Obesity resistance and increased hepatic expression of catabolism-related mRNAs in Cnot3 +/− mice
2011; Springer Nature; Volume: 30; Issue: 22 Linguagem: Inglês
10.1038/emboj.2011.320
ISSN1460-2075
AutoresMasahiro Morita, Yuichi Oike, Takeshi Nagashima, Tsuyoshi Kadomatsu, Mitsuhisa Tabata, Toru Suzuki, Takahisa Nakamura, Nobuaki Yoshida, Mariko Okada, Tadashi Yamamoto,
Tópico(s)Genomics and Chromatin Dynamics
ResumoArticle6 September 2011free access Obesity resistance and increased hepatic expression of catabolism-related mRNAs in Cnot3+/− mice Masahiro Morita Masahiro Morita Division of Oncology, Department of Cancer Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Yuichi Oike Yuichi Oike PRESTO, Japan Science Technology Agency, Saitama, Japan Department of Molecular Genetics, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan Search for more papers by this author Takeshi Nagashima Takeshi Nagashima Laboratory for Cellular System Modeling, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan Search for more papers by this author Tsuyoshi Kadomatsu Tsuyoshi Kadomatsu Department of Molecular Genetics, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan Search for more papers by this author Mitsuhisa Tabata Mitsuhisa Tabata Department of Molecular Genetics, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan Search for more papers by this author Toru Suzuki Toru Suzuki Division of Oncology, Department of Cancer Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Takahisa Nakamura Takahisa Nakamura Division of Oncology, Department of Cancer Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Nobuaki Yoshida Nobuaki Yoshida Laboratory of Gene Expression and Regulation, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Mariko Okada Mariko Okada Laboratory for Cellular System Modeling, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan Search for more papers by this author Tadashi Yamamoto Corresponding Author Tadashi Yamamoto Division of Oncology, Department of Cancer Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan Cell Signal Unit, Okinawa Institute of Science and Technology, Okinawa, Japan Search for more papers by this author Masahiro Morita Masahiro Morita Division of Oncology, Department of Cancer Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Yuichi Oike Yuichi Oike PRESTO, Japan Science Technology Agency, Saitama, Japan Department of Molecular Genetics, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan Search for more papers by this author Takeshi Nagashima Takeshi Nagashima Laboratory for Cellular System Modeling, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan Search for more papers by this author Tsuyoshi Kadomatsu Tsuyoshi Kadomatsu Department of Molecular Genetics, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan Search for more papers by this author Mitsuhisa Tabata Mitsuhisa Tabata Department of Molecular Genetics, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan Search for more papers by this author Toru Suzuki Toru Suzuki Division of Oncology, Department of Cancer Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Takahisa Nakamura Takahisa Nakamura Division of Oncology, Department of Cancer Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Nobuaki Yoshida Nobuaki Yoshida Laboratory of Gene Expression and Regulation, Institute of Medical Science, University of Tokyo, Tokyo, Japan Search for more papers by this author Mariko Okada Mariko Okada Laboratory for Cellular System Modeling, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan Search for more papers by this author Tadashi Yamamoto Corresponding Author Tadashi Yamamoto Division of Oncology, Department of Cancer Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan Cell Signal Unit, Okinawa Institute of Science and Technology, Okinawa, Japan Search for more papers by this author Author Information Masahiro Morita1, Yuichi Oike2,3, Takeshi Nagashima4, Tsuyoshi Kadomatsu3, Mitsuhisa Tabata3, Toru Suzuki1, Takahisa Nakamura1, Nobuaki Yoshida5, Mariko