Loss of mitochondrial protease OMA1 alters processing of the GTPase OPA1 and causes obesity and defective thermogenesis in mice
2012; Springer Nature; Volume: 31; Issue: 9 Linguagem: Inglês
10.1038/emboj.2012.70
ISSN1460-2075
AutoresPedro M. Quirós, Andrew Ramsay, David Sala, Erika Fernández‐Vizarra, Francisco Rodríguez, Juan R. Peinado, María Soledad Fernández‐García, José A. Vega, José Antonio Enrı́quez, António Zorzano, Carlos López‐Otín,
Tópico(s)Metabolism and Genetic Disorders
ResumoArticle20 March 2012free access Loss of mitochondrial protease OMA1 alters processing of the GTPase OPA1 and causes obesity and defective thermogenesis in mice Pedro M Quirós Pedro M Quirós Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Instituto Universitario de Oncología, Universidad de Oviedo, Oviedo, Spain Search for more papers by this author Andrew J Ramsay Andrew J Ramsay Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Instituto Universitario de Oncología, Universidad de Oviedo, Oviedo, Spain Search for more papers by this author David Sala David Sala Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Spain Search for more papers by this author Erika Fernández-Vizarra Erika Fernández-Vizarra IIS Aragón, Unidad de Investigación Traslacional I+CS, Hospital Universitario Miguel Servet, Zaragoza, Spain Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Zaragoza, Zaragoza, Spain Search for more papers by this author Francisco Rodríguez Francisco Rodríguez Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Instituto Universitario de Oncología, Universidad de Oviedo, Oviedo, Spain Search for more papers by this author Juan R Peinado Juan R Peinado Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Instituto Universitario de Oncología, Universidad de Oviedo, Oviedo, SpainPresent address: Departamento de Ciencias Médicas, Facultad de Medicina, Universidad de Castilla-La Mancha, Ciudad Real, Spain Search for more papers by this author Maria Soledad Fernández-García Maria Soledad Fernández-García Servicio de Anatomía Patológica, Hospital Universitario Central de Asturias, Oviedo, Spain Search for more papers by this author José A Vega José A Vega Departamento de Morfología y Biología Celular, Facultad de Medicina, Universidad de Oviedo, Oviedo, Spain Search for more papers by this author José A Enríquez José A Enríquez Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Zaragoza, Zaragoza, Spain Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain Search for more papers by this author Antonio Zorzano Antonio Zorzano Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Spain Search for more papers by this author Carlos López-Otín Corresponding Author Carlos López-Otín Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Instituto Universitario de Oncología, Universidad de Oviedo, Oviedo, Spain Search for more papers by this author Pedro M Quirós Pedro M Quirós Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Instituto Universitario de Oncología, Universidad de Oviedo, Oviedo, Spain Search for more papers by this author Andrew J Ramsay Andrew J Ramsay Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Instituto Universitario de Oncología, Universidad de Oviedo, Oviedo, Spain Search for more papers by this author David Sala David Sala Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Spain Search for more papers by this author Erika Fernández-Vizarra Erika Fernández-Vizarra IIS Aragón, Unidad de Investigación Traslacional I+CS, Hospital Universitario Miguel Servet, Zaragoza, Spain Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Zaragoza, Zaragoza, Spain Search for more papers by this author Francisco Rodríguez Francisco Rodríguez Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Instituto Universitario de Oncología, Universidad de Oviedo, Oviedo, Spain Search for more papers by this author Juan R Peinado Juan R Peinado Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Instituto Universitario de Oncología, Universidad de Oviedo, Oviedo, SpainPresent address: Departamento de Ciencias Médicas, Facultad de Medicina, Universidad de Castilla-La Mancha, Ciudad Real, Spain Search for more papers by this author Maria Soledad Fernández-García Maria Soledad Fernández-García Servicio de Anatomía Patológica, Hospital Universitario Central de Asturias, Oviedo, Spain Search for more papers by this author José A Vega José A Vega Departamento de Morfología y Biología Celular, Facultad de Medicina, Universidad de Oviedo, Oviedo, Spain Search for more papers by this author José A Enríquez José A Enríquez Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Zaragoza, Zaragoza, Spain Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain Search for more papers by this