Inactivation of the Mitochondrial Carrier SLC25A25 (ATP-Mg²⁺/P_i Transporter) Reduces Physical Endurance and Metabolic Efficiency in Mice^*

Gene Targeting

The Cre/loxP system was used to generate mice with a global Slc25a25 deletion (Fig. 2A). A floxed Slc25a25 allele was created by introducing loxP sites into the introns flanking exons 2 and 3, with the third loxP site flanking a neo cassette (G418 resistance) as a selectable marker. Targeted C57BL/6J embryonic stem cells carrying the conditional floxed allele were introduced into C57BL/6J-Tyr c-2J host blastocyts. Chimeric progeny were crossed with C57BL/6J-Tyrc-2J mice to generate progeny heterozygous for the targeted allele. The presence of the 3 loxP sites was validated by genomic PCR (Fig. 2B). Mice with a global deletion of Slc25a25 were generated by breeding Slc25a25^fl/fl to EIIa-Cre mice that express Cre under the control of the EIIa promoter leading to expression of Cre recombinase in preimplantation embryos. The expression of genes under control of the adenoviral EIIa promoter is known to be restricted to the oocytes and preimplantation stages of the embryo (12), thereby leading to excision of exons 2 and 3 in the germline and all subsequent tissues and progeny. Progeny carrying the complete deletion were backcrossed to wild type C57BL/6J (WT) mice to ensure germline transmission of the mutation and removal of the EIIa-Cre transgene.

Microarray Analysis

Total RNA isolated from gastrocnemius red muscle of leptin-treated and untreated (PBS) Ucp1^−/−·Lep^−/− mice were used. RNA isolation was performed as previously described (13). Mice were kept at 28 °C for 1 week, and then ambient temperature was reduced 2 °C daily. Mice received leptin (1 μg/g/day in divided doses at 8:30 a.m. and 4:30 p.m.) from 20 °C until 16 °C. Animals were sacrificed 24 h after ambient temperature reached 16 °C. Quality of RNA was determined using the Agilent 2100 Bioanalyzer. RNA integrity number of samples ranged from 8.1 to 8.8. Each RNA sample was labeled and hybridized to the Applied Biosystems Mouse Genome Survey Microarray platform. Each microarray contained ∼34,000 features that include a set of about 1,000 controls. Each microarray used 32,096 probes to interrogate 32,281 curated genes representing 44,996 transcripts. Labeling and hybridization were performed as described previously (14). Signal intensities across microarrays were normalized by trimmed means. Features with signal/noise values >3 and quality FLAG values <5000 were considered “detected” and subjected to analysis of variance with p value of 0.01 using Spotfire Decision Site software (Spotfire, Somerville, MA). The complete list of genes with statistically significant (p < 0.05) levels of induction by leptin is shown in Table 1.

Molecular Cloning and DNA Sequence of Mouse Skeletal Muscle Slc25a25

cDNA was prepared from leptin-treated Ucp1^−/−·Lep^−/− quadriceps muscle. Slc25a25 NCBI reference sequence NM146118 was used to design primers for PCR using cDNA prepared from Ucp1^−/−·Lep^−/− quadriceps muscle. Final reaction products were phenol-chloroform extracted and ethanol-precipitated. DNA was cut with appropriate restriction enzymes, gel-purified, and subcloned to the pCI-neo mammalian expression vector (Promega, Madison, WI). Nucleotide sequence was verified and aligned with mouse major isoform (NM146118). Slc25a25 sequence from Ucp1^−/−·Lep^−/− quadriceps muscle corresponded to NCBI reference sequence AK132201, which lacks exon 6 and corresponds to the human SCaMC-2b spliced variant.

Animals and Study Design

Experiments were carried out on C57BL/6J (WT) and Slc25a25^−/− male mice maintained in a temperature-controlled room (28 °C) with a 12-h light/12-h dark cycle and fed rodent chow (PicoLab Rodent Diet 20 with 12 kcal % fat, LabDiet, Richmond, IN). Mice carrying a conditional floxed Slc25a25 allele were generated by the Pennington Transgenic Core. All animal experiments were approved by the Pennington Biomedical Research Center Institutional Animal Care and Use Committee in accordance with National Institutes of Health guidelines for care and use of laboratory animals.

Dietary Studies

Eight-week-old Slc25a25 WT and knock-out male mice previously maintained at 28 °C and fed chow diet (PicoLab Rodent Diet 20 with 12 kcal % fat) were single-housed, fed a high fat diet (D12331 with 58 kcal % fat, Research Diets Inc., New Brunswick, NJ), and kept at 20 °C for 10 weeks. Mice were subsequently maintained at 28 °C for another 10 weeks. In another study, two groups of 8-week-old WT mice were fed chow and a high fat diet at 23 °C for 30 weeks.