Okada4 and Tadashi Yamamoto 1,6 1Division of Oncology, Department of Cancer Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan 2PRESTO, Japan Science Technology Agency, Saitama, Japan 3Department of Molecular Genetics, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan 4Laboratory for Cellular System Modeling, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan 5Laboratory of Gene Expression and Regulation, Institute of Medical Science, University of Tokyo, Tokyo, Japan 6Cell Signal Unit, Okinawa Institute of Science and Technology, Okinawa, Japan *Corresponding author. Division of Oncology, Department of Cancer Biology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Tel.: +81 35 449 5301; Fax: +81 35 449 5413; E-mail: [email protected] The EMBO Journal (2011)30:4678-4691https://doi.org/10.1038/emboj.2011.320 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 Obesity is a life-threatening factor and is often associated with dysregulation of gene expression. Here, we show that the CNOT3 subunit of the CCR4–NOT deadenylase complex is critical to metabolic regulation. Cnot3+/− mice are lean with hepatic and adipose tissues containing reduced levels of lipids, and show increased metabolic rates and enhanced glucose tolerance. Cnot3+/− mice remain lean and sensitive to insulin even on a high-fat diet. Furthermore, introduction of Cnot3 haplodeficiency in ob/ob mice ameliorated the obese phenotype. Hepatic expression of most mRNAs is not altered in Cnot3+/− vis-à-vis wild-type mice. However, the levels of specific mRNAs, such as those coding for energy metabolism-related PDK4 and IGFBP1, are increased in Cnot3+/− hepatocytes, having poly(A) tails that are longer than those seen in control cells. We provide evidence that CNOT3 is involved in recruitment of the CCR4–NOT deadenylase to the 3′ end of specific mRNAs. Finally, as CNOT3 levels in the liver and white adipose tissues decrease upon fasting, we propose that CNOT3 responds to feeding conditions to regulate deadenylation-specific mRNAs and energy metabolism. Introduction Posttranscriptional mechanisms are important for various biological events, and their dysregulation is linked to a variety of disorders, including cancer, diabetes, and neuronal defects. Among the posttranscriptional controls of gene expression, regulation of mRNA stability is vitally important, as it determines the availability of mRNAs for translation. Indeed, recent microarray analyses show that nearly half of the changes in gene expression in response to cellular signalling occur at the level of mRNA decay (Fan et al, 2002; Cheadle et al, 2005). Most mRNAs have a poly(A) tail at their 3′ ends, which plays important roles in the regulation of translation and degradation of mRNAs. Once poly(A) tail shortening takes place, being catalysed by deadenylases, mRNA decay from either the 5′ or the 3′ end proceeds (Garneau et al, 2007). In addition, the 3′ untranslated region (3′UTR) of mRNAs has been implicated in the regulation of mRNA decay. RNA-binding proteins that interact with sequences at the 3′UTR, such as AU-rich element (ARE) and the microRNA-binding sites (Garneau et al, 2007; Filipowicz et al, 2008), interact with the CCR4–NOT deadenylase complex (Lykke-Andersen and Wagner, 2005; Belloc and Mendez, 2008; Fabian et al, 2009), suggesting that the proteins in the CCR4–NOT complex is important in controlling gene expression and thus various biological activities. The CCR4–NOT complex is a large (>2 MDa) multi-subunit protein complex conserved from yeast to humans and serves as a major deadenylase (Collart and Timmers, 2004). In yeast, two components of the complex, Ccr4p and Caf1p, possess deadenylase activity (Tucker et al, 2001). The mammalian orthologues of Ccr4p are CNOT6 and CNOT6L, and those of Caf1p are CNOT7 and CNOT8 (Dupressoir et al, 2001; Yamashita et al, 2005; Morita et al, 2007; Aslam et al, 2009). Recent structural analyses of the CNOT6L complexed with nucleotides revealed a deadenylase mechanism involving a pentacovalent phosphate