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Choline contributes to the biogenesis of methyl groups, neurotransmitters, and cell membranes. Our genome-wide association study (GWAS) of circulating choline in 2228 college students found that alleles in SLC25A48 (rs6596270) influence choline concentrations in men (p = 9.6 × 10 -8 ), but not women. Previously, the subcellular location and function of SLC25A48 were unknown. Using super-resolution immunofluorescence microscopy, we localized SLC25A48 to the inner mitochondrial membrane. Our results suggest that SLC25A48 transports choline across the inner mitochondrial membrane., Competing Interests: Declaration of competing interest The authors have no competing interests to declare., (Published by Elsevier Inc.)

Cachexia syndrome, leading to reduced skeletal muscle and fat mass, is highly prevalent in cancer patients, resulting in further negative implications for these patients. To date, there is no approved therapy for cachexia syndrome. The objective of this study was to establish an in vitro model of cancer cachexia in mature human skeletal muscle myotubes, with the intention of exploiting the cell model to assess potential cachexia therapeutics, specifically cannabinoid related drugs. Having cultured and differentiated primary human muscle myoblasts to mature myotubes, we successfully established two cancer cachexia models using conditioned media (CM) from human colon adenocarcinoma (SW480) and from non-small-cell lung carcinoma (H1299) cultured cells. The cancer-CM-induced extensive myotube degeneration, demonstrated by a significant reduction in mature myotube diameter, which progressed over the period studied. Myotube degeneration is a characteristic feature of cancer cachexia and was used in this study as an index of cachexia. Expression of cannabinoid 1 and 2 receptors (CB 1 R and CB 2 R) was confirmed in the mature human skeletal muscle myotubes. Subsequently, the effect of cannabinoid compounds on this myotube degeneration were assessed. Tetrahydrocannabinol (THC), a partial CB 1 R/CB 2 R agonist, and JWH133, a selective CB 2 R agonist, proved efficacious in protecting mature human myotubes from the deleterious effects of both (SW480 and H1299) cancer cachexia conditions. ART27.13, a full, peripherally selective CB 1 R/CB 2 R agonist, currently being trialled in cancer cachexia (IRAS ID 278450, REC 20/NE/0198), was also significantly protective against myotube degeneration in both (SW480 and H1299) cancer cachexia conditions. Furthermore, the addition of the CB 2 R antagonist AM630, but not the CB 1 R antagonist Rimonabant, abolished the protective effect of ART27.13. In short, we have established a convenient and robust in vitro model of cancer-induced human skeletal muscle cachexia. The data obtained using the model demonstrate the therapeutic potential of ART27.13 in cancer-induced cachexia prevention and provides evidence indicating that this effect is via CB 2 R, and not CB 1 R.

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., (© 2023. The Author(s).)

Background: Thoroughbred racehorse performance is largely influenced by a major quantitative trait locus at the myostatin ( MSTN ) gene which determines aptitude for certain race distances due to a promoter region insertion mutation influencing functional phenotypes in skeletal muscle. To develop an in vitro system for functional experiments we established three novel equine skeletal muscle cell lines reflecting the variation in phenotype associated with MSTN genotype (CC/II, CT/IN and TT/NN for SNP g.66493737C > T/SINE insertion 227 bp polymorphism). Primary equine skeletal muscle myoblasts, isolated from Thoroughbred horse gluteus medius , were conditionally immortalised and evaluated to determine whether cell phenotype and metabolic function were comparable to functional characteristics previously reported for ex vivo skeletal muscle isolated from Thoroughbred horses with each genotype., Results: Primary myoblasts conditionally immortalised with the temperature sensitive SV40TtsA58 lentivirus vector successfully proliferated and could revert to their primary cell phenotype and differentiate into multinucleated myotubes. Skeletal muscle fibre type, MSTN gene expression, mitochondrial abundance, and mitochondrial function of the three MSTN genotype cell lines, were consistent with equivalent characterisation of ex vivo skeletal muscle samples with these genotypes. Furthermore, addition of coenzyme Q 10 (CoQ 10 ) to the cell lines improved mitochondrial function, an observation consistent with ex vivo skeletal muscle samples with these genotypes following supplementation with CoQ 10 in the diet., Conclusions: The observation that the phenotypic characteristics and metabolic function of the cells lines are equivalent to ex vivo skeletal muscle indicates that this in vitro system will enable efficient and cost-effective analyses of equine skeletal muscle for a range of different applications including understanding metabolic function, testing of nutritional supplements, drug test development and gene doping test development. In the multi-billion-euro international Thoroughbred horse industry research advances in the biological function of skeletal muscle are likely to have considerable impact. Furthermore, this novel genotype-specific system may be adapted and applied to human biomedicine to improve understanding of the effects of myostatin in human physiology and medicine., Competing Interests: EWH is a shareholder in and Chief Scientific Officer of Plusvital Ltd., an equine nutrition and genetic testing company. Plusvital Ltd. has been granted a licence for commercial use of the data that is contained within multiple granted patents and patent applications including (patent reference numbers): EP2352850, JP5667057, US8771943, AU2009290452, NZ591711, US9249470 and US2016215335. EWH is named on these patents. MFR, NGBN, MGM and RKP declare that they have no competing interests. Other than EWH, Plusvital Ltd. played no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript., (© 2022 The Authors.)

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., (© 2022. The Author(s).)

The cannabinoid 1 (CB 1 ) 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, CB 1 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 CB 1 receptor also plays an important role in the gut-brain axis control of appetite and satiety. The combined effect of peripheral CB 1 activation is to promote appetite, energy storage, and energy preservation (and the opposite is true for CB 1 antagonists). Therefore, the next generation of CB 1 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 CB 1 antagonists) and pain conditions (peripherally restricted CB 1 agonists and FAAH inhibitors). This review will discuss the mechanisms by which peripheral CB 1 receptors regulate body weight, and the therapeutic utility of peripherally restricted drugs in the management of body weight and beyond.

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.