Phenotypes of Energy Balance

Body composition was analyzed by NMR (Minispec body composition analyzer, Bruker Optics, Billerica, MA). Oxygen consumption, carbon dioxide production, and physical activity of individual mice were measured in a 16-chamber Oxymax lab animal monitoring system CLAMS (Columbus Instruments, Columbus, OH). Glucose tolerance and insulin tolerance tests were performed after an overnight fast as previously described (13).

Cold Exposure

Slc25a25 WT and knock-out mice maintained at 28 °C were single-housed in cages with corncob bedding and exposed to ambient temperature of 4 °C, and body temperature was measured with a rectal probe (TH-8, Physitemp Instruments Inc., Clifton, NJ).

Assessment of Exercise Endurance

Untrained, age- and body weight-matched male mice were used in forced exercise endurance testing using a six-lane motorized rodent treadmill (Columbus Instruments, Columbus, OH). Mice were acclimatized to the treadmill for 4 days at a velocity of 8 m/min for 15 min. On day 5, exercise endurance was tested at an initial velocity of 8 m/min for 1 min after which velocity was increased at 2-m/min increments every minute until a maximum velocity of 14 m/min. Exhaustion was determined as the point at which the animal would repeatedly fall onto the metal grid despite gentle tapping on the back of the mouse.

Quantitative Reverse Transcription-PCR

Total RNA was prepared from tissues homogenized in TRI reagent (Molecular Research Center Inc., Cincinnati, OH) as described previously (15). The gastrocnemius is divided into 2 parts, the outer gastrocnemius white and the inner gastrocnemius red. The gastrocnemius white is located on the superficial lateral and medial portions, whereas the red part is in the deep medial and lateral portions. Quantitative reverse transcription-PCR was performed using total RNA with specific primers and probes designed using Primer-Express^TM, version 2.0.0 (Applied Biosystems, Foster City, CA). TaqMan probes were used for quantification of genes using the TaqMan one-step reverse transcriptase PCR master mixture (Applied Biosystems). All of gene expression data were normalized to the level of cyclophilin B for mouse studies, RPLPO for human studies, or total nanograms of input RNA/reaction. Primer and probe sequences are available upon request. Probes were designed from sequences in the C-terminal region to detect all 4 isoforms of human SLC25A25 (hSCaMC-2a to 2d).

Mouse Embryonic Fibroblasts

Mouse embryonic fibroblasts (MEF) were generated from Slc25a25 WT and knock-out E13.5 embryos. Carcasses were eviscerated, decapitated, and finely minced. Tissue from each embryo was digested with 0.05% trypsin for 15 min at 37 °C, washed with 10% FBS/DMEM, centrifuged at 1,000 × g for 5 min, and the supernatant discarded. Cell pellet was re-suspended and cultured in DMEM plus 10% FBS and penicillin-streptomycin. Genotype of each embryo was validated by genomic PCR. Primary MEF up to passage 7 were used for experiments.

Calcium Mobilization Assay

MEF were seeded at a density of 30,000 cells/well and incubated overnight at 37 °C. Medium was replaced with Hanks' balanced salt solution buffer and an equal volume of loading buffer with fluorescent dye (FLIPR Calcium Assay Kit, Molecular Devices, Sunnyvale, CA) and 25 mm probenecid was added. Cell plates were then incubated for 1 h at 37 °C and then for 45 min at RT protected from light. Changes in cytoplasmic calcium concentration in response to 300 nm bradykinin, 1 μm thapsigargin, and 5 μm ionomycin were recorded as relative fluorescence units using the FlexStation fluorescence plate reader (Molecular Devices). Probenecid, bradykinin, thapsigargin, and ionomycin were from Sigma.

