A study protocol to investigate if acipimox improves muscle function and sarcopenia: an open-label, uncontrolled, before-and-after experimental medicine feasibility study in community-dwelling older adults.

INTRODUCTION: Sarcopenia is the age-associated loss of muscle mass and strength. Nicotinamide adenine dinucleotide (NAD) plays a central role in both mitochondrial function and cellular ageing processes implicated in sarcopenia. NAD concentrations are low in older people with sarcopenia, and increasing skeletal muscle NAD concentrations may offer a novel therapy for this condition. Acipimox is a licensed lipid-lowering agent known to act as an NAD precursor. This open-label, uncontrolled, before-and-after proof-of-concept experimental medicine study will test whether daily supplementation with acipimox improves skeletal muscle NAD concentrations. METHODS AND ANALYSIS: Sixteen participants aged 65 and over with probable sarcopenia will receive acipimox 250 mg and aspirin 75 mg orally daily for 4 weeks, with the frequency of acipimox administration being dependent on renal function. Muscle biopsy of the vastus lateralis and MRI scanning of the lower leg will be performed at baseline before starting acipimox and after 3 weeks of treatment. Adverse events will be recorded for the duration of the trial. The primary outcome, analysed in a per-protocol population, is the change in skeletal muscle NAD concentration between baseline and follow-up. Secondary outcomes include changes in phosphocreatine recovery rate by 31P magnetic resonance spectroscopy, changes in physical performance and daily activity (handgrip strength, 4 m walk and 7-day accelerometry), changes in skeletal muscle mitochondrial respiratory function, changes in skeletal muscle mitochondrial DNA copy number and changes in NAD concentrations in whole blood as a putative biomarker for future participant selection. ETHICS AND DISSEMINATION: The trial is approved by the UK Medicines and Healthcare Products Regulatory Agency (EuDRACT 2021-000993-28) and UK Health Research Authority and Northeast - Tyne and Wear South Research Ethics Committee (IRAS 293565). Results will be made available to participants, their families, patients with sarcopenia, the public, regional and national clinical teams, and the international scientific community. PROTOCOL: Acipimox feasibility study Clinical Trial Protocol V.2 2/11/21. TRIAL REGISTRATION NUMBER: The ISRCTN trial database (ISRCTN87404878).

Acipimox in Mitochondrial Myopathy (AIMM): study protocol for a randomised, double-blinded, placebo-controlled, adaptive design trial of the efficacy of acipimox in adult patients with mitochondrial myopathy.

BACKGROUND: Mitochondrial disease is a heterogenous group of rare, complex neurometabolic disorders. Despite their individual rarity, collectively mitochondrial diseases represent the most common cause of inherited metabolic disorders in the UK; they affect 1 in every 4300 individuals, up to 15,000 adults (and a similar number of children) in the UK. Mitochondrial disease manifests multisystem and isolated organ involvement, commonly affecting those tissues with high energy demands, such as skeletal muscle. Myopathy manifesting as fatigue, muscle weakness and exercise intolerance is common and debilitating in patients with mitochondrial disease. Currently, there are no effective licensed treatments and consequently, there is an urgent clinical need to find an effective drug therapy. AIM: To investigate the efficacy of 12-week treatment with acipimox on the adenosine triphosphate (ATP) content of skeletal muscle in patients with mitochondrial disease and myopathy. METHODS: AIMM is a single-centre, double blind, placebo-controlled, adaptive designed trial, evaluating the efficacy of 12 weeks' administration of acipimox on skeletal muscle ATP content in patients with mitochondrial myopathy. Eligible patients will receive the trial investigational medicinal product (IMP), either acipimox or matched placebo. Participants will also be prescribed low dose aspirin as a non-investigational medical product (nIMP) in order to protect the blinding of the treatment assignment. Eighty to 120 participants will be recruited as required, with an interim analysis for sample size re-estimation and futility assessment being undertaken once the primary outcome for 50 participants has been obtained. Randomisation will be on a 1:1 basis, stratified by Fatigue Impact Scale (FIS) (dichotomised as < 40, ≥ 40). Participants will take part in the trial for up to 20 weeks, from screening visits through to follow-up at 16 weeks post randomisation. The primary outcome of change in ATP content in skeletal muscle and secondary outcomes relating to quality of life, perceived fatigue, disease burden, limb function, balance and walking, skeletal muscle analysis and symptom-limited cardiopulmonary fitness (optional) will be assessed between baseline and 12 weeks. DISCUSSION: The AIMM trial will investigate the effect of acipimox on modulating muscle ATP content and whether it can be repurposed as a new treatment for mitochondrial disease with myopathy. TRIAL REGISTRATION: EudraCT2018-002721-29 . Registered on 24 December 2018, ISRCTN 12895613. Registered on 03 January 2019, https://www.isrctn.com/search?q=aimm.

MYC sensitises cells to apoptosis by driving energetic demand.

The MYC oncogene is a potent driver of growth and proliferation but also sensitises cells to apoptosis, which limits its oncogenic potential. MYC induces several biosynthetic programmes and primary cells overexpressing MYC are highly sensitive to glutamine withdrawal suggesting that MYC-induced sensitisation to apoptosis may be due to imbalance of metabolic/energetic supply and demand. Here we show that MYC elevates global transcription and translation, even in the absence of glutamine, revealing metabolic demand without corresponding supply. Glutamine withdrawal from MRC-5 fibroblasts depletes key tricarboxylic acid (TCA) cycle metabolites and, in combination with MYC activation, leads to AMP accumulation and nucleotide catabolism indicative of energetic stress. Further analyses reveal that glutamine supports viability through TCA cycle energetics rather than asparagine biosynthesis and that TCA cycle inhibition confers tumour suppression on MYC-driven lymphoma in vivo. In summary, glutamine supports the viability of MYC-overexpressing cells through an energetic rather than a biosynthetic mechanism.

