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Thoroughbred horses are finely-tuned athletes with a high aerobic capacity relative to skeletal muscle mass, attributable to centuries of genetic selection for speed and stamina. Polymorphisms in the myostatin gene (MSTN), a pronounced inhibitor of skeletal muscle growth, have been shown to almost singularly account for gene-based race distance aptitude in racehorses. In Thoroughbreds, two MSTN polymorphisms, a single nucleotide variation in the first intron (SNP g.66493737C>T) and a non-coding transposable element within the promoter region (a 227 bp SINE insertion) are of particular interest. Until now, it has not been clear which of these variants affect skeletal muscle phenotypes or whether both can impact racing performance. In a large cohort of Thoroughbreds, we observed a complete concordance between the SNP and the SINE insertion. By means of in vitro assays in C2C12 myoblasts, we isolated the SNP variant from the SINE polymorphism and showed the latter is exclusively responsible for adversely affecting transcription initiation and gene expression thereby limiting myostatin protein production. Mapping the MSTN transcription start site in horse skeletal muscle likewise revealed anomalous transcription initiation in the presence of the SINE insertion. Our data provides mechanistic evidence that the SINE insertion uniquely accounts for the MSTN \"speed gene\" effect on race distance aptitude in the Thoroughbred horse.

The cannabinoid 1 (CB1) receptor regulates appetite and body weight; however, unwanted central side effects of both agonists (in wasting disorders) or antagonists (in obesity and diabetes) have limited their therapeutic utility. At the peripheral level, CB1 receptor activation impacts the energy balance of mammals in a number of different ways: inhibiting satiety and emesis, increasing food intake, altering adipokine and satiety hormone levels, altering taste sensation, decreasing lipolysis (fat break down), and increasing lipogenesis (fat generation). The CB1 receptor also plays an important role in the gut–brain axis control of appetite and satiety. The combined effect of peripheral CB1 activation is to promote appetite, energy storage, and energy preservation (and the opposite is true for CB1 antagonists). Therefore, the next generation of CB1 receptor medicines (agonists and antagonists, and indirect modulators of the endocannabinoid system) have been peripherally restricted to mitigate these issues, and some of these are already in clinical stage development. These compounds also have demonstrated potential in other conditions such as alcoholic steatohepatitis and diabetic nephropathy (peripherally restricted CB1 antagonists) and pain conditions (peripherally restricted CB1 agonists and FAAH inhibitors). This review will discuss the mechanisms by which peripheral CB1 receptors regulate body weight, and the therapeutic utility of peripherally restricted drugs in the management of body weight and beyond.

In an endeavour to understand metastasis from oral squamous cell carcinomas, we characterised the metastatic potential of a human tongue derived cell line (SCC-4 cells) and compared this phenotype to pre-cancerous dysplastic oral keratinocyte (DOK) cells derived from human tongue and primary gingival keratinocytes (PGK). We demonstrate that SCC-4 cells constitutively synthesize and release significant amounts of IL-6, a process that is enhanced by the addition of the TLR2/TLR6 agonist, Pam2CSK4. The expression of TLR2/6 and IL-6Ra/gp130 receptors was also confirmed in SCC-4 cells. Cancerous SCC-4 human tongue cells also have a classic EMT profile, unlike precancerous human tongue DOK cells. We also established that IL-6 is driving anoikis resistance in an autocrine fashion and that anti-IL-6 neutralising antibodies, anti-IL-6 receptor antibodies and anti-TLR2 receptor antibodies inhibit anoikis resistance in cancerous SCC-4 human tongue cells . The data suggest a promising role for anti-IL-6 receptor antibody and anti-TLR2 receptor antibody treatment for oral cancer.

The selection of cattle with enhanced feed efficiency is of importance with regard to reducing feed costs in the beef industry. Global transcriptome profiling was undertaken on liver and skeletal muscle biopsies from Simmental heifers and bulls divergent for residual feed intake (RFI), a widely acknowledged feed efficiency phenotype, in order to identify genes that may be associated with this trait. We identified 5 genes (adj. p < 0.1) to be differentially expressed in skeletal muscle between high and low RFI heifers with all transcripts involved in oxidative phosphorylation and mitochondrial homeostasis. A total of 11 genes (adj. p < 0. 1) were differentially expressed in liver tissue between high and low RFI bulls with differentially expressed genes related to amino and nucleotide metabolism as well as endoplasmic reticulum protein processing. No genes were identified as differentially expressed in either heifer liver or bull muscle analyses. Results from this study show that the molecular control of RFI in young cattle is modified according to gender, which may be attributable to differences in physiological maturity between heifers and bulls of the same age. Despite this we have highlighted a number of genes that may hold potential as molecular biomarkers for RFI cattle.