author Antonio Zorzano Antonio Zorzano Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Spain Search for more papers by this author Carlos López-Otín Corresponding Author Carlos López-Otín Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Instituto Universitario de Oncología, Universidad de Oviedo, Oviedo, Spain Search for more papers by this author Author Information Pedro M Quirós1, Andrew J Ramsay1, David Sala2,3,4, Erika Fernández-Vizarra5,6, Francisco Rodríguez1, Juan R Peinado1, Maria Soledad Fernández-García7, José A Vega8, José A Enríquez6,9, Antonio Zorzano2,3,4 and Carlos López-Otín 1 1Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Instituto Universitario de Oncología, Universidad de Oviedo, Oviedo, Spain 2Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain 3Departament de Bioquímica i Biologia Molecular, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain 4CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Instituto de Salud Carlos III, Spain 5IIS Aragón, Unidad de Investigación Traslacional I+CS, Hospital Universitario Miguel Servet, Zaragoza, Spain 6Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Zaragoza, Zaragoza, Spain 7Servicio de Anatomía Patológica, Hospital Universitario Central de Asturias, Oviedo, Spain 8Departamento de Morfología y Biología Celular, Facultad de Medicina, Universidad de Oviedo, Oviedo, Spain 9Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid, Spain *Corresponding author. Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Oviedo, 33006 Oviedo, Spain. Tel.: +34 985 104201; Fax: +34 985 103564; E-mail: [email protected] The EMBO Journal (2012)31:2117-2133https://doi.org/10.1038/emboj.2012.70 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Mitochondria are dynamic subcellular organelles that convert nutrient intermediates into readily available energy equivalents. Optimal mitochondrial function is ensured by a highly evolved quality control system, coordinated by protein machinery that regulates a process of continual fusion and fission. In this work, we provide in vivo evidence that the ATP-independent metalloprotease OMA1 plays an essential role in the proteolytic inactivation of the dynamin-related GTPase OPA1 (optic atrophy 1). We also show that OMA1 deficiency causes a profound perturbation of the mitochondrial fusion–fission equilibrium that has important implications for metabolic homeostasis. Thus, ablation of OMA1 in mice results in marked transcriptional changes in genes of lipid and glucose metabolic pathways and substantial alterations in circulating blood parameters. Additionally, Oma1-mutant mice exhibit an increase in body weight due to increased adipose mass, hepatic steatosis, decreased energy expenditure and impaired thermogenenesis. These alterations are especially significant under metabolic stress conditions, indicating that an intact OMA1-OPA1 system is essential for developing the appropriate adaptive response to different metabolic stressors such as a high-fat diet or cold-shock. This study provides the first description of an unexpected role in energy metabolism for the metalloprotease OMA1 and reinforces the importance of mitochondrial quality control for normal metabolic function. Introduction Mitochondria are fundamental and highly dynamic organelles of eukaryotic cells that play critical roles in energy generation, control of intermediate metabolism, homeostasis of intracellular calcium and regulation of apoptosis events (Mammucari and Rizzuto, 2010; Wallace et al, 2010). Additionally, mitochondria are also the primary source of endogenous reactive oxygen species (Orrenius et al, 2007). Due to these multiple functions, it is not surprising that mitochondrial alterations have been associated with many pathological conditions including cancer, neurodegenerative disorders and cardiovascular diseases (Green and Kroemer, 2004; Wallace, 2005; Chen and Chan, 2009; Fulda et al, 2010). To maintain their functional activities, mitochondria have developed a quality control mechanism, in which several proteolytic enzymes play important roles (Tatsuta and Langer, 2008). These mitochondrial proteases, together with some mitochondrial chaperones, monitor the folding and assembly of mitochondrial proteins and selectively degrade misfolded and non-assembled polypeptides from the organelle (Voos, 2009). Among the proteolytic enzymes implicated in the mitochondrial quality control system, there are several ATP-dependent proteases such as Yme1L and m-AAA proteases located in the inner membrane, or ClpP and Lonp1 that carry out their functions in the matrix (Koppen and Langer, 2007). The functional relevance of these mitochondrial enzymes is underscored by the finding