The hepatic compensatory response to elevated systemic sulfide promotes diabetes.

Impaired hepatic glucose and lipid metabolism are hallmarks of type 2 diabetes. Increased sulfide production or sulfide donor compounds may beneficially regulate hepatic metabolism. Disposal of sulfide through the sulfide oxidation pathway (SOP) is critical for maintaining sulfide within a safe physiological range. We show that mice lacking the liver- enriched mitochondrial SOP enzyme thiosulfate sulfurtransferase (Tst-/- mice) exhibit high circulating sulfide, increased gluconeogenesis, hypertriglyceridemia, and fatty liver. Unexpectedly, hepatic sulfide levels are normal in Tst-/- mice because of exaggerated induction of sulfide disposal, with associated suppression of global protein persulfidation and nuclear respiratory factor 2 target protein levels. Hepatic proteomic and persulfidomic profiles converge on gluconeogenesis and lipid metabolism, revealing a selective deficit in medium-chain fatty acid oxidation in Tst-/- mice. We reveal a critical role of TST in hepatic metabolism that has implications for sulfide donor strategies in the context of metabolic disease.

Fatty acid synthase cooperates with protrudin to facilitate membrane outgrowth of cellular protrusions.

Cellular protrusion formation capacity is a key feature of developing neurons and many eukaryotic cells. However, the mechanisms underlying membrane growth in protrusion formation are largely unclear. In this study, photo-reactive unnatural amino acid 3-(3-methyl-3H-diazirin-3-yl)-propamino-carbonyl-Nε-l-lysine was incorporated by a genetic code expansion strategy into protrudin, a protein localized in acidic endosomes and in the endoplasmic reticulum, that induces cellular protrusion and neurite formation. The modified protrudin was used for covalent trapping of protrudin-interacting proteins in living cells. Fatty acid synthase (FASN), which synthesizes free fatty acids, was identified to transiently interact with protrudin. Further characterization revealed a unique cooperation mechanism in which protrudin cooperates with FASN to facilitate cellular protrusion formation. This work reveals a novel mechanism involved in protrusion formation that is dependent on transient interaction between FASN and protrudin, and establishes a creative strategy to investigate transient protein-protein interactions in mammalian cells.

The Succinated Proteome of FH-Mutant Tumours.

Inherited mutations in the Krebs cycle enzyme fumarate hydratase (FH) predispose to hereditary leiomyomatosis and renal cell cancer (HLRCC). Loss of FH activity in HLRCC tumours causes accumulation of the Krebs cycle intermediate fumarate to high levels, which may act as an oncometabolite through various, but not necessarily mutually exclusive, mechanisms. One such mechanism, succination, is an irreversible non-enzymatic modification of cysteine residues by fumarate, to form S-(2-succino)cysteine (2SC). Previous studies have demonstrated that succination of proteins including glyceraldehyde 3-phosphate dehydrogenase (GAPDH), kelch-like ECH-associated protein 1 (KEAP1) and mitochondrial aconitase (ACO2) can have profound effects on cellular metabolism. Furthermore, immunostaining for 2SC is a sensitive and specific biomarker for HLRCC tumours. Here, we performed a proteomic screen on an FH-mutant tumour and two HLRCC-derived cancer cell lines and identified 60 proteins where one or more cysteine residues were succinated; 10 of which were succinated at cysteine residues either predicted, or experimentally proven, to be functionally significant. Bioinformatic enrichment analyses identified most succinated targets to be involved in redox signaling. To our knowledge, this is the first proteomic-based succination screen performed in human tumours and cancer-derived cells and has identified novel 2SC targets that may be relevant to the pathogenesis of HLRCC.

Prolyl hydroxylase domain enzymes: important regulators of cancer metabolism.

The hypoxia-inducible factor (HIF) prolyl hydroxylase domain enzymes (PHDs) regulate the stability of HIF protein by post-translational hydroxylation of two conserved prolyl residues in its α subunit in an oxygen-dependent manner. Trans-4-prolyl hydroxylation of HIFα under normal oxygen (O2) availability enables its association with the von Hippel-Lindau (VHL) tumor suppressor pVHL E3 ligase complex, leading to the degradation of HIFα via the ubiquitin-proteasome pathway. Due to the obligatory requirement of molecular O2 as a co-substrate, the activity of PHDs is inhibited under hypoxic conditions, resulting in stabilized HIFα, which dimerizes with HIFß and, together with transcriptional co-activators CBP/p300, activates the transcription of its target genes. As a key molecular regulator of adaptive response to hypoxia, HIF plays important roles in multiple cellular processes and its overexpression has been detected in various cancers. The HIF1α isoform in particular has a strong impact on cellular metabolism, most notably by promoting anaerobic, whilst inhibiting O2-dependent, metabolism of glucose. The PHD enzymes also seem to have HIF-independent functions and are subject to regulation by factors other than O2, such as by metabolic status, oxidative stress, and abnormal levels of endogenous metabolites (oncometabolites) that have been observed in some types of cancers. In this review, we aim to summarize current understandings of the function and regulation of PHDs in cancer with an emphasis on their roles in metabolism.