Compensatory growth (CG) is a naturally accelerated growth which occurs upon realimentation, following a prior period of dietary restriction. The process is harnessed worldwide as a management practice to reduce feed costs in beef cattle production. The objective of this study was to assess the potential contribution of hepatic cellular mitochondrial capacity to CG through global hepatic oxidative phosphorylation gene expression analyses as well as functional mitochondrial enzyme activity assays. Holstein-Friesian bulls were separated into two groups: (i) restricted feed allowance for 125 days (Period 1) (RES; n = 30) followed by ad-libitum feeding for 55 days (Period 2) or (ii) ad-libitum access to feed throughout (Periods 1 and 2) (ADLIB; n = 30). At the end of each period, 15 animals from each treatment group were slaughtered and hepatic tissue was collected. Tissue samples were subjected to RNAseq and spectrophotometric analysis for the functional assessment of mitochondria. RES and ADLIB groups grew at 0.6 kg/day and 1.9 kg/day, respectively, during Period 1. During Period 2, the RES group underwent CG growing at 2.5 kg/day, with ADLIB animals gaining 1.4 kg/day. Oxidative phosphorylation genes were differentially expressed in response to both dietary restriction and CG. Spectrophotometric assays indicated that mitochondrial abundance was greater in animals undergoing dietary restriction at the end of Period 1 and subsequently reduced during realimentation (P < 0.02). Results indicate that mitochondrial capacity may be enhanced during dietary restriction to more effectively utilize diet-derived nutrients. However, enhanced mitochondrial capacity does not appear to be directly contributing to CG in cattle., (Copyright © 2021. Published by Elsevier B.V.)

We compared the activity of complex 1, complex 2, and the expression of the complex 1 subunit, NDUFA9, in isolated brown adipose tissue mitochondria from wild type and mitochondrial uncoupling protein 1 (UCP1) knockout mice. Direct spectrophotometric measurement revealed that complex 2 activity was similar, but complex 1 activity was greater (~2.7 fold) in isolated mitochondria from wild-type mice compared to UCP1 knockout mice, an observation endorsed by greater complex 1 subunit expression (NDUFA9) in mitochondria of wild-type mice. We also measured reactive oxygen species (ROS) production by isolated brown adipose mitochondria respiring on succinate, without rotenone, thus facilitating reverse electron flow through complex 1. We observed that reverse electron flow in isolated mitochondria from wild-type mice, with UCP1 inhibited, produced significantly greater (~1.6 fold) ROS when compared with isolated brown adipose mitochondria from UCP1 knockout mice. In summary, we demonstrate that ROS production by succinate-driven reverse electron flow can occur in brown adipose tissue mitochondria and is a good index of complex 1 activity.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Clinical and experimental data suggest that pathogenesis in anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis is driven by ANCA-mediated activation of neutrophils and monocytes. While the role of neutrophils has been extensively investigated, the function of monocytes remains relatively understudied. We have previously demonstrated that stimulation of monocytes with anti-myeloperoxidase (MPO), but not anti-proteinase-3 (PR3), antibodies results in production of the pro-inflammatory cytokine IL-1β. Changes in cellular metabolism, particularly a switch to glycolysis, have recently been linked to activation of immune cells and production of IL-1β. Therefore, we investigated the metabolic profile of monocytes following ANCA stimulation. We found a significant increase in glucose uptake in anti-MPO stimulated monocytes. Interestingly, both anti-MPO and anti-PR3 stimulation resulted in an immediate increase in glycolysis, measured by Seahorse extracellular flux analysis. However, this increase in glycolysis was sustained (for up to 4 h) in anti-MPO- but not anti-PR3-treated cells. In addition, only anti-MPO-treated cells exhibited increased oxidative phosphorylation, a metabolic response that correlated with IL-1β production. These data indicate that monocyte metabolism is altered by ANCA, with divergent responses to anti-MPO and anti-PR3 antibodies. These metabolic changes may underlie pathologic immune activation in ANCA associated vasculitis, as well as potentially contributing to the differing clinical phenotype between PR3- and MPO-ANCA positive patients. These metabolic pathways may therefore be potential targets for therapeutic intervention., (Copyright © 2020 O'Brien, White, Wyse, Leacy, Porter, Little and Hickey.)

Cellular mitochondrial function has been suggested to contribute to variation in feed efficiency (FE) among animals. The objective of this study was to determine mitochondrial abundance and activities of various mitochondrial respiratory chain complexes (complex I (CI) to complex IV (CIV)) in liver and muscle tissue from beef cattle phenotypically divergent for residual feed intake (RFI), a measure of FE. Individual DM intake (DMI) and growth were measured in purebred Simmental heifers (n = 24) and bulls (n = 28) with an initial mean BW (SD) of 372 kg (39.6) and 387 kg (50.6), respectively. All animals were offered concentrates ad libitum and 3 kg of grass silage daily, and feed intake was recorded for 70 days. Residuals of the regression of DMI on average daily gain (ADG), mid-test BW0.75 and backfat (BF), using all animals, were used to compute individual RFI coefficients. Animals were ranked within sex, by RFI into high (inefficient; top third of the population), medium (middle third of population) and low (efficient; bottom third of the population) terciles. Statistical analysis was carried out using the MIXED procedure of SAS v 9.3. Overall mean ADG (SD) and daily DMI (SD) for heifers were 1.2 (0.4) and 9.1 (0.5) kg, respectively, and for bulls were 1.8 (0.3) and 9.5 (1.02) kg, respectively. Heifers and bulls ranked as high RFI consumed 10% and 15% more (P < 0.05), respectively, than their low RFI counterparts. There was no effect of RFI on mitochondrial abundance in either liver or muscle (P > 0.05). An RFI × sex interaction was apparent for CI activity in muscle. High RFI animals had an increased activity (P < 0.05) of CIV in liver tissue compared to their low RFI counterparts; however, the relevance of that observation is not clear. Our data provide no clear evidence that cellular mitochondrial function within either skeletal muscle or hepatic tissue has an appreciable contributory role to overall variation in FE among beef cattle.