Cellular Metabolism

The XF24 Analyzer (Seahorse Bioscience, Billerica, MA) was used to measure the oxygen consumption rate in a 24-well format (16). Culture medium was replaced with unbuffered DMEM supplemented with 25 mm glucose, 0.5 mm glutamine, and 1 mm pyruvate 1 h prior to assay. Optimum number of cells for bioenergetic experiments was initially determined with MEF seeded at different densities (25,000, 30,000, 35,000, and 40,000 cells/well). We used 30,000 cells/well for subsequent experiments. Mitochondrial function in MEF was determined through sequential addition of 5 μm oligomycin, 1 μm carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), and 1.5 μm antimycin A, 1 μm rotenone. This allowed determination of basal oxygen consumption, oxygen consumption linked to ATP synthesis, non-ATP linked oxygen consumption (proton leak), uncoupled respiration, and non-mitochondrial oxygen consumption (17, 18). Oligomycin-sensitive respiration is calculated from the mean of the four measurements after addition of oligomycin. ATP synthesis-linked oxygen consumption is the difference between basal respiration and oligomycin-sensitive respiration. Proton leak is the difference between oligomycin-sensitive respiration and respiration after injection of antimycin A and rotenone. Non-mitochondrial respiration is residual respiration obtained from the mean of least three points after addition of antimycin A and rotenone. Uncoupled respiration was calculated as the mean of the first three points after FCCP addition. Protein content in each well was measured to confirm an equal number of cells per well. Protein concentrations were determined by bicinchoninic acid assay (Sigma). Oligomycin, FCCP, antimycin A, and rotenone were from Sigma.

ATP Measurements

ATP levels in wild type and SLC25A25-deficient MEF were determined using an ATPlite Luminescence Assay System (PerkinElmer Life Sciences). Cells were seeded at a density of 30,000 cells per well in unbuffered DMEM supplemented with 25 mm glucose, 0.5 mm glutamine, and 1 mm pyruvate, and incubated in a non-CO₂ incubator at 37 °C for 1 h. Cells were then exposed to the following compounds for 1 min: 5 μm oligomycin, 1 μm FCCP, 100 mm 2-deoxy-d-glucose, 2 μm rotenone, and 25 μm carboxyatractyloside. Cells were lysed and cellular ATP content was measured. Luminescence intensity of each well was measured using a Berthold EG&G MicroLumat Plus DL Ready plate-reading luminometer.

Cell Viability

The viability of the MEF following a 1-min exposure to test compounds was assessed using the CellTiter-Blue Cell Viability Assay (Promega, Madison, WI). The fluorescence intensity of each well was measured using a FlexStation fluorescence plate reader (Molecular Devices).

Muscle Fiber-type Analysis

Skeletal muscle fiber-type was analyzed using a immunohistochemistry technique modified from Bajpeyi et al. (19). In brief, soleus muscle were collected, cleaned, and blotted dry prior to mounting in a mixture of optimal cutting temperature compound and tragacanth powder and then frozen in isopentane cooled over liquid nitrogen. Twelve-μm sections were processed for immunohistochemical staining. Muscle sections were incubated in blocking buffer (donkey serum) for 2 h followed by overnight incubation at 4 °C in primary antibodies (1:200, v/v) specific for myosin slow muscle (MAB1628, Chemicon) and laminin (AB2500, Abcam Inc., Cambridge, MA). After washing with phosphate-buffered saline, sections were incubated in the secondary antibodies (Alexa Fluor 680 donkey anti-mouse and Alexa Fluor 350 donkey anti-rat at 1:200 and 1:500 (v/v) dilution, respectively, from Invitrogen) for 2 h. After further washing, the sections were mounted in permount and coverglass was applied. Images were taken using confocal microscope (Zeiss 510 META, Carl Zeiss, Thornwood, NY) and type I fibers were counted. The relative numbers of the different fiber types were quantified by counting 2–3 sections from each sample containing a minimum of 50 muscle fibers.

Statistical Analysis

The data are expressed as the mean ± S.E. Unpaired t test was used to compare differences between groups. Analysis of variance with the Bonferroni post hoc test was used when more than two groups were compared (Statview, version 5.0.1; SAS Institute Inc., Cary, NC).

Microarray and Gene Expression Analysis of Skeletal Muscle for Genes Associated with Induction of Thermogenesis

