SLC25A48 influences plasma levels of choline and localizes to the inner mitochondrial membrane.

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.

The Peripheral Cannabinoid Receptor Type 1 (CB1) as a Molecular Target for Modulating Body Weight in Man.

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.

Neuromedin U alters bioenergetics and expands the cancer stem cell phenotype in HER2-positive breast cancer.

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.

Excess visceral adiposity induces alterations in mitochondrial function and energy metabolism in esophageal adenocarcinoma.

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.

A role for ubiquitinylation and the cytosolic proteasome in turnover of mitochondrial uncoupling protein 1 (UCP1).

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).

Conditionally immortalised equine skeletal muscle cell lines for in vitro analysis.

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 Q10 (CoQ10) to the cell lines improved mitochondrial function, an observation consistent with ex vivo skeletal muscle samples with these genotypes following supplementation with CoQ10 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.

Cancer-Cachexia-Induced Human Skeletal Muscle Myotube Degeneration Is Prevented via Cannabinoid Receptor 2 Agonism In Vitro.

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 (CB1R and CB2R) 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 CB1R/CB2R agonist, and JWH133, a selective CB2R 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 CB1R/CB2R 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 CB2R antagonist AM630, but not the CB1R 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 CB2R, and not CB1R.

Effect of breed and diet on the M. longissimus thoracis et lumborum transcriptome of steers divergent for residual feed intake.

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.

Absence of mitochondrial uncoupling protein 3: effect on thymus and spleen in the fed and fasted mice.

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.

Studies on the function and regulation of mitochondrial uncoupling proteins.

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.

Evidence of a role for interleukin-6 in anoikis resistance in oral squamous cell carcinoma.

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 role of UCP 1 in production of reactive oxygen species by mitochondria isolated from brown adipose tissue.

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.

Absence of mitochondrial uncoupling protein 1 affects apoptosis in thymocytes, thymocyte/T-cell profile and peripheral T-cell number.

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.

The Use of Reactive Oxygen Species Production by Succinate-Driven Reverse Electron Flow as an Index of Complex 1 Activity in Isolated Brown Adipose Tissue Mitochondria.

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.

An examination of skeletal muscle and hepatic tissue transcriptomes from beef cattle divergent for residual feed intake.

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.

Control of choline oxidation in rat kidney mitochondria.

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.

Pro-inflammatory Stimulation of Monocytes by ANCA Is Linked to Changes in Cellular Metabolism.

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-1ß. Changes in cellular metabolism, particularly a switch to glycolysis, have recently been linked to activation of immune cells and production of IL-1ß. 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-1ß 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.

UCP3 reciprocally controls CD4+ Th17 and Treg cell differentiation.

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.

Identification of serine phosphorylation in mitochondrial uncoupling protein 1.

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.

Mitochondrial uncoupling protein 1 expression in thymocytes.

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.