transition (Wang et al, 2010). In contrast to the enzymatic subunits, the function of the non-deadenylase subunits, CNOT1–3, CNOT9, and CNOT10, is elusive. Some of them are implied to be involved in the control of deadenylase activity (Tucker et al, 2002; Temme et al, 2010). In Drosophila, miRNA-dependent deadenylation is suppressed by CNOT1 depletion (Behm-Ansmant et al, 2006) and CNOT2 depletion affects the length of mRNA poly(A) tails (Temme et al, 2004). Slight poly(A) tail lengthening is seen in Not3 mutants (Tucker et al, 2002). Furthermore, Drosophila NOT3 recruits the CCR4–NOT deadenylase to its target mRNA (Chicoine et al, 2007). In yeast, the CCR4–NOT complex plays important roles in cell growth, glucose metabolism, and DNA damage response (Collart, 2003). The mammalian CCR4–NOT complex is also suggested to be relevant to biological functions. Knockdown of the expression of the enzymatic subunit, CNOT6, CNOT6L, CNOT7, or CNOT8, reduces cell growth (Morita et al, 2007; Aslam et al, 2009; Mittal et al, 2011). Knockdown of CNOT2 induces apoptotic cell death (Ito et al, 2011). CNOT3 depletion in embryonic stem cell results in differentiation into trophectoderm lineage (Hu et al, 2009). Cnot7-knockout mice are viable, but defective in spermatogenesis, resulting in male sterility (Berthet et al, 2004; Nakamura et al, 2004). CNOT7-knockout mice also have bone-mass increases that are due to enhanced bone formation (Washio-Oikawa et al, 2007). These intriguing findings provide a glimpse into the physiological importance of the CCR4–NOT deadenylase and direct evidence for the involvement of the 3′UTR and CCR4–NOT-mediated deadenylation in these biological phenomena is to be provided. In this study, we addressed the biological significance of CNOT3 and found that mice haplodeficient in Cnot3 are lean due to poor fat accumulation. We provide evidence that CNOT3 is involved in the regulation of the CCR4–NOT-mediated deadenylation of some specific, but not all, mRNAs that are involved in energy metabolism. We also found that the expression of the CNOT3 protein, but not other subunits of the CCR4–NOT complex, is lowered in the liver and white adipose tissues of fasted mice compared with that in the fed mice. Taken together, we propose that CNOT3 could function in sensing nutrients and alter the deadenylase activity of the CCR4–NOT complex to control the length of the poly(A) tails and eventually expression of mRNAs coding for proteins relevant to the energy metabolism. Results CNOT3 reduction leads to leanness and diminishes liver and adipose tissue weight Northern blot analyses showed that Cnot3 was expressed well in the various tissues examined except that its expression was very low in muscle (Figure 1A). The data suggest that CNOT3 plays roles in various tissues. To dissect the physiological roles of CNOT3, we produced mice lacking the Cnot3 gene. The success of the procedure was confirmed by Southern blot and PCR analysis (Supplementary Figure S1). As described recently by others (Neely et al, 2010), Cnot3−/− mice did not develop past embryonic day 6.5 (Supplementary Table SI). Cnot3+/− mice were alive and fertile, and the expression levels of CNOT3 in Cnot3+/− mice were half of that in wild-type mice (Figure 1B). This decrease did not affect the expression levels of the other components, such as CNOT1, 6L, and 7. Cnot3+/− mice were smaller than their wild-type littermates. The difference in body weight between wild-type and Cnot3+/− mice was apparent in the newborn mice (Figure 1C) and remained throughout development (Figure 1D). At 12 weeks of age, Cnot3+/− mice weighed ∼20% less than wild-type mice (Figure 1E, left). The nose–anus length of Cnot3+/− mice was also reduced by about 5% at 12 weeks of age compared with wild-type mice (Figure 1E, right). Dissection of Cnot3+/− mice revealed a reduction in the size of almost all organs (Supplementary Figure S2); however, when the organ weights were normalized to body weight, the differences disappeared in most organs except for