Obesity resistance and increased hepatic expression of catabolism-related mRNAs in Cnot3+/- mice - PubMed (original) (raw)

Obesity resistance and increased hepatic expression of catabolism-related mRNAs in Cnot3+/- mice

Masahiro Morita et al. EMBO J. 2011.

Abstract

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.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1

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.

Figure 2

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.

Figure 3

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.

Figure 4

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<0.001.

Figure 5

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.

Figure 6

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+/− hepatocytes. _n_=3 for each genotype. (D) Half-life of the reporter mRNA. Wild-type and Cnot3+/− hepatocytes transfected with a reporter plasmid harbouring the 3′UTR of Pdk4 mRNA were incubated with actinomycin D (5 μg/ml) for the indicated time length. The level of the reporter mRNA was determined by the qPCR method and normalized to that of Hprt mRNA, and plotted semilogarithmically. (E) Luciferase assay (top) with reporter plasmids (bottom) harbouring the various segments of Pdk4 3′UTR in wild-type and Cnot3+/− hepatocytes. ARE, GRE, and possible miRNA-binding sites were indicated. miRanda-mirSVR algorithm provided by microRNA.org was used for the possible miRNA-binding site. (F) Association of CNOT3 with the 3′UTR of Pdk4 mRNA. Cell lysates from HepG2 cells transfected with the reporter plasmids harbouring the 3′UTR of Pdk4 were immunoprecipitated with anti-CNOT3 and control-IgG antibodies. The immunoprecipitates were analysed by RT–PCR using primers specific for the Firefly luciferase mRNA. (G) Association of CNOT6L with Pdk4 mRNA in a CNOT3-dependent manner. Proteins in the hepatocyte lysates prepared from wild-type and Cnot3+/− mice were immunoprecipitated with the monoclonal anti-CNOT6L and control-IgG antibodies. RT–PCR analysis of the immunoprecipitates was performed with primers specific to Pdk4 mRNA. All values represent mean±s.e.m. *P<0.05.

Figure 7

Figure 7

Alternation of CNOT3 expression level by responding to nutrient status and identification of CNOT3-regulated mRNAs. (A) Reduced expression of CNOT3 in the liver and WAT of fasted mice. Western blots of CNOT3 and other CCR4–NOT subunits expression in the liver, WAT, brain, and pancreas of mice fed ad libitum, 24 h fasted, and 24 h re-fed. (B) Quantification of the data is shown in (A). _Ad libitum_-fed, open columns; 24 h fasted, filled columns; and 24 h re-fed mice, semifilled columns. Levels were normalized to α-tubulin. _n_=3 for each condition. All values are mean±s.e.m. *P<0.01 versus fed mice. (C) Levels of CNOTs in the CCR4–NOT complex. Immunoblotting of the anti-CNOT7 immunoprecipitates prepared from the liver of mice fed ad libitum, 24 h fasted and 24 h re-fed. (D) Identification of genes regulated by CNOT3. Global gene expression profiling of liver RNAs was assessed for 8-week-old mice. Note that the data shown in Figure 5 were from the analysis of 12-week-old mice. Comparisons were made between wild-type and Cnot3+/− mice (circled red) and between feeding and fasted wild-type mice (circled blue). (E) Real-time PCR analysis of selected genes in the livers of 8-week-old wild-type and Cnot3+/− mice under fasting or feeding conditions (_n_=3–5 for each genotype). Data represent mean values±s.e.m. *P<0.05; **P<0.01 and ***P<0.001.

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