Due to limited availability of pharmacological therapies, triple-negative breast cancer (TNBC) is the subtype with worst outcome. We hypothesised that 2-Deoxy-D-Glucose (2-DG), a glucose analogue, may hold potential as a therapy for particularly aggressive TNBC. We investigated 2-DG's effects on TNBC cell line variants, Hs578T parental cells and their isogenic more aggressive Hs578Ts(i) 8 variant, using migration, invasion and anoikis assays. We assessed their bioenergetics by Seahorse. We evaluated metabolic alterations using a Seahorse XF Analyzer, citrate synthase assay, immunoblotting and flow cytometry. We assessed the cancer stem cell (CSC) phenotype of the variants and 2-DG's effects on CSCs. 2-DG significantly inhibited migration and invasion of Hs578Ts(i) 8 versus Hs578T and significantly decreased their ability to resist anoikis. Investigating 2-DG's preferential inhibitory effect on the more aggressive cells, we found Hs578Ts(i) 8 also had significantly decreased oxidative phosphorylation and increased glycolysis compared to Hs578T. This is likely due to mitochondrial dysfunction in Hs578Ts(i) 8 , shown by their significantly decreased mitochondrial membrane potential. Furthermore, Hs578Ts(i) 8 had a significantly increased proportion of cells with CSC phenotype, which was significantly decreased by 2-DG. 2-DG may have benefit as a therapy for TNBC with a particularly aggressive phenotype, by targeting increased glycolysis. Studies of more cell lines and patients' specimens are warranted.

Brown adipose tissue (BAT) and brown in white (brite) adipose tissue, termed also beige adipose tissue, are major sites of mammalian nonshivering thermogenesis. Mitochondrial uncoupling protein 1 (UCP1), specific for these tissues, is the key factor for heat production. Recent molecular aspects of UCP1 structure provide support for the fatty acid cycling model of coupling, i.e. when UCP1 expels fatty acid anions in a uniport mode from the matrix, while uncoupling. Protonophoretic function is ensured by return of the protonated fatty acid to the matrix independent of UCP1. This mechanism is advantageous for mitochondrial uncoupling and compatible with heat production in a pro-thermogenic environment, such as BAT. It must still be verified whether posttranslational modification of UCP1, such as sulfenylation of Cys253, linked to redox activity, promotes UCP1 activity. BAT biogenesis and UCP1 expression, has also been linked to the pro-oxidant state of mitochondria, further endorsing a redox signalling link promoting an establishment of pro-thermogenic state. We discuss circumstances under which promotion of superoxide formation exceeds its attenuation by uncoupling in mitochondria and throughout point out areas of future research into UCP1 function., (Copyright © 2018. Published by Elsevier B.V.)

[This corrects the article DOI: 10.1186/s40104-018-0282-9.].

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., Competing Interests: EWH is Chief Science Officer of Plusvital Ltd. Plusvital Ltd. has been granted a licence for commercial use of the data that is contained within multiple granted patents and patent applications including (patent reference numbers): EP2352850, JP5667057, US8771943, AU2009290452, NZ591711, US9249470 and US2016215335. EWH is named on these patents. Plusvital Ltd. had no part in the research contained in this manuscript. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Glycerol-3-phosphate is an excellent substrate for FAD-linked mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) in brown adipose tissue mitochondria and is regularly used as the primary substrate to measure oxygen consumption and reactive oxygen consumption by these mitochondria. mGPDH converts cytosolic glycerol-3-phosphate to dihydroxyacetone phosphate, feeding electrons directly from the cytosolic side of the mitochondrial inner membrane to the CoQ-pool within the inner membrane. mGPDH activity is allosterically activated by calcium, and when calcium chelators are present in the mitochondrial preparation medium and/or experimental incubation medium, calcium must be added to insure maximal mGPDH activity. It was demonstrated that in isolated brown adipose tissue mitochondria (1) mGPDH enzyme activity is maximal at free calcium ion concentrations in the 350 nM-1 μM range, (2) that ROS production also peaks in the 10-100 nM range in the presence of a UCP1 inhibitory ligand (GDP) but wanes with further increasing calcium concentration, and (3) that oxygen consumption rates peak in the 10-100 nM range with rates being maintained at higher calcium concentrations. This article provides easy-to-follow protocols to facilitate the measurement of mGPDH-dependent UCP1 activity in the presence of calcium for isolated brown adipose tissue mitochondria.

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.

Phenformin, a member of the biguanides class of drugs, has been reported to be efficacious in cancer treatment. The focus of the current study was to establish whether there were direct effects of phenformin on the metabolism and bioenergetics of neuroblastoma SH-SY5Y cancer cells. Cell viability was assessed using the alamar blue assay, flow cytometry analysis using propidium iodide and annexin V stain and poly (ADP-ribose) polymerase analysis. Cellular and mitochondrial oxygen consumption was determined using a Seahorse Bioscience Flux analyser and an Oroboros Oxygraph respirometer. Cells were transfected using electroporation and permeabilized for in situ mitochondrial functional analysis using digitonin. Standard protocols were used for immunoblotting and proteins were separated on denaturing gels. Phenformin was effective in reducing the viability of SH-SY5Y cells, causing G 1 cell cycle arrest and inducing apoptosis. Bioenergetic analysis demonstrated that phenformin significantly decreased oxygen consumption in a dose- and time-dependent manner. The sensitivity of oxygen consumption in SH-SY5Y cells to phenformin was circumvented by the expression of NADH-quinone oxidoreductase 1, a ubiquinone oxidoreductase, suggesting that complex I may be a target of phenformin. As a result of this inhibition, adenosine monophosphate protein kinase is activated and acetyl-coenzyme A carboxylase is inhibited. To the best of our knowledge, the current study is the first to demonstrate the efficacy and underlying mechanism by which phenformin directly effects the survival of neuroblastoma cancer cells.