the liver and adipose tissues (Figure 1F). In Cnot3+/− mice, hepatic lipids accumulated poorly (Figure 1G) and the adipocytes in white and brown adipose tissues (WAT and BAT, respectively) were smaller than those in wild-type mice (Figure 1H and I). Histological analysis revealed that almost all tissues, including the thyroid, pituitary, adrenal gland, growth plate, and salivary gland, were normal (unpublished observation). Therefore, CNOT3 might have a specific function in the liver and adipose tissues. Figure 1.Leanness and reduced lipid content in Cnot3+/− mice. (A) Expression of Cnot3 mRNA in mouse tissue. A membrane filter containing mRNAs from multiple mouse tissues (Clontech) was hybridized with a probe specific for Cnot3. Actin-specific probe was used as a loading control. (B) Immunoblotting of CNOT3, CNOT6L, CNOT7, and CNOT1 protein in the Cnot3+/− livers. (C) Gross appearance of 2-week-old Cnot3+/− mice and wild-type littermates. (D) Growth curve of wild-type and Cnot3+/− mice from 3 to 8 weeks after birth. n=10 for each genotype. (E) Comparison of body weight (left) and body length (right) of 12-week-old Cnot3+/− mice and wild-type littermates. (F) The relative weight of the indicated organs. The weight of the organ was normalized to body weight. n=8–10 for each genotype. (G) Lipid levels in the liver. (Left panel) Liver triglyceride levels of 12-week-old wild-type and Cnot3+/− mice. Triglycerides in the homogenized liver were extracted with 2-propanol and measured with a Triglyceride E-Test Kit. n=5 for each genotype. (Right panel) Sudan Black B staining of liver sections from 12-week-old wild-type and Cnot3+/− mice. (H) Histological analysis of and cell size distribution in epididymal WATs of 12-week-old wild-type and Cnot3+/− mice. (I) Histological analysis of BATs of 12-week-old wild-type and Cnot3+/− mice. All values represent mean±s.e.m. *P<0.05; **P<0.01 and ***P<0.001. Download figure Download PowerPoint Metabolic balance is disordered in Cnot3+/− mice The rate of food intake per day appeared to be slightly higher in Cnot3+/− mice than in wild-type mice, but the difference was not significant (Figure 2A). Therefore, it appears that nutrients are burned more efficiently in Cnot3+/− mice than in wild-type mice. Consistent with this notion, whole-body oxygen consumption was higher in Cnot3+/− mice during dark and light periods than in wild-type mice (Figure 2B and C). In all, 24 h oxygen consumption rates were 20% higher in Cnot3+/− mice than in wild-type mice (1.611±0.088 l/kg0.75/h and 1.347±0.035 l/kg0.75/h, respectively). There was no significant difference in rectal temperature (Figure 2D). These results suggest that the leanness of Cnot3+/− mice results from an enhanced metabolic rate. Figure 2.Increased glucose homeostasis, insulin sensitivity, and metabolic rates in Cnot3+/− mice. (A) Average daily food intake normalized to body weight. Daily food intake per mouse was measured over 7 days. n=10 for each genotype. (B, C) Oxygen consumption (VO2) over 24 h (B) and average VO2 (C) of wild-type and Cnot3+/− mice. The data were normalized to body weight0.75. n=5 for each genotype. (D) Rectal temperatures of wild-type and Cnot3+/− mice. n=5 for each genotype. (E–G) Blood tests. Blood glucose levels (E), serum triglyceride concentrations (F), and serum insulin concentrations (G) in fed or fasted wild-type and Cnot3+/− mice. n=6–13 for each genotype. (H, I) Glucose tolerance tests. Mice were deprived of food for 16 h before the experiment. Blood glucose levels (H) and serum insulin levels (I) in wild-type and Cnot3+/− mice were measured at the indicated times following intraperitoneal injection of glucose. n=8–10 for each genotype. (J) Insulin tolerance tests. Blood glucose levels in wild-type and Cnot3+/− mice were measured at the indicated times following intraperitoneal injection of insulin. n=12 for each genotype. (K) Immunoblotting of phospho (Ser-473) and total Akt protein in the liver and WAT of wild-type and Cnot3+/− mice, with or without insulin