that deficiencies in m-AAA proteases SPG7 and AFG3L2 are responsible for important neurodegenerative diseases such as hereditary spastic paraplegia or a dominant form of spinocerebellar ataxia (Casari et al, 1998; Di Bella et al, 2010). In addition to the key role of mitochondrial proteases in the quality control system characteristic of these organelles, these enzymes may also regulate the activity of some mitochondrial proteins through their ability to perform highly specific reactions of proteolytic processing, which contribute to the maturation or inactivation of these substrates. This may be the case of OMA1, a proteolytic enzyme located in the inner mitochondrial membrane, which was first identified in yeast as a protease with overlapping activities with m-AAA proteases (Kaser et al, 2003). OMA1 is a zinc metalloprotease of the M48 family, which exhibits multiple transmembrane domains and significant amino-acid sequence similarity with FACE1/ZMPSTE24, a protease involved in the processing of prelamin-A and whose mutations cause premature ageing disorders (Navarro et al, 2005; Varela et al, 2005, 2008). Yeast OMA1 seems to play a role in mitochondrial quality control through a mechanism similar to that of m-AAA proteases, although in an ATP-independent manner. This activity has been proposed to be part of a salvage system of quality control, reminiscent of that performed in Escherichia coli by HtpX, a stress protease with overlapping activities with the AAA protease FtsH (Shimohata et al, 2002). Recently, a series of in vitro studies have permitted the conclusion that OMA1, in collaboration with m-AAA protease isoenzymes, contributes to the proteolytic processing of OPA1 (optic atrophy 1), a dynamin-related GTPase involved in mitochondrial inner membrane fusion as well as in the regulation of mitochondrial morphology and in the protection of cells from apoptosis (Ehses et al, 2009; Head et al, 2009). To further characterize the in vivo role of this mitochondrial metalloprotease, we have generated mutant mice deficient in OMA1. These mice are viable and fertile, but exhibit a marked obesity with metabolic alterations, reduced energy expenditure and altered thermogenic response. Furthermore, we have found that OMA1 plays an essential and non-redundant role in the in vivo proteolytic inactivation of the GTPase OPA1. Finally, we describe studies of mitochondrial activity and function in brown fat tissue and primary adipocytes deficient in OMA1. On the basis of these findings, we propose that OMA1 participates in mitochondrial quality control regulating mitochondrial dynamics, and its absence induces mitochondrial dysfunctions, which have an important impact on mouse metabolic homeostasis. Results Generation of mice deficient in OMA1 To evaluate the in vivo roles of OMA1 mitochondrial protease, we engineered a targeting vector to generate a null allele of Oma1. This vector was designed to replace exon 2 of the Oma1 gene with a neo cassette (Supplementary Figure S1A). The linearized construct was electroporated into G4 embryonic stem cells and after homologous recombination, we obtained seven positive targeted clones that were used to generate chimeric mice. These mice were then bred to C57BL/6 mice to generate heterozygous mice. After intercrossing these heterozygous mice, we generated Oma1−/− animals at the expected Mendelian ratio. Homozygosity for the mutation was confirmed by Southern blot and PCR analysis (Supplementary Figure S1B and C). Despite the Oma1 deficiency, these mutant mice developed normally, with males and females being fertile, and their survival rates indistinguishable from those of their wild-type littermates (Supplementary Figure S2A). During the time period we studied the animals, we did not observe any physical indicators of neurological abnormalities. Moreover, histopathological analysis of the brain, specifically the hippocampus, from 18-month-old mice did not reveal any neuronal loss, nor increment in ubiquitin staining in Oma1-deficient mice compared with their littermates controls. Finally, all samples analysed for both genotypes displayed changes consistent with normal ageing (Supplementary Figure S2B). These findings demonstrate that OMA1 is dispensable for embryonic and adult mouse development, as well as for normal growth and fertility, possibly due to functional redundancy with other peptidases present in the inner membrane of mitochondria. Diet-induced obesity in Oma1−/− mice In the course of phenotypic characterization of Oma1-deficient mice, we noticed a significant increase in the body weight of Oma1−/− mice as compared with their wild-type littermates kept on standard chow (Supplementary Figure S2C). This observation