Acquired cisplatin resistance is a common feature of tumours following cancer treatment with cisplatin and also of non-small cell lung cancer (H1299) and mesothelioma (P31) cell lines exposed to cisplatin. To elucidate the cellular basis of acquired cisplatin resistance, a comprehensive bioenergetic analysis was undertaken. We demonstrate that cellular oxygen consumption was significantly decreased in cisplatin resistant cells and that the reduction was primarily due to reduced mitochondrial activity as a result of reduced mitochondrial abundance. The differential mitochondrial abundance was supported by data showing reduced sirtuin 1 (SIRT1), peroxisome-proliferator activator receptor-γ co-activator 1-alpha (PGC1α), sirtuin 3 (SIRT3) and mitochondrial transcription factor A (TFAM) protein expression in resistant cells. Consistent with these data we observed increased reactive oxygen species (ROS) production and increased hypoxia inducible factor 1-alpha (HIF1α) stabilization in cisplatin resistant cells when compared to cisplatin sensitive controls. We also observed an increase in AMP kinase subunit α2 (AMPKα2) transcripts and protein expression in resistant H1299 cells. mRNA expression was also reduced for cisplatin resistant H1299 cells in these genes, however the pattern was not consistent in resistant P31 cells. There was very little change in DNA methylation of these genes, suggesting that the cells are not stably reprogrammed epigenetically. Taken together, our data demonstrate reduced oxidative metabolism, reduced mitochondrial abundance, potential for increased glycolytic flux and increased ROS production in acquired cisplatin resistant cells. This suggests that the metabolic changes are a result of reduced SIRT3 expression and increased HIF-1α stabilization., Competing Interests: CONFLICTS OF INTEREST The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Neuromedin U (NmU) is a neuropeptide belonging to the neuromedin family. Recently, we reported a significant association between NmU and breast cancer, particularly correlating with increased aggressiveness, resistance to HER2-targeted therapies and overall significantly poorer outcome for patients, although the mechanism through which it exerts this effect remained unexplained. Investigating this, here we found that ectopic over-expression of NmU in HER2-positive breast cancer cells induced aberrant metabolism, with increased glycolysis, likely due to enhanced pyruvate dehydrogenase kinase activity. Similar results were observed in HER2-targeted drug-resistant cell variants, which we had previously shown to display increased levels of NmU. Overexpression of NmU also resulted in upregulation of epithelial-mesenchymal transition markers and increased IL-6 secretion which, together with aberrant metabolism, have all been associated with the cancer stem cell (CSC) phenotype. Flow cytometry experiments confirmed that NmU-overexpressing and HER2-targeted drug-resistant cells showed an increased proportion of cells with CSC phenotype (CD44 + /CD24 - ). Taken together, our results report a new mechanism of action for NmU in HER2-overexpressing breast cancer that enhances resistance to HER2-targeted drugs through conferring CSC characteristics and expansion of the CSC phenotype., (© 2017 UICC.)

Metformin is the main drug of choice for treating type 2 diabetes, yet the therapeutic regimens and side effects of the compound are all undesirable and can lead to reduced compliance. The aim of this study was to elucidate the mechanism of action of two novel compounds which improved glucose handling and weight gain in mice on a high-fat diet. Wildtype C57Bl/6 male mice were fed on a high-fat diet and treated with novel, anti-diabetic compounds. Both compounds restored the glucose handling ability of these mice. At a cellular level, these compounds achieve this by inhibiting complex I activity in mitochondria, leading to AMP-activated protein kinase activation and subsequent increased glucose uptake by the cells, as measured in the mouse C2C12 muscle cell line. Based on the inhibition of NADH dehydrogenase (IC50 27µmolL(-1)), one of these compounds is close to a thousand fold more potent than metformin. There are no indications of off target effects. The compounds have the potential to have a greater anti-diabetic effect at a lower dose than metformin and may represent a new anti-diabetic compound class. The mechanism of action appears not to be as an insulin sensitizer but rather as an insulin substitute., (© 2016 Society for Endocrinology.)

Over several years we have provided evidence that uncoupling protein 1 (UCP1) is present in thymus mitochondria. We have demonstrated the conclusive evidence for the presence of UCP1 in thymus mitochondria and we have been able to demonstrate a GDP-sensitive UCP1-dependent proton leak in non-phosphorylating thymus mitochondria. In this chapter, we show how to detect UCP1 in mitochondria isolated from whole thymus using immunoblotting. We show how to measure GDP-sensitive UCP1-dependent oxygen consumption in non-phosphorylating thymus mitochondria and we show that increased reactive oxygen species production occurs on addition of GDP to non-phosphorylating thymus mitochondria. We conclude that reactive oxygen species production rate can be used as a surrogate for detecting UCP1 catalyzed proton leak activity in thymus mitochondria.

Background: Visceral obesity has a strong association with both the incidence and mortality of esophageal adenocarcinoma (EAC). Alterations in mitochondrial function and energy metabolism is an emerging hallmark of cancer, however, the potential role that obesity plays in driving these alterations in EAC is currently unknown., Methods: Adipose conditioned media (ACM) was prepared from visceral adipose tissue taken from computed tomography-determined viscerally-obese and non-obese EAC patients. Mitochondrial function in EAC cell lines was assessed using fluorescent probes, mitochondrial gene expression was assessed using qPCR-based gene arrays and intracellular ATP levels were determined using a luminescence-based kit. Glycolysis and oxidative phosphophorylation was measured using Seahorse XF technology and metabolomic analysis was performed using 1H NMR. Expression of metabolic markers was assessed in EAC tumor biopsies by qPCR., Results: ACM from obese EAC patients significantly increased mitochondrial mass and mitochondrial membrane potential in EAC cells, which was significantly associated with visceral fat area, and was coupled with a significant decrease in reactive oxygen species. This mitochondrial dysfunction was accompanied by altered expression of 19 mitochondrial-associated genes and significantly reduced intracellular ATP levels. ACM from obese EAC patients induced a metabolic shift to glycolysis in EAC cells, which was coupled with significantly increased sensitivity to the glycolytic inhibitor 2-deoxyglucose. Metabolomic profiling demonstrated an altered glycolysis and amino acid-related signature in ACM from obese patients. In EAC tumors, expression of the glycolytic marker PKM2 was significantly positively associated with obesity., Conclusion: This study demonstrates for the first time that ACM from viscerally-obese EAC patients elicits an altered metabolic profile and can drive mitochondrial dysfunction and altered energy metabolism in EAC cells in vitro. In vivo, in EAC patient tumors, expression of the glycolytic enzyme PKM2 is positively associated with obesity.