stimulation. *Unspecific signals. All values represent mean±s.e.m. *P<0.05 and **P<0.01. Download figure Download PowerPoint We then examined glucose and lipid metabolism in Cnot3+/− mice and found significant decreases in blood glucose under fasting conditions (Figure 2E) and serum triglyceride levels under feeding and fasting conditions (Figure 2F), in comparison with wild-type mice. By contrast, no significant differences were detected in the serum insulin levels of wild-type and Cnot3+/− mice under the same conditions (Figure 2G). A glucose tolerance test revealed that the blood glucose levels of Cnot3+/− mice remained significantly lower than those of wild-type mice after glucose administration (Figure 2H). The insulin response to glucose was virtually the same between wild-type and Cnot3+/− mice (Figure 2I), indicating that the control of insulin levels was normal in Cnot3+/− mice. These data suggest that insulin sensitivity is increased in Cnot3+/− mice. Indeed, an insulin tolerance test revealed a greater decrease in blood glucose levels in Cnot3+/− mice than in wild-type mice in response to insulin (Figure 2J). Moreover, insulin-stimulated Akt phosphorylation in the liver and WAT of Cnot3+/− mice was increased relative to wild-type mice (Figure 2K). Thus, we conclude that Cnot3+/− mice exhibit enhanced glucose tolerance and that signalling downstream of insulin receptor is enhanced. Cnot3+/− mice are resistant to high-fat diet-induced obesity As the above data suggest that Cnot3+/− mice are protected against diet-induced obesity, we challenged Cnot3+/− mice with a high-fat (32% wt/wt fat) diet for 12 weeks. As shown in Figure 3, Cnot3+/− mice were resistant to high-fat diet (HFD)-induced obesity; they were less obese than wild-type mice (Figure 3A) and the weight accumulation of Cnot3+/− mice was significantly reduced compared with that of wild-type mice (Figure 3B) after the period of HFD feeding. The net weight gain of wild-type and Cnot3+/− mice was 23.0±0.4 and 13.5±1.0 g, respectively (Figure 3C). Notably, the weight of the liver, WAT, and BAT of the Cnot3+/− mice was less than that of wild-type mice (Figure 3D). Poor fat accumulation in the liver, white adipose tissue, and brown adipose tissue was observed in Cnot3+/− mice fed a HFD (Figure 3E). Moreover, the development of fatty livers was much less significant in Cnot3+/− mice than in wild-type mice (Figure 3F). Macroscopic and computed tomographic analyses showed that both visceral and subcutaneous fat depots were greatly decreased in Cnot3+/− mice relative to wild-type mice fed HFDs (Figure 3G). In addition, blood glucose levels in Cnot3+/− mice were lower than those in wild-type mice on a HFD (Figure 3H). Blood glucose levels remained lower in Cnot3+/− mice than in wild-type mice in both glucose tolerance tests (Figure 3I) and insulin tolerance tests (Figure 3J), suggesting that Cnot3+/− mice are insulin sensitive even on a HFD. Figure 3.Resistance to diet-induced obesity and related metabolic disorder in Cnot3+/− mice. (A) Gross appearance of 20-week-old Cnot3+/− mice and their wild-type littermates after HFD feeding. (B) Growth curves of wild-type and Cnot3+/− mice during HFD feeding. n=16–20 for each genotype. HFD feeding started at 8 weeks of age. (C) Changes in body weight of wild-type and Cnot3+/− mice. n=16–20 for each genotype. (D) The relative weights of the indicated organs from wild-type and Cnot3+/− mice. n=6 for each genotype. (E) Histological analysis of the liver, epididymal WATs, and BATs of wild-type and Cnot3+/− mice. (F) Liver morphology of wild-type and Cnot3+/− mice at the end of HFD feeing. (G) CT scan analysis of wild-type and Cnot3+/− mice. (H) Blood glucose levels of wild-type and Cnot3+/− mice after 16 h of HFD feeding or fasting at the end of the HFD feeding. n=9–10 for each genotype. (I, J) Glucose and insulin tolerance tests. Blood glucose levels in wild-type and Cnot3+/− mice were measured at each indicated time point following intraperitoneal injection of glucose or insulin. n=8–10 for each