encouraged us to evaluate a putative role for OMA1 in adipose tissue biology. Accordingly, wild-type and Oma1 knockout mice were fed a high-fat diet and total body weights were determined for 24 weeks. We started our study with 4-week-old mice, which displayed similar weights and adipose deposit composition in males and females of each genotype (Supplementary Figure S2D). At 8 weeks, we observed significant weight differences in Oma1−/− males when compared with controls, and these marked changes continued until the end of the experiment. In the case of Oma1−/− females, there was also an increase of weight when compared with the corresponding controls, but lower than in male mice (Figure 1A and B). Statistical analysis revealed that the gain of weight observed at the end of the experiment was significantly higher in both male and female mice deficient in Oma1 than in their littermate controls (Figure 1C). Figure 1.Increase of body weight and fat content in Oma1−/− mice after diet-induced obesity. (A) Photograph of representative Oma1+/+ and Oma1−/− mice after high-fat diet induced obesity. (B) Body weight curves of males and females of Oma1+/+ (♦) and Oma1−/− (▪) mice (n=8–12). (C) Lean body mass in the same animals. (D) Gonadal and subscapular fat mass and (E) gonadal and subscapular fat mass as a percentage of total body weight of the same animals at the end of the experiment. (F) H&E sections of gonadal WAT and skin of Oma1+/+ and Oma1−/− mice. Original magnifications WAT: × 200; skin: × 40. Scale bar: 60 μm. (G) Mean of adipocyte area in gonadal WAT and skin. Results are mean±s.e.m. (n=6–12). *P<0.05; **P<0.01; ***P<0.001. Download figure Download PowerPoint To determine whether the increase of body weight observed in Oma1-deficient mice was due to an increase in fat content, gonadal and subscapular fat pad deposits were separately weighted. We only detected an increase of gonadal fat pads weight and their weight normalized to total body weight in Oma1-null mice (Figure 1D and E), whereas subscapular fat pads and their weight relative to total body weight did not reach statistical significant differences. Histological analysis of white adipose tissue (WAT) from gonadal and subcutaneous deposits showed a marked adipocyte hypertrophy in Oma1−/− mice (Figure 1F), which was confirmed by morphometric measurement of adipocyte area (Figure 1G). Analysis of brown adipose tissue (BAT) of Oma1-deficient mice did not show any significant alteration in fat content (Supplementary Figure S3A), indicating that the obesity-induced phenotype was only related to WAT hypertrophy. Interestingly, those Oma1−/− mice exhibiting the most severe obesity phenotype often presented granuloma-like lesions in the intestinal mesenteric fat. Further histological analysis of these granulomatous lesions revealed the presence of fat necrosis foci containing foamy histiocytes and cholesterol crystals (Supplementary Figure S3B). Similar lesions have been described previously as crown-like structures, which appear to manifest as a result of increased inflammation and adipocyte cell death (Cinti et al, 2005). All these findings suggest that the absence of OMA1 protease leads to profound alterations in WAT homeostasis after nutritional challenges. Metabolic changes in Oma1−/− mice To further evaluate the phenotypic alterations observed in Oma1−/− mice, we performed an analysis of biochemical parameters and metabolic enzymes in the liver of these mutant mice under both standard and high-fat diet. First, and because in many animal models of obesity blood glucose tends to be above normal due to obesity-induced insulin resistance, we measured blood glucose levels in Oma1−/− adult mice, but we did not observe any differences with control mice in both standard and high-fat diet conditions (Figure 2A). Furthermore, analysis of liver parameters did not reveal significant differences in levels of hepatic transaminases under standard chow diet. However, under high-fat diet, we observed an increment in the levels of both Ala- and Asp-transaminases (Figure 2B). Total levels of cholesterol in mutant mice did not exhibit significant differences with those of control animals under chow and high-fat diet (Figure 2C). Nevertheless, levels of triglycerides were significantly higher in Oma1−/− mice compared with littermate controls, in both standard and high-fat diet (Figure 2D). Consistent with this, Oil Red O staining revealed a marked steatosis in the liver of Oma1−/− mice maintained on a normal chow diet, which increased significantly under a high-fat diet (Figure 2E). Levels of free fatty acids were comparable between genotypes on both diets (Figure 2F). Interestingly, analysis of insulin and