Moreover, comparative enzymology of 11β-HSD1-dependent oxidoreduction of 7-oxoLCA revealed that the guinea-pig enzyme is devoid of 7-oxoLCA oxidoreductase activity. Unlike in other species, 7-oxoLCA and its glycine conjugate are major bile acids in guinea-pigs. In conclusion, the oxidoreduction of 7-oxoLCA and its conjugated metabolites are catalyzed by 11β-HSD1, and the lack of this activity leads to the accumulation of these bile acids in guinea-pigs and 11β-HSD1-deficient mice. Thus, 7-oxoLCA and its conjugates may serve as biomarkers of impaired 11β-HSD1 activity.