genotype. All values represent mean±s.e.m. *P<0.05; **P<0.01 and **P<0.01. Download figure Download PowerPoint The obese phenotype of ob/ob mice is ameliorated by the reduction of CNOT3 Given that suppressed CNOT3 expression had an anti-obesity effect, we addressed whether introduction of CNOT3 haplodeficiency (Cnot3+/−) into ob/ob mice could improve the obese phenotype. Note that ob/ob mice showed stronger hepatic expression of CNOT3 compared with wild-type mice (Figure 4A). As expected, ob/ob,Cnot3+/− mice were less obese than ob/ob mice: body weight, glucose tolerance, and insulin sensitivity were all ameliorated in ob/ob,Cnot3+/− mice (Figure 4B–D) compared with ob/ob mice. In ob/ob,Cnot3+/− mice, oxygen consumption rate was increased (Figure 4E) and respiratory quotient was lower (Figure 4F) compared with ob/ob mice, indicating greater utilization of fat versus carbohydrates as an energy source. Little difference was observed in locomotor activity (Figure 4G) or food intake (Figure 4H) between ob/ob and ob/ob,Cnot3+/− mice. Thus, we conclude that reduction of the CNOT3 expression in ob/ob mice ameliorates their obese phenotype. Figure 4.Improvement of obesity and insulin resistance in ob/ob;Cnot3+/− mice. (A) Increased expression of CNOT3 in the liver of ob/ob mice. Immunoblotting of CNOT3 and CNOT6L in wild-type and ob/ob mice (left), and quantification of the data (right). Levels were normalized to α-tubulin. n=3 for each genotype. (B) Decreased body weight in 12-week-old ob/ob,Cnot3+/− mice. n=10 for ob/ob mice. n=4 for ob/ob,Cnot3+/− mice. (C, D) Glucose (C) and insulin (D) tolerance tests. Blood glucose levels were measured at each indicated time point following intraperitoneal glucose or insulin injection. n=6 for ob/ob mice. n=4 for ob/ob,Cnot3+/− mice. (E–H) Comparison of average VO2 (E), respiratory quotient (F), locomotor activity (G), and average daily food intake (H) between ob/ob and ob/ob,Cnot3+/− mice. VO2 were normalized to body weight0.75. Respiratory quotient was calculated by carbon dioxide production/oxygen consumption. Daily food intake per mouse was measured over 7 days. All values represent mean±s.e.m. *P<0.05; **P<0.01 and ***P 2.0-fold compared with those in wild-type mice (Figure 5A and B). Thus, CNOT3 appeared to regulate the CCR4–NOT deadenylase activity for a fraction, but not all, of the liver mRNAs. Of ∼23 000 mRNA transcripts, ∼250 were up-regulated >2.0-fold while 20 were down-regulated >2.0-fold in the liver of Cnot3+/− mice compared with that of wild-type mice (Figure 5B). The genes whose expression was altered are listed in Supplementary Table SII. Figure 5.Deadenylase regulates genes involved in metabolism in the liver. (A) A scatter plot of mRNA expression values in the livers isolated from 12-week-old wild-type (x axis) and Cnot3+/− (y axis) mice. n=2 for each genotype. (B) A proportion of each group was categorized as a fraction of the fold change in Cnot3+/− livers relative to wild-type livers. Genes displaying a fold change of −1.5 to +1.5 were considered within the normal range. (C) Fold change in expression values of genes related to fatty acid oxidation, lipogenesis, oxidative phosphorylation (OXPHOS), ketone body synthesis, cholesterol metabolism, growth regulation, and glucose metabolism from the microarray data shown in (A) and listed in Supplementary Figure S2. (D) Real-time PCR analysis of selected genes involved in fatty acid oxidation, oxidative phosphorylation, cholesterol metabolism, and growth regulation in the livers of wild-type and Cnot3+/− mice. Hprt mRNA levels were used for normalization. n=3–5 for each genotype. All values represent mean±s.e.m. *P<0.05 and **P<0.01. Download figure Download PowerPoint KEGG (Kyoto Encyclopedia of Genes and Genomes) Pathway analysis revealed that the largest proportion of altered genes was involved in metabolic processes, and genes involved in lipid metabolism were the most enriched (19 genes, P (raw)=0.04). As shown in Figure 5C, the genes up-regulated in Cnot3+/− mice include