Improving cattle feed efficiency through selection of residual feed intake (RFI) is a widely accepted approach to sustainable beef production. A greater understanding of the molecular control of RFI in various breeds offered contrasting diets is necessary for the accurate identification of feed efficient animals and will underpin accelerated genetic improvement of the trait. The aim of this study was to determine genes and biological processes contributing to RFI across varying breed type and dietary sources in skeletal muscle tissue. Residual feed intake was calculated in Charolais and Holstein–Friesian steers across multiple dietary phases (phase-1: high concentrate (growing-phase); phase-2: zero-grazed grass (growing-phase); phase-3: high concentrate (finishing-phase). Steers divergent for RFI within each breed and dietary phase were selected for muscle biopsy collection, and muscle samples subsequently subjected to RNAseq analysis. No gene was consistently differentially expressed across the breed and diet types examined. However, pathway analysis revealed commonality across breeds and diets for biological processes including fatty acid metabolism, immune function, energy production and muscle growth. Overall, the lack of commonality of individual genes towards variation in RFI both within the current study and compared to the published literature, suggests other genomic features warrant further evaluation in relation to RFI.

Variation in the myostatin (MSTN) gene has been reported to be associated with race distance, body composition and skeletal muscle fibre composition in the horse. The aim of the present study was to test the hypothesis that MSTN variation influences mitochondrial phenotypes in equine skeletal muscle. Mitochondrial abundance and skeletal muscle fibre types were measured in whole muscle biopsies from the gluteus medius of n = 82 untrained (21 ± 3 months) Thoroughbred horses. Skeletal muscle fibre type proportions were significantly (p < 0.01) different among the three MSTN genotypes and mitochondrial content was significantly (p < 0.01) lower in the combined presence of the C-allele of SNP g.66493737C>T (C) and the SINE insertion 227 bp polymorphism (I). Evaluation of mitochondrial complex activities indicated higher combined mitochondrial complex I+III and II+III activities in the presence of the C-allele / I allele (p ≤ 0.05). The restoration of complex I+III and complex II+III activities following addition of exogenous coenzyme Q1 (ubiquinone1) (CoQ1) in vitro in the TT/NN (homozygous T allele/homozygous no insertion) cohort indicated decreased coenzyme Q in these animals. In addition, decreased gene expression in two coenzyme Q (CoQ) biosynthesis pathway genes (COQ4, p ≤ 0.05; ADCK3, p ≤ 0.01) in the TT/NN horses was observed. This study has identified several mitochondrial phenotypes associated with MSTN genotype in untrained Thoroughbred horses and in addition, our findings suggest that nutritional supplementation with CoQ may aid to restore coenzyme Q activity in TT/NN horses.

Background/Objectives: Oral squamous cell carcinoma (OSCC) is a major cause of human cancer. The enzyme, nicotinamide N-methyltransferase (NNMT), is overexpressed in a variety of human cancers, including OSCC. Our objective was to target NNMT with novel inhibitors and determine their anti-cancer efficacy while shedding light on their possible mechanism of action. Methods: We identified two small molecule inhibitors of NNMT (AG-670 and AO-022) based on a pharmacophore of the in silico nicotinamide binding site. These inhibitors were investigated for (i) potency to inhibit the activity of the isolated NNMT enzyme (EC50 values), (ii) cytotoxicity (IC50 values) against the human OSCC cell line, SCC-4, and (iii) ability to affect cellular energy metabolism, as measured by oxygen consumption, in SCC-4 cells (plus dysplastic oral keratinocytes (DOK) cells and breast cancer MCF-7 cells). Immunoblotting was used to determine whether NNMT was expressed in the aforementioned cells. Results: NNMT is expressed in SCC-4 and DOK cells (and primary human oral keratinocytes) but not MCF 7 cells. The NNMT inhibitors inhibit isolated NNMT enzyme activity and were cytotoxic to SCC-4 cells (EC50 and IC50 values in the micromolar range). Sublethal doses of the inhibitors were demonstrated to inhibit in situ mitochondrial oxygen consumption in SCC-4 and DOK cells but not in MCF-7 cells. It was demonstrated that the NNMT inhibitors do not directly inhibit mitochondrial electron transport chain activity. Thus, we deduce that the NNMT inhibitors affect mitochondrial activity indirectly via NNMT. Conclusions: It is concluded that NNMT is a potential drug target for oral cancer.