The body temperature of Ucp1^−/− or Lep^−/− mice drops 10 °C within 3 h when they are acutely exposed to an ambient temperature of 5 °C. If the ambient temperature is gradually reduced about 2 degrees/day both mutant mice can tolerate the cold at 5 °C for weeks (20, 21). Double mutant Ucp1^−/−· Lep^−/− mice are not able to tolerate the cold under this protocol of gradually reducing the ambient temperature; however, they can be rescued, if they are administered leptin as the ambient temperature is gradually reduced (22). A microarray analysis was performed on skeletal muscle RNA from Ucp1^−/−·Lep^−/− mice with and without treatment with leptin to identify inducible thermogenic pathways. Table 1 provides a list of 29 genes with significant levels of induction. That 5 of these genes are associated with Ca²⁺ metabolism in skeletal muscle underscores the pivotal role for this metabolism in the induction of thermogenesis by leptin. We selected Slc25a25, which encodes a ATP-Mg²⁺/inorganic phosphate carrier located in the inner mitochondrial membrane, for further analysis, because it is structurally analogous to UCP1 and may have a related transport function. Up-regulation of Slc25a25 (ATP-Mg²⁺/P_i carrier) consistently occurs in skeletal muscle of other cold-adapted UCP1-deficient mouse models, including, induction in Ucp1^−/−·Lep^−/− (Fig. 1A), Dbh^−/− (dopamine β-hydroxylase) (Fig. 1B), and Gdm^−/−·Ucp1^−/− (Gdm; mitochondrial glycerol-3-phosphate dehydrogenase) mice (Fig. 1C). SLC25A25 is thought to maintain ATP homeostasis by functioning as a Ca²⁺-regulated shuttle of ATP-Mg²⁺ and P_i across the inner mitochondrial membrane (11), however, the induction of Slc25a25 expression under severe cold stress suggests that SLC25A25 is able to contribute to thermogenesis in skeletal muscle and inguinal fat. How this thermogenic activity is achieved by the putative function of SLC25A25 as an inner mitochondrial solute transporter is not understood.

The DNA sequence analysis of the Slc25a25 isoform induced in muscle was performed on a cDNA cloned from leptin-treated Ucp1^−/−·Lep^−/− quadriceps muscle. The Slc25a25 isoform overexpressed in skeletal muscle of UCP1-deficient mouse models corresponded to the human SCaMC-2b spliced variant lacking exon 6.

Generation and Validation of Global Slc25a25 Knock-out Mice

To determine the role of Slc25a25 in overall energy homeostasis, we used the Cre/loxP system to generate mice with a global Slc25a25 deletion as described under “Experimental Procedures” (Fig. 2A). Mice with a global deletion of Slc25a25 were generated by breeding Slc25a25^fl/fl to EIIa-Cre mice leading to Slc25a25 excision in the germline and all subsequent tissues and progeny (Fig. 2B). At 28 °C ambient temperature, newborn Slc25a25^−/− progeny are viable, born at the expected Mendelian ratio and fertile. Gene expression for Slc25a25 in liver, quadriceps, and inguinal fat measured by qRT-PCR showed similar levels in expression in WT and Slc25a25^fl/fl mice, intermediate expression in Slc25a25^+/− mice, and absence of expression in Slc25a25^−/− mice (Fig. 2C). Importantly, expression in each tissue was proportional to the number of intact alleles.

Body Weight Regulation and Metabolic Efficiency in Slc25a25^−/− Mice

At weaning (24 days of age) there was no difference in body weight or composition in Slc25a25^+/− or Slc25a25^−/− mice raised by mothers fed a chow diet (∼12 Kcal % fat) at 28 °C ambient temperature. During the subsequent 5 weeks of development, mutant mice began to show slight, but significant, reductions in both fat and fat-free (lean) mass (Fig. 3A). Mice at 8 weeks of age were then fed a high fat diet and maintained first at 20 °C and then at 28 °C (Fig. 3B). We predicted that if SLC25A25 is a part of a thermogenic mechanism that is involved in the regulation of body temperature, reductions in adiposity in the KO mice will be robust at 20 °C and then disappear when the ambient temperature is increased to 28 °C, as we previously observed for Ucp1^−/− mice (10, 13). Similar to the Ucp1^−/− mice, at 20 °C fat mass gain was much lower in Slc25a25^−/− than WT mice; however, the phenotype of Slc25a25^−/− mice deviated from Ucp1^−/− mice in 2 ways (Fig. 3B). First, fat mass remained much lower in Slc25a25^−/− mice when the ambient temperature was increased to 28 °C and second, lean mass was lower in the Slc25a25^−/− mice at both 20 and 28 °C. Ucp1^−/− mice did not have reduced lean mass (13).

Comparisons of body weight and composition of wild type and Slc25a25^−/− mice suggested that the capacity for growth and maintenance of both fat and fat-free mass is attenuated when the mice are placed on a high fat diet. To assess this phenotype further we calculated metabolic efficiency from body weight and food intake data (Table 2). Food intake was about 10% higher in WT than KO mice at 28 °C but not different at 20 °C. Metabolic efficiency increased 2.1- and 2.2-fold for WT and KO, respectively, in the transition from 20 to 28 °C, indicating that the effects of ambient temperature were similar for WT and KO mice; however, at 20 °C metabolic efficiency was 1.45-fold greater in WT than in KO mice and at 28 °C it was 1.35-fold higher in Wt than KO mice. This suggests that inactivation of Slc25a25 reduces metabolic efficiency and leads to a reduction in both fat and fat-free mass. Whether metabolic efficiency is reduced in aging mice on a low fat diet is not known, but should be investigated. Young adult Slc25a25^−/− mice maintained a normal body temperature when acutely exposed to the cold at 4 °C (data not shown), indicating that SLC25A25 is not required for the normal regulation of body temperature. At 18 weeks, described in Fig. 3B, glucose tolerance and insulin tolerance tests showed no difference in insulin resistance between wild type and Slc25a25^−/− mice (data not shown).