lipid catabolism-related genes such as Acot9 (acyl-CoA thioesterase 9), Aldh1b1 (aldehyde dehydrogenase 1 family, member B1), Cpt1b (carnitine palmitoyltransferase 1b), Hsd17b6 (17-β hydroxysteroid dehydrogenase 6), Lepr (leptin receptor), and Pdk4 (pyruvate dehydrogenase kinase 4). By contrast, lipogenic genes such as Elovl6 (elongation of long chain fatty acids family member 6), Scd1 (sterol O-acyltransferase 1), and Srebf1 (sterol regulatory element binding transcription factor 1) were down-regulated in Cnot3+/− mice. These results indicate that increased fat oxidation and decreased lipogenesis contribute to poor fat accumulation in Cnot3+/− mice. Glycolytic genes such as Aldoc (aldolase C) and Hk2/3 (hexokinase 2/3) were also up-regulated in Cnot3+/− mice (Figure 5C). Furthermore, energy consumption-related genes, such as Cox6b2 (cytochrome c oxidase subunit VIb polypeptide 2), Pgc1α (peroxisome proliferative activated receptor, γ, coactivator 1 α), and Ucp2 (uncoupling protein 2), as well as the metabolism and growth regulatory gene Igfbp1 (insulin-like growth factor binding protein 1) were up-regulated in Cnot3+/− mice (Figure 5C). We then validated the data by quantitative RT–PCR analysis and confirmed that the expression levels of Pdk4, Cpt1b, Hsd17b6, Aldh1b1, Pgc1α, Ucp2, Lepr, Igfbp1, and Soat1 were significantly increased in the livers of Cnot3+/− mice relative to control mice (Figure 5D). We tentatively concluded that altered expression of these genes underlies the leanness of Cnot3+/− mice. Control of poly(A) tail length by CNOT3 involves the 3′UTR of target mRNAs To determine whether up-regulation of the mRNAs in Cnot3+/− mice is caused by malfunction of the CCR4–NOT deadenylase, we measured the length of the poly(A) tails of mRNAs whose hepatic expression was augmented in Cnot3+/− mice. Among the up-regulated mRNAs, we focused on two mRNA species: Pdk4 and Igfbp1 mRNAs. PDK4 (pyruvate dehydrogenase kinase 4) inhibits pyruvate dehydrogenase activity, which is often correlated with enhanced utilization of fatty acids (Sugden and Holness, 2003). IGFBP1 is involved in the control of mitogenic and metabolic actions (Siddals et al, 2002), and elevated expression of IGFBP1 produces a lean phenotype (Rajkumar et al, 1995). The increase in Pdk4 and Igfbp1 mRNA levels in the livers of Cnot3+/− mice was confirmed by northern blot (Figure 6A). To demonstrate that down-regulation of CNOT3 led the inhibition of the poly(A) tail shortening of the Igfbp1 and Pdk4 mRNAs, we prepared their 3′ poly(A)-containing portions by utilizing RNase H and short stretches of nucleotide sequences that were complementary to the mRNAs' sequences (Figure 6B, left). Northern blot analyses of the enriched samples revealed that the poly(A) tails of the two mRNA species from Cnot3+/− mice were longer than those from wild-type mice (Figure 6B, right). After removal of the poly(A) tails, the lengths of the Igfbp1 or Pdk4 mRNA samples from Cnot3+/− mice were virtually the same as that from wild-type mice. As a control, we tested Gapdh mRNA, a transcript whose expression is not affected by the Cnot3 haplodeficiency, and found that there was virtually no difference in the length of poly(A) tail between wild-type and Cnot3+/− mice (Figure 6B). Figure 6.CNOT3 reduction affects the poly(A) tail length of mRNAs involved in lipid metabolism and growth. (A) Northern blot of Igfbp1 and Pdk4 mRNAs from the livers of wild-type and Cnot3+/− mice. (B) Comparison of the poly(A) tail lengths of the Pdk4, Igfbp1, and Gapdh mRNAs between wild-type and Cnot3+/− mice. RNAs were prepared from the livers. The experimental procedure (left) and northern blot data (right) are presented. Completely deadenylated mRNAs were prepared by RNase H treatment in the presence of oligo(dT). Estimated length of the poly(A) tails is indicated on the right of each panel. (C) Luciferase assay with reporter plasmids harbouring the 3′UTRs of Pdk4, Igfbp1, or Lpl mRNA in wild-type and Cnot3+/−
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