leptin plasma levels showed different patterns under standard and high-fat diet. Thus, under standard rodent chow, both insulin and leptin plasma levels were reduced in mutant mice. However, under high-fat diet, insulin levels did not show differences despite the increment levels in control and mutant mice. Furthermore, leptin levels were significant enhanced as expected in an obese mouse model (Figure 2G and H). Figure 2.Lipid metabolism and glucose tolerance alterations in Oma1−/− mice under standard chow and high-fat diet. Analysis of serum and plasma parameters of Oma1+/+ and Oma1−/− mice (12–20 weeks old, n=6–12 for each group). Mice were fed on standard chow and high-fat diet, and analysis was determined after an overnight fast. (A) Blood glucose levels in Oma1+/+ and Oma1−/− mice. (B) Serum levels of alanine aminotransaminase (ALT) and aspartate aminotransaminase (AST), (C) cholesterol and (D) triglycerides. (E) Oil Red O staining of histological sections of liver from Oma1+/+ and Oma1−/− mice under chow diet (CD) and high-fat diet (HFD). Scale bar: 20 μm. Levels of (F) free fatty acids, (G) insulin and (H) leptin in plasma of Oma1+/+ and Oma1−/− mice. (I) IPGTT and (J) IPITT in wild-type and Oma1−/− male mice (8–12 weeks old, n=6–8). AUC, area under the curve (arbitrary units). Bars represent mean values±s.e.m. ns denotes no significant difference. *P<0.05. Download figure Download PowerPoint The absence of hyperglycaemia in Oma1-deficient mice, together with their low insulin levels under standard chow diet, suggested the possibility of an increased insulin sensitivity of Oma1−/− mice. Accordingly, intraperitoneal glucose tolerance tests (IPGTTs) revealed that Oma1−/− mice showed improved glucose clearance compared with control mice (Figure 2I). Intraperitoneal insulin tolerance tests (IPITTs) confirmed that Oma1−/− mice presented increased insulin sensitivity (Figure 2J), which is consistent with their low insulin levels. Remarkably, these advantages in glucose metabolism were lost when Oma1-deficient mice were fed a high-fat diet. Thus, the metabolic tests in mutant mice showed a decrease in glucose tolerance and the same insulin insensitivity than control mice (Figure 2I and J, lower panels). Insulin tolerance was also analysed at 8 weeks, with animals displaying similar insulin responses to those observed at 12 weeks (data not shown). In addition, obese Oma1−/− mice showed the same higher levels of insulin than controls (Figure 2F), which explains the observed insulin insensitivity. To further investigate these metabolic alterations observed in Oma1-deficient mice, we performed oligonucleotide-based microarrays to analyse transcriptional changes in adipose tissue from Oma1−/− mice kept under high-fat diet. As can be seen in Supplementary Table S1, we found a marked up-regulation in the expression of genes encoding proteins related with lipid metabolism and transport such as fatty-acid binding protein 1 (FABP1) and several apolipoproteins and cytochromes. Further, bioinformatic analysis revealed that a number of the up-regulated genes in Oma1-deficient mice are associated with two metabolic pathways (peroxisome proliferator-activated receptors α and γ, and retinol pathway), which can be related to the obesity phenotype observed in these mutant mice (Supplementary Figure S4). Collectively, these results indicate that mutant mice deficient in OMA1 mitochondrial protease exhibit significant alterations in lipid metabolism, characterized by an increase in triglycerides and hepatic steatosis, as well as an improved glucose clearance likely derived from an increased sensitivity to insulin. Energy balance and thermogenesis are altered in Oma1-deficient mice To determine the possible causes of the obesity and metabolic dysfunction observed in Oma1-deficient mice, we performed analyses of their food-intake and energy balance in mice maintained on a control chow diet. We did not find significant differences in food-intake of Oma1−/− mice relative to control littermates (Figure 3A). However, indirect calorimetry studies indicated that Oma1-deficient mice showed reduced oxygen consumption and CO2 production, which correlated with a significant reduction in heat production in these mice (Figure 3B–D). These alterations occurred in the absence of changes in respiratory quotient values (Figure 3E) and could not be explained by changes in ambulation (Figure 3F). Thus, we decided to study thermogenic activity of BAT under basal and cold-stress conditions. As expected, wild-type mice showed an increase in body temperature at night under basal conditions (22°C), which was not observed in Oma1-deficient mice (Figure 3G). To further evaluate their adaptive