Indirect Calorimetry in Slc25a25 Knock-out Mice Indicates Reduced Metabolic Efficiency in the Context of Diet-induced Obesity

As shown in Fig. 3A, slight, but significant reductions, in fat mass can be detected in Slc25a25^−/− mice fed a low-fat chow diet. Energy expenditure in high fat fed mice was not different in KO and wild type mice maintained at 28 °C, but upon the shift to 4 °C oxygen consumption increased about 300% in both genotypes (Fig. 4A), consistent with indistinguishable cold-sensitive phenotypes in wild type and KO mice, that is, mice of both genotypes housed at 28 °C were able to maintain body temperature when acutely exposed to an ambient temperature of 5 °C for up to 17 h.⁵ The data on RER showed the expected reduction (increased fat oxidation) in mice when maintained at 4 °C, but no biologically significant difference in RER between wild type and Slc25a25^−/− mice. It is unlikely that the slight increase observed in heterozygote mice only has any biological significance (Fig. 4A). On the other hand, oxygen consumption was slightly higher in Slc25a25^−/− mice fed a high fat diet at 4 °C, and at 20 and 28 °C than in wild-type mice during long-term feeding of a high fat diet (Fig. 4B). This is different from the response of Ucp1^−/−, which showed differences in diet-induced obesity at 4 and 20 °C, but not at 28 °C, and no genotype-dependent differences in oxygen consumption until the temperature was well below 20 °C (13, 23). Accordingly, between 28 and 20 °C the effects of inactivation of Slc25a25 on energy expenditure are stronger than those seen in Ucp1^−/− mice where oxygen consumption does not exceed that of control mice until the ambient temperature is reduced to about 12 °C (23). Whatever physiological pathway SLC25A25 is involved in, it does not seem to be directly associated with thermogenesis regulating body temperature. Furthermore, the levels of Ucp1 mRNA in interscapular brown adipose tissue from wild type, Slc25a25^+/−, and Slc25a25^−/− mice expressed as arbitrary units relative to cyclophilin mRNA were 14.1 ± 0.4 (n = 9), 13.6 ± 0.9 (n = 9), and 12.7 ± 0.9 (n = 11), respectively; differences were not statistically significant.

Altered Exercise Endurance in Slc25a25 Knock-out Mice

The initial induction of Slc25a25 in skeletal muscle of cold-stressed mice, the slight reduction in lean mass under low fat chow diet, and the increased oxygen consumption per g of lean mass in Slc25a25^−/− mice suggested that skeletal muscle function was perturbed. The impact of the deletion in Slc25a25 on skeletal muscle function was determined by subjecting Slc25a25^−/− mice to a forced treadmill exercise test. Mice lacking Slc25a25 expression showed less than half the running capacity of WT mice and heterozygotes had an intermediate phenotype (Fig. 5A). Fiber-type analysis in soleus muscle, composed of oxidative fibers, collected from mice 1 week after the endurance test showed no difference in quantity of type 1 and type 2 fibers (Fig. 5B) indicating that loss of SLC25A25 did not affect skeletal fiber type distribution. Mitochondrial content estimated by citrate synthase activity in mitochondrial preparations isolated from the gastronemius and quadriceps skeletal muscle was not different between the wild type and knock-out mice (data not shown). Immunoblot analysis of the composition of the respiratory complexes from mitochondria isolated from the quadriceps femoris with the MS604 antibody mixture from Mitosciences, Inc. (Eugene, OR) showed no differences in composition between wild type and mutant mice when fed a low fat chow diet or a high fat diet (data not shown). The hearts of SLC25A25-deficient mice have normal size (% of body weight: 0.65 ± 0.02 in WT versus 0.64 ± 0.03 in Slc25a25^−/−) and do not show evidence of hypertrophy. Thus disruption of Slc25a25 function leads to a compromised muscle function under conditions of forced physical exertion. At present we do not know whether the difference in physical endurance is a consequence of skeletal or heart muscle function.