Mitochondrial glutathione (mtGSH) supports iron-sulfur cluster (ISC) stability in the electron transport chain (ETC). Here we have investigated the role of the mtGSH transporter SLC25A40 in macrophage activation. SLC25A40 is present in both murine and human macrophages and its expression was increased by LPS treatment. Reducing SLC25A40 expression using siRNA destabilized ISC-rich ETC proteins and elevated mitochondrial and cellular reactive oxygen species (ROS). It also induced expression of the genes Gclc and Gclm , which are involved in GSH biosynthesis. SLC25A40 deficiency also diminished IL-1β and IL-10 production at the transcriptional level in response to LPS. As a result, the production of mature IL-1β was decreased following activation of NLRP3 by nigericin or ATP, with no effect on pyroptosis. Depleting mtGSH with mitochondrially-targeted CDNB phenocopied these defects, whereas supplementation with a cell-permeable GSH ester partially restored pro-IL-1β production. Together, these data identify SLC25A40 as a key regulator that sustains ETC integrity to promote cytokine production, revealing a previously unrecognized role for the SLC25A40-mtGSH axis in coupling mitochondrial redox control to macrophage activation.

Uncoupling proteins (UCPs) are members of the mitochondrial anion carrier superfamily that can mediate the transfer of protons into the mitochondrial matrix from the intermembrane space. We have previously reported UCP3 expression in thymocytes, mitochondria of total splenocytes and splenic lymphocytes. Here, we demonstrate that Ucp3 is expressed in peripheral naive CD4 + T cells at the mRNA level before being markedly downregulated following activation. Non-polarized, activated T cells (Th0 cells) from Ucp3 -/- mice produced significantly more IL-2, had increased expression of CD25 and CD69 and were more proliferative than Ucp3 +/+ Th0 cells. The altered IL-2 expression observed between T cells from Ucp3 +/+ and Ucp3 -/- mice may be a factor in determining differentiation into Th17 or induced regulatory (iTreg) cells. When compared to Ucp3 +/+ , CD4 + T cells from Ucp3 -/- mice had increased FoxP3 expression under iTreg conditions. Conversely, Ucp3 -/- CD4 + T cells produced a significantly lower concentration of IL-17A under Th17 cell-inducing conditions in vitro . These effects were mirrored in antigen-specific T cells from mice immunized with KLH and CT. Interestingly, the altered responses of Ucp3 -/- T cells were partially reversed upon neutralisation of IL-2. Together, these data indicate that UCP3 acts to restrict the activation of naive T cells, acting as a rheostat to dampen signals following TCR and CD28 co-receptor ligation, thereby limiting early activation responses. The observation that Ucp3 ablation alters the Th17:Treg cell balance in vivo as well as in vitro suggests that UCP3 is a potential target for the treatment of Th17 cell-mediated autoimmune diseases.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype characterized by high recurrence rates and limited treatment options due to the absence of hormone receptors. Despite advancements in breast cancer research, effective therapies for TNBC remain inadequate, highlighting the need to elucidate subtype-specific metabolic vulnerabilities. TNBC cells exhibit a strong dependence on the exogenous amino acids cystine and glutamine, yet the interplay between these metabolic dependencies remains poorly understood. Here, we demonstrate that TNBC cells exhibit sensitivity to individual nutrient deprivation but can survive dual cystine and glutamine deprivation via distinct mechanisms. Exogenous glutamine primarily fuels glutamine anaplerosis, supporting TNBC cell proliferation. Notably, when exogenous glutamine is absent, restricted cystine uptake restores intracellular glutamate levels, fulfilling metabolic demands and sustaining TNBC cell growth. Under cystine deprivation, inhibition of glutaminolysis rescues TNBC cells by mitigating lipid peroxidation and reducing ROS production, whereas supplementation with the TCA cycle intermediates ɑ-ketoglutarate (ɑ-KG) and succinate induces profound cell death in both TNBC and luminal breast cancer cells under glutaminolysis blockade. Collectively, these findings highlight the metabolic interdependence of glutamine and cystine in TNBC, providing mechanistic insights into potential metabolic-targeted and dietary interventions for TNBC therapy.