We previously demonstrated that uncoupling protein 1 activity, as measured in isolated brown adipose tissue mitochondria (and as a native protein reconstituted into liposome membranes), was not activated by the non-flippable modified saturated fatty acid, glucose-O-ω-palmitate, whereas activity was stimulated by palmitate alone (40 nM free final concentration). In this study, we investigated whether fatty acid chain length had any bearing on the ability of glucose-O-ω-fatty acids to activate uncoupling protein 1. Glucose-O-ω-saturated fatty acids of various chain lengths were synthesized and tested for their potential to activate GDP-inhibited uncoupling protein 1-dependent oxygen consumption in brown adipose tissue mitochondria, and the results were compared with equivalent non-modified fatty acid controls. Here we demonstrate that laurate (12C), palmitate (16C) and stearate (18C) could activate GDP-inhibited uncoupling protein 1-dependent oxygen consumption in brown adipose tissue mitochondria, whereas there was no activation with glucose-O-ω-laurate (12C), glucose-O-ω-palmitate (16C), glucose-O-ω-stearate (18C), glucose-O-ω-arachidate (20C) or arachidate alone. We conclude that non-flippable fatty acids cannot activate uncoupling protein 1 irrespective of chain length. Our data further undermine the cofactor activation model of uncoupling protein 1 function but are compatible with the model that uncoupling protein 1 functions by flipping long-chain fatty acid anions.

We have previously shown that uncoupling protein 1 is present in thymus and has a role in T-cell development. As reactive oxygen species have been implicated in T-cell development, we set out to determine whether uncoupling protein 1 had the potential to regulate reactive oxygen species production in mitochondria isolated from thymus. This was achieved by inhibiting proton leak through uncoupling protein 1 using the purine nucleotide GDP and through ablation of uncoupling protein 1, measuring the amplex red sensitive reactive oxygen species production by mitochondria. In this work we demonstrate, for the first time, that uncoupling protein 1 has the potential to regulate reactive oxygen species production in thymus mitochondria. We also show that reverse electron transport is possible in thymus mitochondria respiring on succinate and glycerol-3-phosphate. The implications of this regulatory role for uncoupling protein 1 are discussed in the context of thymus function. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

In this study we show that mitochondrial uncoupling protein 1 (UCP1) in brown adipose tissue (BAT) and thymus mitochondria can be ubiquitinylated and degraded by the cytosolic proteasome. Using a ubiquitin conjugating system, we show that UCP1 can be ubiquitinylated in vitro. We demonstrate that UCP1 is ubiquitinylated in vivo using isolated mitochondria from brown adipose tissue, thymus and whole brown adipocytes. Using an in vitro ubiquitin conjugating-proteasome degradation system, we show that the cytosolic proteasome can degrade UCP1 at a rate commensurate with the half-life of UCP1 (i.e. 30-72h in brown adipocytes and ~3h, in thymocytes). In addition, we demonstrate that the cytoplasmic proteasome is required for UCP1 degradation from mitochondria that the process is inhibited by the proteasome inhibitor MG132 and that dissipation of the mitochondrial membrane potential inhibits degradation of UCP1. There also appears to be a greater amount of ubiquitinylated UCP1 associated with BAT mitochondria from cold-acclimated animals. We have also identified (using immunoprecipitation coupled with mass spectrometry) ubiquitinylated proteins with molecular masses greater than 32kDa, as being UCP1. We conclude that there is a role for ubiquitinylation and the cytosolic proteasome in turnover of mitochondrial UCP1. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012)., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Mitochondrial uncoupling proteins are members of the SLC25 family of solute carriers. Models of mitochondrial transporter function predict that uncoupling proteins are solute carriers. Evidence in the literature suggests that uncoupling proteins can transport protons, fatty acid anions, chloride anions, and recently the dicarboxylate succinate. Studies have also demonstrated that UCPs can be covalently modified and in some instances this covalent modification is needed to affect uncoupling function. The current evidence from functional analyses of mammalian uncoupling proteins is summarized in this chapter.

Previous researchers have demonstrated that 3,4-methylenedioxymethamphetamine (MDMA) induced hyperthermia, in skeletal muscle of animals, is uncoupling protein 3 (UCP3) dependent. In light of our investigations that in vivo phosphorylation of UCP1 is augmented under conditions of cold-acclimation, we set out to investigate whether (a) UCP3 was phosphorylated in vivo and (b) whether in vivo phosphorylation of UCP3 resulted in increased proton leak following MDMA administration to animals. Our data demonstrate that MDMA treatment (but not PBS treatment) of animals results in both in vivo serine and tyrosine phosphorylation of UCP3 in skeletal muscle mitochondria, isolated in the presence of phosphatase inhibitors to preserve in vivo phosphorylation. In addition, proton leak is only increased in skeletal muscle mitochondria isolated from MDMA treated animals (in the presence of phosphatase inhibitors) and the increased proton leak is due to phosphorylated UCP3. UCP3 abundance in skeletal muscle mitochondria is unaffected by MDMA administration. Preservation of UCP3 phosphorylation and increased proton leak is lost when skeletal muscle mitochondria are isolated in the absence of phosphatase inhibitors. We conclude that MDMA treatment of animals increases proton leak in skeletal muscle mitochondria by activating UCP3 through in vivo covalent modification of UCP3 by phosphorylation. Furthermore, we deduce that the MDMA induced hyperthermia in skeletal muscle is due to increased proton leak in vivo as a result of activation of UCP3 through phosphorylation., (Copyright © 2011 Elsevier B.V. and Mitochondria Research Society. All rights reserved.)

Mitochondrial uncoupling protein 3 (UCP3) is constitutively expressed in mitochondria from thymus and spleen of mice, and confocal microscopy has been used to visualize UCP3 in situ in mouse thymocytes. UCP3 is present in mitochondria of thymus and spleen up to at least 16 weeks after birth, but levels decrease by a half in thymus and a fifth in spleen after three weeks, probably reflecting the suckling to weaning transition. UCP3 protein levels increase approximately 3-fold in thymus on starvation, but expression levels in spleen were unaffected by starvation. Lack of UCP3 had little effect on thymus mass or thymocyte number. However, lack of UCP3 affected spleen mass and splenocyte number (in the fasted state) and results in reduced CD4+ single positive cell numbers and reduced double negative cells in the thymus, but as a 2-fold increase in the proportion of CD4(+), CD8(+) and DP cells in spleen. Starvation attenuates these proportionate differences in the spleen. A lack of UCP3 had no apparent effect on basal oxygen consumption of thymocytes or splenocytes or on oxygen consumption due to mitochondrial proton leak. Splenocytes from UCP3 knock-out mice are also more resistant to apoptosis than those from wild-type mice. Overall we can conclude that UCP3 affects thymocyte and spleen cell profiles in the fed and fasted states., (2011 Elsevier B.V. All rights reserved.)