SLC25A25-deficient Mouse Embryonic Fibroblasts Show Reduced Ca²⁺ Flux and ATP Content

Based upon the fact that SLC25A25 has Ca²⁺-binding EF-hand domains and its ATP-Mg²⁺/P_i activity is regulated by Ca²⁺ (24), we next compared Ca²⁺ mobilization in MEF from WT and Slc25a25^−/− mice. Cells were exposed to 300 nm bradykinin, an agonist of phospholipase C-coupled bradykinin receptor, to trigger Ca²⁺ release from the endoplasmic reticulum (ER). Bradykinin application induced a Ca²⁺ flux that was significantly less in mutant than WT MEF (area under the curve: 1,141,495 relative fluorescence units in WT versus 1,044,953 relative fluorescence units in Slc25a25^−/−, p < 0.05), indicating a difference in the filling state of the ER Ca²⁺ stores (Fig. 6A). Ca²⁺ flux in response to thapsigargin (Fig. 6A), a SERCA inhibitor, and ionomycin (Fig. 6A), an ionophore that induces pore formation in the endoplasmic reticulum membrane, did not show differences between WT and mutant Slc25a25 MEF. Our study shows that the genetic manipulation of the transporter does not have any effect on calcium entry but does have an effect on ER calcium accumulation.

To identify bioenergetic defects incurred by inactivation of Slc25a25, we used an assay for mitochondrial function previously described (17, 18). MEF established from Slc25a25 WT and knock-out mice were exposed to oligomycin (inhibitor of ATP synthase), FCCP (a protonophore and uncoupler of electron transport), antimycin A/rotenone (mitochondrial complex III/complex I inhibitor, respectively). The rate of basal respiration was 11% lower in Slc25a25^−/− MEF compared with WT (Fig. 6B). SLC25A25-deficient MEF, in an effort to compensate for lower basal respiration, had a significantly higher rate of glycolysis as shown by extracellular acidification rate measurements (Fig. 6B). Similarly, the oligomycin-sensitive oxygen consumption rate, which corresponds to respiration linked to ATP synthesis was also 10% less in SLC25A25-deficient MEF (Fig. 6, B and C) compared with WT. Respiration associated with proton leak and FCCP-stimulated maximal respiratory rate was indistinguishable between WT and SLC25A25-deficient MEF; however, non-mitochondrial respiration was 19% lower in mutant MEF (Fig. 6, B and C) compared with WT. Measurement of cellular ATP levels showed that the basal ATP levels are 11% lower in mutant MEF (Fig. 6D). Inhibition of ATP synthesis by oligomycin treatment and glycolysis by 2-deoxyglucose treatment showed that ATP levels are significantly lower in Slc25a25-deficient MEF (Fig. 6D). Treatment of cells with inhibitors did not reduce cell viability and the activity of the mitochondrial marker, citrate synthase, was similar. These findings suggest that overall cellular respiration in mutant MEF is significantly reduced and that these cells are unable to compensate for sufficient ATP production by increasing glycolysis in the face of reduced mitochondrial respiration. Therefore, disruption of Slc25a25 leads to altered cellular metabolism characterized by reduction in total mitochondrial and non-mitochondrial respiration, which leads to reduction in ATP levels. This may have significant implications in the ability of SLC25A25-deficient mice to produce sufficient ATP under conditions of increased energy demand such as skeletal muscle contraction and thus is consistent with the reduced endurance phenotype earlier described. ATP-Mg²⁺/P_i carriers possess Ca²⁺-binding motifs facing the extramitochondrial space, which allows regulation of the carrier transport activity by cytosolic Ca²⁺ without the requirement of Ca²⁺ entry in the organelle (25). In SLC25A25-deficient cells, the reduced Ca²⁺ flux paralleled the decrease in the capacity for ATP synthesis.

Reduced Viability of Slc25a25^−/−·Gdm^−/− Double Mutant Mice

Given the striking similarities in phenotypes of Slc25a25^−/− and Gdm^−/− mice, their Ca²⁺ binding domains and locations in the mitochondria, we used a number of different breeding strategies (Slc25a25^+/−/Gdm^+/− × Slc25a25^+/−/Gdm^+/−, Slc25a25^+/−/Gdm^+/− × Slc25a25^−/−/Gdm^+/−, Slc25a25^+/−/Gdm^−/− × Slc25a25^+/−/Gdm^−/− and Slc25a25^−/−/Gdm^+/− × Slc25a25^−/−/Gdm^+/−) to generate Slc25a25^−/−·Gdm1^−/− mice. Only 8 double mutants were obtained out of the expected 17 mice from a total of 159 offspring produced (χ square test, p = 1.5 × 10⁻⁸). Three of the 8 viable double mutants had observable abnormalities: 1 had a unilateral cataract, another was runted, and 1 was found dead shortly after weaning. The reduction in viability of the Slc25a25^−/−·Gdm1^−/− suggests that inactivation of both genes in a mouse disrupts mitochondrial function to a degree that survival is affected.