thermogenic response, Oma1+/+ and Oma1−/− mice were subjected to cold exposure at 4°C monitoring their temperature for 12 h. As can be seen in Figure 3H, Oma1−/− mice showed a significant decrease in body core temperature when compared with controls from 3 to 12 h. We analysed UCP1 protein levels after cold-stress but we did not find any differences, indicating that the observed failure in thermogenic response was not due to a decrease in UCP1-mediated uncoupling (data not shown). However, histological analysis of BAT showed that while control mice depleted lipid droplets under cold-stress, Oma1-deficient animals retained most of them (Figure 3I and J). Taken together, these results indicate that Oma1 ablation causes reduced energy expenditure under stress conditions, which may explain the susceptibility of these mice to obesity and their impaired adaptive thermogenic response. Figure 3.Decrease of energy expenditure and impaired thermogenesis in Oma1−/− mice. (A) Food-intake of 2-month-old Oma1+/+ and Oma1−/− mice kept on standard rodent chow (n=6). (B) Whole body oxygen consumption (ACC O2), (C) carbon dioxide production (ACC CO2), (D) heat production, (E) respiratory quotient (RQ) and (F) ambulatory movement of 2-month-old Oma1+/+ and Oma1−/− mice during dark period of 3 days kept on standard rodent chow (n=6–8). (G) Basal rectal temperature of Oma1+/+ and Oma1−/− mice measured during daytime (1000 hours) and night (2200 hours) for 3 consecutive days (n=5). (H) Body temperature curve of 2-month-old Oma1+/+ and Oma1−/− mice exposed to 4°C during 12 h (n=6). (I) Representation of BAT histology stained with H&E of Oma1-deficient mice and controls before and after cold exposure for 3 h. Scale bar: 50 μm. (J) The cumulative area occupied by all lipid droplets in each of the tissue sections was determined using image J and presented as a percentage of the total area analysed (n=4). *P<0.05; **P<0.01; ***P<0.001. Download figure Download PowerPoint Next, we examined whether reduced respiration of Oma1-deficient mice could be explained by alterations in the expression of genes coding for mitochondrial proteins or for the nuclear co-activator PGC1α, an essential transcriptional regulator of mitochondria and oxidative metabolic programmes (Finck and Kelly, 2006; Uldry et al, 2006; Fernandez-Marcos and Auwerx, 2011). To this end, we obtained tissues from wild-type and Oma1-deficient mice chronically subjected to a high-fat diet (20 weeks) and analysed the expression of selected genes by real-time qPCR. BAT samples showed reduced expression of β-oxidation genes, Cpt1b and Vlcad in the Oma1-deficient group (Table I). Livers from Oma1-deficient mice showed a reduced expression of Ndufa9, Uqcrc2 and CoxIV (Table I). Furthermore, we detected increase in Pgc1a and decrease in Pgc1b, together with an increase in lipogenic genes Fasn and Scd1 in Oma1-deficient mice compared with controls (Table I). In addition, we also detected reduced expression of Sdha and Atp5a1 in skeletal muscle from Oma1-null mice (Supplementary Table S2). Collectively, these data indicate that Oma1 deficiency causes a reduced expression of nuclear genes encoding mitochondrial proteins, decrease in β-oxidation genes and increase in lipogenic genes, which may explain the low oxygen consumption and obesity phenotype of these animals. Notably, Oma1 deletion also led to a marked decrease in the expression of genes encoding proteins involved in mitochondrial fusion (Mfn2 or Opa1) or in mitochondrial fission (Drp1). In fact, Mfn2 was markedly down-regulated in BAT and WAT from Oma1-null mice, whereas Opa1 was down-regulated in BAT, WAT and skeletal muscle (Table I; Supplementary Table S2). Additionally, Drp1 was down-regulated in BAT, skeletal muscle and liver from Oma1-deficient mice (Table I; Supplementary Table S2). Table 1. Expression of mitochondrial genes in BAT and liver of Oma1+/+ and Oma1−/− under high-fat diet Brown adipose tissue Liver Oma1+/+ Oma1−/− Oma1+/+ Oma1−/− Mfn2 1±0.18 0.55±0.09* 1±0.25 0.64±0.11 Opa1 1±0.37 0.47±0.05 1±0.12 0.94±0.30 Drp1 1±0.17 0.56±0.07* 1±0.21 0.41±0.07* Ndufa9 1±0.30 0.93±0.07 1±0.11 0.67±0.14* SdhA 1±0.42 0.64±0.10 1±0.14 1.18±0.33 Uqcrc2 1±0.39 0.84±0.08 1±0.16 0.61±0.12* CoxIV 1±0.29 1.13±0.12 1±0.05 0.77±0.09* Atp5a1 1±0.35 0.73±0.05 1±0.12 0.91±0.18 Pgc1a 1±0.11 0.82±0.24 1±0.01 1.62±0.19* Pgc1b 1±0.16 0.64±0.12 1±0.19 0.53±0.07* Mcad 1±0.17 0.83±0.13 1±0.08 0.82±0.17 Vlcad 1±0.14 0.65±0.65* 1±0.11 0.92±0.14 Cpt1a 1±0.15 1.07±0.21 Cpt1b 1±0.08 0.67±0.13* Cpt2 1±0.11 0.82±0.24 1±0.10 0.84±0.11 Fasn 1±0.11 0.82±0.24 1±0.14 2.79±0.45** Acc1 1±0.06 0.91±0.21 1±0.06 1.27±0.13 Scd1 1±0.13 0.81±0.04 1±0.11 2.16±0.47* Gene expression relative to β-actin
Referência(s)