We provide evidence that ablation or inhibition of, uncoupling protein 1 increases the rate of reactive oxygen containing species production by mitochondria from brown adipose tissue, no matter what electron transport chain substrate is used (succinate, glycerol-3-phosphate or pyruvate/malate). Consistent with these data are our observations that (a) the mitochondrial membrane potential is maximal when uncoupling protein 1 is ablated or inhibited and (b) oxygen consumption rates in mitochondria from uncoupling protein 1 knock-out mice, are significantly lower than those from wild-type mice, but equivalent to those from wild-type mice in the presence of GDP. In summary, we show that uncoupling protein 1 can affect reactive oxygen containing species production by isolated mitochondria from brown adipose tissue., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

Our laboratory has previously demonstrated the presence of constitutively expressed mitochondrial uncoupling protein 1 in mouse thymocytes. In our endeavours to understand the role of mitochondrial uncoupling protein 1 in thymocyte function, we compared cell profiles in thymus and spleen of wild-type with those of UCP 1 knock-out mice, which in turn led to comparative investigations of apoptotic potential in thymocytes from these mice. We demonstrate that spleen cell numbers were reduced approximately 3-fold in UCP 1 knock-out mice compared to wild-type mice. We record a halving of CD8 single positive cell numbers in thymus with a significant incremental increase in CD4/CD8 double positives cell numbers in the thymus of UCP 1 knock-out mice compared to wild-type mice. These data are mirrored by an approximate halving of CD8 single positive cell numbers and a doubling of CD4/CD8 double positive cell numbers in the spleen of UCP 1 knock-out mice compared to wild-type mice. These differences are most probably explained by our observations of decreased apoptotic potential and higher ATP levels in thymocytes of UCP 1 knock-out mice when compared to wild-type controls. We conclude that constitutively expressed UCP 1 is a factor in determining T-cell population selection in mice., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Choline is a quaternary amino cationic organic alcohol that is oxidized to betaine in liver and kidney mitochondria. Betaine acts as an intracellular organic osmolyte in the medulla of the kidney. Evidence is provided that kidney mitochondria have a choline transporter in their inner membrane. The transporter has a Km of 173+/-64 microM and a Vmax of 0.4+/-0.1 nmol/min/mg mitochondrial protein (at 10 degrees C). Uptake of choline is not coupled to betaine efflux. Transporter activity demonstrates a dependence on membrane potential and choline transport is inhibited by hemicholinium-3. Steady-state oxygen consumption due to choline oxidation in kidney mitochondria was measurable at 37 degrees C (125+/-6 pmol O2/min/mg mitochondrial protein), in the absence of other mitochondrial electron transport chain substrates and the choline transporter was shown to be the major site of control (96+/-4%) over choline oxidation flux in isolated kidney mitochondria. We conclude that the choline transporter in rat kidney mitochondria is the major site of control over the production of the organic osmolyte, betaine.

We determined the proportion of oxygen consumption due to oxidative phosphorylation by mitochondria in an adrenal gland cell line (Y1 cells). In addition we determined the relative proportion of in situ mitochondrial oxygen consumption attributable to (i) proton leak and (ii) ATP turnover in these cells. This approach allowed use of top-down elasticity analysis to determine control of oxidative phosphorylation by mitochondrial (a) proton leak flux (b) substrate oxidation flux and (c) ATP turnover flux, as a function of changes in in situ mitochondrial membrane potential. Our data show that resting oxygen consumptions rates of Y1 cells to be 87 +/- 7 nmolO/min/10(7) cells of which 38 +/- 3% was not due to oxidative phosphorylation. We demonstrated that mitochondrial proton leak accounted for 7 +/- 3% of total cellular oxygen consumption or 12 +/- 6% of resting mitochondrial oxygen consumption, with ATP turnover accounting for 55 +/- 3% of total cellular oxygen consumption or 78 +/- 6% of mitochondrial oxygen consumption. Control of resting mitochondrial oxygen consumption in Y1 cells was shared by (a) substrate oxidation flux (37 +/- 8%), (b) proton leak flux (15 +/- 8%) and (c) ATP turnover (56 +/- 8%). Our data demonstrate, for the first time, that the majority of oxygen consumption by resting Y1 cells is due to oxidative phosphorylation.

One of the significant naturally occurring short-circuits in the chemiosmotic process is manifest through the activity of uncoupling protein 1, most notably present in the inner membrane of mitochondria in brown adipose tissue. Much has been learnt about the mechanism of action of uncoupling protein 1 since its discovery in the 1970's, yet many areas of controversy surround (i) the mechanism of its action, (ii) novel regulators of its activity, and (iii) the tissue specificity of its expression. In this mini-review, areas of controversy are highlighted from the aforementioned categories using selected key experiments to demonstrate the arguments.

Using an antibody specific and selective to mitochondrial uncoupling protein 1 (UCP1) peptide, this study confirms the observation that UCP 1 is present in thymocytes isolated from UCP 1 wild-type, but not UCP 1 knock-out mice. UCP 1 is also shown to be present in thymocytes isolated from rat. It was also demonstrated that an antibody raised to the full-length UCP 1 protein appears to be non-specific for UCP 1, as it detects protein in UCP 1 wild-type and UCP 1 knock-out mice, protein in mitochondria isolated from brown adipose tissue of both UCP 1 wild-type and UCP 1 knock-out mice, as well as detecting protein in mitochondria isolated from rat spleen, kidney, skeletal muscle and liver, tissues that do not express UCP 1. We were also able to show that CIDEA, a soluble protein with a suggested role in regulating UCP 1 function, is equally abundant in thymocytes from UCP 1 wild-type and UCP 1 knock-out mice. Taken together our data demonstrate that (a) UCP 1 is present in rat and mouse thymocytes, (b) that the antibody to full-length UCP 1 is not specific for UCP 1 and (c) that the absence of UCP 1 does not affect native expression of CIDEA in thymocytes.