The ATP-Mg²⁺/P_i Carrier Function and the SLC25A25 Mitochondrial Carrier

In the 1980s an ATP-Mg²⁺/P_i carrier function, located on the inner mitochondrial membrane, was proposed to be involved in uptake and accumulation of adenine nucleotides in newborn rat liver (26). This ATP-Mg²⁺/P_i carrier catalyzes an electroneutral exchange of ATP-Mg²⁺ for P_i (27) and it is not inhibited by carboxyatractyloside and therefore not a ADP/ATP translocator. Importantly, the ATP transport activity can be activated by cytosolic Ca²⁺ (28, 29) and because variations in the concentration of adenine nucleotides were shown to affect state 3 respiration and ATP synthase-F₀F₁ activity (26), it is plausible that this carrier plays an important function in the regulation of ATP homeostasis during skeletal muscle contraction. Although, a structural analysis of the purified ATP-Mg^2+/P_i carrier from the mitochondrial membrane has not been achieved (30), structural and functional evidence suggested that SLC25A25 has properties consistent with that of the ATP-Mg^2+/P_i carrier (31, 32). SLC25A25 is a member of a family of inner membrane mitochondrial carriers (MC) that function to shuttle metabolites, nucleotides, and cofactors between the cytosol and mitochondria. SLC25A25 belongs to a subgroup of SCaMCs (short calcium-binding MC) having a bipartite structure; the C-terminal half is homologous with MC family members and the N-terminal extension harbors EF-hand motifs (corresponding to α-helices E and F of parvalbumin, the archetypical calcium-binding protein) (11). The extensions, which face the inter-membrane space, enable CaMCs to transduce Ca²⁺ signals without the need of Ca²⁺ uptake into the mitochondrial matrix. These structural properties, together with the fact that SLC25A25 is expressed at much higher levels in skeletal muscle than in liver or adipose tissue suggest an important function in controlling ATP and Ca²⁺ homeostasis in muscle.

Metabolic Inefficiency from Over- and Underexpression of Slc2a25

A challenge toward understanding the function of SLC25A25 in energy metabolism lies in explaining how both overexpression and underexpression of Slc25a25 leads to reduced metabolic efficiency in mice fed an obesogenic diet. Slc25a25 is overexpressed in mouse models of thermogenic deficiency when they are acutely exposed to an ambient temperature of 5 °C and cannot maintain normal body temperature (Fig. 1). We have reasoned that the maintenance of body temperature in the mouse is so critical to its survival that it was necessary to evolve a highly efficient system for the production, regulation, and distribution of heat, that is, brown adipose tissue. Therefore, if brown fat thermogenesis is inactivated by mutations to Ucp1, then alternative thermogenesis must be activated. As Slc25a25, which encodes a mitochondrial membrane protein associated with ATP regulation, was one of the major genes up-regulated in skeletal muscle in three models of cold-sensitive UCP1-deficient mice (Fig. 1), it became a candidate for alternative thermogenesis. To support this correlative data on induced overexpression implicating SLC25A25 in thermogenesis, we sought additional correlative data by producing mice with an inactivated Slc25a25 gene. Similar to Ucp1^−/− mice, Slc25a25^−/− mice are resistant to diet-induced obesity, but unlike Ucp1^−/− mice, resistance to diet-induced obesity is independent of the ambient temperature, that is, Slc25a25^−/− mice are resistant to diet-induced obesity after the ambient temperature was increased to 28 °C (Fig. 3B). Therefore, Slc25a25^−/− are metabolically inefficient (Table 2), but the source of inefficiency is not from a primary function in thermoregulation, because Slc25a25^−/− mice maintain body temperature upon acute exposure to the cold (data not shown). Rather, the role of SLC25A25 in metabolic efficiency is most likely linked to the reduced efficiency of muscle function as evident from the physical endurance test of Slc25a25^−/− mice on a treadmill. Accordingly, in the absence of SLC25A25 the efficiency of ATP production required for skeletal muscle function is diminished with secondary effects on adiposity.