Peroxisome proliferator-activated receptor alpha (PPARalpha) belongs to the nuclear receptor superfamily that regulates multiple target genes involved in lipid metabolism. Cholesterol ester transfer protein (CETP) is a secreted glycoprotein that modifies high-density lipoprotein (HDL) particles. In humans, plasma CETP activity is inversely correlated with HDL cholesterol levels. We report here that PPARalpha agonists increase CETP mRNA, protein and accordingly its activity. In a human CETP transgenic animal model harboring the natural flanking regions (Jiang et al. in J Clin Investigat 90:1290-1295, 1992), both fenofibrate and a specific synthetic PPARalpha agonist LY970 elevated human CETP mRNA in liver, serum protein and CETP activity. In hamsters, the endogenous liver CETP mRNA level and the serum CETP activity were dose-dependently upregulated by fenofibrate. In addition Wy14643, a PPARalpha agonist, also significantly elevated CETP mRNA and activity. In a carcinoma cell line of hepatic origin, HepG2 cells, overexpression of PPARalpha resulted in increased CETP mRNA and agonist treatment further elevated CETP mRNA levels. We conclude that PPARalpha agonists upregulate CETP expression and activity and may play an important role in PPARalpha (agonist mediated HDL cholesterol homeostasis in humans.

Native uncoupling protein 1 was purified from rat brown adipose tissue of cold-acclimated rats and rats kept at room temperature, in the presence of phosphatase inhibitors. The purified protein from cold-acclimated animals was digested with trypsin and immobilized metal affinity chromatography was used to select for phosphopeptides. Tandem mass spectroscopic analysis of the peptides derived from uncoupling protein 1, suggests phosphorylation of serine 3 or 4 and identified phosphorylation of serine 51. Furthermore, we were able to demonstrate that antibodies to phosphoserine detect full-length UCP 1 and that the proportion of phosphoserine on UCP1, purified from cold-acclimated rats, was significantly greater than that on UCP 1 from rats kept at room temperature (90+/-4% compared to 62+/-8%, p=0.013), respectively). We conclude that uncoupling protein 1 is a phosphoprotein and that cold-acclimation increases the proportion of UCP1 that is serine phosphorylated.

Stearoyl-CoA desaturase (SCD) catalyzes the formation of monounsaturated fatty acids from saturated fatty acids. It plays a key role in lipid metabolism and energy expenditure in mammals. In mice, four SCD isoforms (SCD1-4) have been identified. Here we report the identification of cDNA sequences corresponding to SCD1, SCD2 and SCD3 of golden hamster. The deduced amino acid sequences of these hamster SCD (hmSCD) isoforms display a high degree of homologies to their mouse counterparts (mouse SCD). Polyclonal antibodies specific to rodent SCDs detected proteins of predicted size in the human embryonic kidney 293 cells transfected with hmSCD cDNAs. Microsome fractions prepared from these cells also displayed increased SCD activity versus cells transfected with vector alone. Real-time reverse transcription-polymerase chain reaction analysis revealed the highest expression of hmSCD1 in liver and adipose tissue, while the highest hmSCD2 expression was detected in the brain. Very low levels of hmSCD3 mRNA can be detected in the tissues tested. This report is the first description of three SCD isoforms in the hamster and will provide useful tools in the further study of fatty acids metabolism in this species.

The aim of this study was to demonstrate the constitutive expression of mitochondrial uncoupling protein 1 (UCP 1) in pure thymocytes using laser scanning confocal microscopic imagery. To that end we probed thymocytes from UCP 1 knock-out and wild-type mice. Mitochondrial location in thymocytes was determined using Mitotracker Red and the nucleus was labelled using Hoescht stain. We demonstrate that all cells investigated were thymocytes as determined by a monoclonal antibody specific for the thymocyte surface marker Thy 1 (CD90) pre-coupled to a fluorescent labelled (Alexa 448, green). Using a primary peptide antibody specific to UCP 1, and secondary fluorescently labelled (Alexa 647, magenta) antibody, we were able to demonstrate that UCP 1 is associated with mitochondria in thymocytes from UCP 1 wild-type mice but not thymocytes from UCP1-knock-out mice. These are the first images demonstrating the presence of UCP 1 in thymocyte mitochondria, in situ, and the first to clearly demonstrate UCP 1 expression in cells other than brown adipocytes. We conclude that mouse thymocytes contain UCP 1 in their mitochondria.

Uncoupling proteins 1 (UCP1) and 2 (UCP2) belong to the family of mitochondrial anion transporters and share 59% sequence identity with each other. Whereas UCP1 was shown to be responsible for the rapid production of heat in brown adipose tissue, the primary function and transport properties of ubiquitously expressed UCP2 are controversially discussed. Here, for the first time, the activation pattern of the recombinant human UCP2 in comparison to the recombinant human UCP1 are studied using a well-defined system of planar lipid bilayers. It is shown that despite apparently different physiological functions, hUCP2 exhibited its protonophoric function similar to hUCP1--exclusively in the presence of long-chain fatty acids (FA). The calculated hUCP2 transport rate of 4.5 s(-1) is the same order of magnitude, as shown previously for UCP1. It leads to the conclusion that the differences in the activity of both proteins in living mitochondria are based exclusively on their different expression level. Both proteins are activated much more effectively by polyunsaturated than by saturated FA. The proton and total membrane conductances increased in the range palmitic < oleic < eicosatrienoic < linoleic < retinoic < arachidonic acids. The higher uncoupling protein (UCP)-dependent conductance in the presence of polyunsaturated FA is explained on the basis of the FA cycling hypothesis.