The ultimate underlying cause for reduced muscle endurance lies in a reduced capacity for ATP synthesis (33). Indeed our studies on mitochondrial respiration in MEF from wild type and Slc25a25^−/− mice show reduced basal and oligomycin-sensitive respiration in MEF from KO mice, as well as reduced ATP levels estimated by the luciferase/luciferin assay (Fig. 6). Consistent with this reduced ATP synthesis activity, Ca²⁺ cycling is attenuated in MEF of SLC25A25-deficient mice stimulated with bradykinin. During the muscle contraction process in Slc25a25 mutants forced to exercise on a treadmill, it is possible that the reduced capacity for ATP synthesis cannot support activity of SERCA to maintain the sarcoplasmic reticulum Ca²⁺ pool. With time, insufficient Ca²⁺ will be released via the Ca²⁺ release channels impairing cross-bridge cycling causing diminished power output or muscle fatigue. In human patients with reduced Ca²⁺ sensitivity of force, a higher myoplasmic Ca²⁺ concentration is required to activate skeletal muscle fibers at the same level as controls through increased SERCA activity, thereby, diminishing the efficiency of muscle contraction (34).

Although the EF-hand structural domains of SLC25A25 suggest a function in Ca²⁺ metabolism, the reduced levels of ATP may affect muscle performance through reduced myosin activity. If reduced myosin activity occurs, the potential effects of reduced ATP production in SLC25A25-deficient mice may also extend to myosin-dependent skeletal muscle shivering. Shivering is thought to be the first line of defense to avoid a reduction in core temperature upon acute exposure to the cold, with induction of non-shivering thermogenesis subsequently being the main defense during chronic cold exposure (35). Several genetic models of mice with defective brown adipose tissue thermogenesis, e.g. UCP1 (21), acyl-CoA dehydrogenases (36), dopamine β-hydroxylase (37), or leptin (20), are cold intolerant during acute exposure to an ambient temperature of 4 °C, which suggests that shivering is incapable of maintaining body temperature in the acute phase. However, all of these mutants can tolerate the cold if gradually adapted by slowly lowering the ambient temperature (22, 23, 38). So what is the thermogenic mechanism in the brown adipose tissue-deficient cold adapted mice? The induction of Slc25a25 in skeletal muscle of mice chronically exposed to the cold suggests it could contribute to adaptive thermogenesis in muscle. Given that its absence leads to accelerated muscle fatigue, it is worthwhile to consider whether SLC25A25 may be involved not only providing energy for muscle contraction associated with physical activity, but also with that associated with shivering and the possibility that there is an adaptive aspect to shivering associated with chronic exposure to cold.

Similarities between Gdm and Slc25a25

There is a remarkable similarity in the phenotypes of Slc25a25^−/− and Gdm^−/− mice that we and others have described (13, 39). Both mice have reduced lean mass, both maintain body temperature during an acute exposure to the cold at 4 °C, and both show the same adiposity profile during feeding of a high fat diet, first at 20 °C for 10 weeks and then at 28 °C for 10 weeks. Both mutant mice show increased oxygen consumption per g of lean mass when they are cold stressed. In addition, double mutant Slc25a25^−/−·Gdm^−/− mice are obtained below the expected Mendelian ratios in appropriate crosses. One phenotypic difference is that Gdm^−/− mice do not show reduced physical endurance.⁵ There are also striking structural similarities, both proteins are located on the inner mitochondrial where they have EF-hand Ca²⁺-binding domains facing the cytoplasmic space (11, 40). These enzymatic and transport activities and the molecular structures of SLC25A25 and GDM, together with the phenotypes of Slc25a25^−/− mice, suggest a model in which both proteins are located in the inner mitochondrial membranes where their EF-hand Ca²⁺-binding domains sense fluctuating Ca²⁺ and ATP concentrations associated with muscle contraction (Fig. 7).

In summary, the ability of UCP1-deficient mice to adapt to cold led us to search for an alternative thermogenic system. A microarray experiment of RNA from Ucp1^−/− and wild type mice identified Slc25a25 as a promising candidate. The phenotypes of Slc25a25^−/− mice reveals that SLC25A25 is located in the inner mitochondria where its absence perturbs both mitochondrial respiration and Ca²⁺ cycling in the ER with effects on skeletal muscle function. Furthermore, the loss of SLC25A25 activity has effects on systemic energy balance as indicated by the resistance of Slc25a25^−/− mice to diet-induced obesity. These studies on a role for mitochondrial Ca²⁺ proteins in muscle dovetail with a recently described role for an ATP-sensitive K⁺ channel protein (Kir6.2) in energy homeostasis as evidence by a resistance to obesity and reduced muscle endurance in the Kir6.2-deficient mice (41, 42). These phenotypes of Slc25a25^−/− and Kir6.2^−/− mice point to a pivotal role for these proteins in systemic energy balance as well as muscle function.