Mitochondrial uncoupling protein 1 is usually associated with brown adipose tissue but has recently been discovered in rat and mouse thymus. We wished to establish whether there was a thermogenic role for UCP 1 in thymus and thus examined the effect of 5 weeks cold-acclimation on rat thymus tissue abundance, thymocyte oxygen consumption, thymus mitochondrial abundance, uncoupling protein 1 expression and function. We found that thymocytes from cold-acclimated rats had oxygen consumption rates 8 times less than those from rats held at room temperature and that thymocytes from cold-acclimated rats or rats kept at room temperature were noradrenaline insensitive. In addition, we found that thymus tissue or mitochondrial abundance was not increased after cold-acclimation. However uncoupling protein 1 expression per unit mass of mitochondria was increased after cold-acclimation, as determined by immunoblotting (approximately 1.7-fold) and GDP binding (approximately 1.5-fold). Consistent with our protein expression data, we also observed an increased, state 4 (approximately 1.5-fold), GDP-inhibitable (approximately 1.3-fold) and palmitate activatable (approximately 1.6-fold) oxygen consumption rates in isolated thymus mitochondria. However, extrapolation of our data showed that cold-acclimation only increased the amount of UCP 1 per gram of thymus tissue approximately 1.2-fold. Taken together, we conclude that UCP 1 does not have a thermogenic role in thymus.

Electrophysiological characterisation of the vast number of annotated channel and transport proteins in the postgenomic era would be greatly facilitated by the introduction of rapid and robust methods for the functional incorporation of membrane proteins into defined lipid bilayers. Here, we describe an automated technique for reconstitution of membrane proteins into lipid bilayer membranes, which substantially reduces both the reconstitution time and the amount of protein required for the membrane formation. The method allows the investigation of single protein channels as well as insertion of multiple copies (approximately 10(7)) into a single bilayer. Despite a comparatively large membrane area (up to 300 microm diameter), the high stability of the membrane permits the application of transmembrane voltages up to 300 mV. This feature is especially important for studies of inner membrane mitochondrial proteins, since they act at potentials up to approximately 200 mV under physiological conditions. It is a combination of these advantages that enables the detailed investigation of the minuscule single protein conductances typical for proton transporters. We have applied the new technique for the reconstitution and electrophysiological characterisation of human recombinant uncoupling protein 1, hUCP1, that has been overexpressed in E. coli and purified from inclusion bodies. We demonstrate that hUCP1 activity in the presence of fatty acids is comparable to the activity of UCP1 isolated from brown adipose tissue.

There has been a resurgence of interest in mitochondrial uncoupling protein 1 due to a desire to understand the regulation of the prominent role it plays in control of metabolic flux in brown adipose tissue and non-shivering thermogenesis, combined with the fact that UCP 1 acts as a paradigm for other novel less abundant uncoupling proteins. In this manuscript, we review the recent evidence for detection, purification, identification and function of UCP 1 in thymus mitochondria. In addition, we review the two proposed mechanisms for fatty acid dependent UCP 1 activity, namely (a) the flippase (flip-flop) model and (b) the cofactor/activation model, and the implication for these models of recent data showing that glucose-O-omega-palmitate cannot facilitate UCP 1 activity.

Native uncoupling protein 1 (UCP 1) was purified from rat mitochondria by hydroxyapatite chromatography and identified by peptide mass mapping and tandem mass spectrometry. Native and expressed UCP 1 were reconstituted into liposomes, and proton flux through UCP 1 was shown to be fatty acid-dependent and GDP-sensitive. To investigate the mechanism of action of UCP 1, we determined whether hydrophilic modification of the omega-carbon of palmitate effected its transport function. We show that proton flux was greater through native UCP 1-containing proteoliposomes when facilitated by less hydrophilically modified palmitate (palmitate > omega-methoxypalmitate > omega-hydroxypalmitate with little or no proton flux due to glucose-O-omega-palmitate or undecanesulfonate). We show that non-proton-dependent charge transfer was greater when facilitated by less hydrophilically modified palmitate (palmitate/undecanesulfonate > omega-methoxypalmitate > omega-hydroxypalmitate, with no non-proton-dependent charge transfer flux due to glucose-O-omega-palmitate). We show that the GDP-inhibitable oxygen consumption rate in brown adipose tissue mitochondria was reversed by palmitate (as expected) but not by glucose-O-omega-palmitate. Our data are consistent with the model that UCP 1 flips long-chain fatty acid anions and contradict the "cofactor" model of UCP 1 function.

Glucose-O-omega-palmitic acid is an amphipathic molecule that is useful as a tool for studying the mechanism of mitochondrial uncoupling proteins. The synthesis of this glycolipid is described herein. The study of the reaction of a series of glycosyl donors with appropriate acceptors derived from 16-hydroxyhexadecanoic acid showed that a glycosyl trichloroacetimidate donor was more efficient than thioglycoside, glycosyl halide and glycosyl acetate donors for synthesis of this glycolipid.

We present evidence that rat and mouse thymi contain mitochondrial uncoupling protein (UCP 1). Reverse transcriptase-PCR detected RNA transcripts for UCP 1 in whole thymus and in thymocytes. Furthermore, using antibodies to UCP 1 the protein was also detected in mitochondria isolated from whole thymus and thymocytes but not in thymus mitochondria from UCP 1 knock-out mice. Evidence for functional UCP 1 in thymus mitochondria was obtained by a comparative analysis with the kinetics of GDP binding in mitochondria from brown adipose tissue. Both tissues showed equivalent B(max) and K(D) values. In addition, a large component of the nonphosphorylating oxygen consumption by thymus mitochondria was inhibited by GDP and subsequently stimulated by addition of nanomolar concentrations of palmitate. UCP 1 was purified from thymus mitochondria by hydroxyapatite chromatography. The isolated protein was identified by peptide mass mapping and tandem mass spectrometry by using MALDI-TOF and LC-MS/MS, respectively. We conclude that the thymus contains a functioning UCP 1 that has the capacity to regulate metabolic flux and production of reactive oxygen-containing molecules in the thymus.

Thymi were dissected from rats and connective tissue was removed. Mitochondria were purified from isolated thymocytes and immunoblot analysis was performed using an antibody specific for uncoupling protein 1, which detected a 32.5 kDa protein associated with mitochondria from the thymocytes. This implies that rat thymocytes contain uncoupling protein 1.