Cyclin Y Is Involved in the Regulation of Adipogenesis and Lipid Production

Abstract

A new member of the cyclin family cyclin Y (CCNY) is involved in the regulation of various physiological processes. In this study, the role of CCNY in energy metabolism was characterized. We found that compared with wild-type (WT) mice, Ccny knockout (KO) mice had both lower body weight and lower fat content. The Ccny KO mice also had a higher metabolic rate, resisted the stress of a high-fat diet, and were sensitive to calorie restriction. The expression levels of UCP1 and PGC1α were significantly higher in the brown adipose tissue (BAT) of the Ccny KO mice than that of the WT littermate controls, whereas there was no significant difference in BAT weight between the WT and the Ccny KO mice. In addition, the down-regulation of Ccny resulted in suppression of white adipocyte differentiation both in vivo and in vitro, while the expression of Ccny was up-regulated by C/EBPα. Furthermore, both hepatocytes and HepG2 cells that were depleted of Ccny were insensitive to insulin stimulation, consistent with the significant inhibition of insulin sensitivity in the liver of the Ccny KO mice, but no significant changes in WAT and muscle, indicating that CCNY is involved in regulating the hepatic insulin signaling pathway. The hepatic insulin resistance generated by Ccny depletion resulted in down-regulation of the sterol-regulatory element-binding protein (SREBP1) and fatty acid synthase (FASN). Together, these results provide a new link between CCNY and lipid metabolism in mice, and suggest that inhibition of CCNY may offer a therapeutic approach to obesity and diabetes.

Fig 1. Ccny KO mice show dysfunctional lipid metabolism.

The mice received a normal chow diet, and we measured parameters related to lipid homeostasis at the indicated times. (A) Growth curves of wild-type (WT) and Ccny KO mice (n = 7 male WT and n = 10 male Ccny KO; n = 7 female WT and n = 4 female Ccny KO). (B) Nuclear magnetic resonance analysis of the body fat and lean mass in 16-week-old Ccny KO mice and their littermate controls (n = 7 male WT and n = 10 male Ccny KO; n = 7 female WT and n = 4 female Ccny KO). (C) H&E staining of white adipose tissue in 16-week-old mice (left panel) and measurements of the adipocyte size (>500 cells per genotype) (right panel). (D) The levels of triglycerides, cholesterol, free fatty acids (FFAs), and insulin in fasting plasma and liver of the mice were measured in 16-week-old mice (n = 6 males per genotype). (E) Glucose tolerance test (GTT) (n = 6 males per genotype) (left), the area under the curve (AUC) (right). (F) Insulin tolerance test (ITT) (n = 7–8 males per genotype) (left), the area under the curve (AUC) (right). *, P<0.05; **, P<0.01; ***, P<0.001 WT vs Ccny KO. Error bars, S. E.

Fig 2. Increased expression of thermogenic genes in Ccny KO mice.

(A) mRNA level of thermogenic genes in BAT and WAT (n = 4–5 females per genotype). (B) The protein level of UCP1 and PGC1α in BAT (n = 4–5 females per genotype). *, P<0.05; **, P<0.01; ***, P<0.001 WT vs Ccny KO. Error bars, S. E.

Fig 3. Ccny KO mice are resistant to a high-fat diet (HFD) and sensitive to calorie restriction.

The mice received either HFD or calorie restriction. (A) The body weight curve of 8-week-old mice treated with 12 weeks HFD (n = 6–7 males per genotype). (B) Body fat contents of the 12-week-HFD mice (n = 6–7 males per genotype). Body fat content: retroperitoneal fat (retroperi), inguinal fat, periadrenal fat and armpit fat. (C) The fasting plasma levels of the triglycerides, cholesterol, free fatty acids (FFAs) were measured in 12-week-HFD mice (n = 6–7 males per genotype). (D-F) The mice were treated with calorie restriction. (D) The body weights of 13-week-old mice treated with a 10% reduced food intake were measured at the indicated times (n = 6–8 males per genotype); (E) Body fat contents (n = 6–8 males per genotype); (F) Fasting plasma levels of triglycerides, cholesterol, and free fatty acids (FFAs) (n = 6–8 males per genotype). *, P<0.05; **, P<0.01 WT vs Ccny KO. Error bars, S. E.

Fig 4. Ccny deficiency impairs adipogenesis in vivo and in vitro.

(A) C57BL/6 mice (8 weeks old) were fed a HFD and normal diet (ND) for 13 weeks. The mRNA levels of Ccny and PPARγ in the white adipocyte tissue were detected. **, P<0.01 HFD vs ND. (B) Protein levels of CCNY and PPARγ in the primary pre-adipocytes and adipocytes of C57B6 mice. (C) The stromal vascular cells isolated from adipose tissue in Ccny KO and WT mice were differentiated into adipocytes. The cells were stained with oil red O on day 8. (D, E) The mRNA and protein levels of CCNY, PPARγ, C/EBPα, and aP2 were detected by real-time PCR and Western blot analysis, respectively. *, P<0.05. (F) 3T3-L1 cells before the MDI induction (D0) and 8 days after the MDI induction (D8). The protein levels of CCNY, PPARγ, C/EBPα and aP2 were detected by Western blot analysis (right) and quantified from three independent experiments (left). **, P<0.01 D8 vs D0. (G) 3T3-L1 cells subjected to siRNA treatments were stained with oil red O on day 8 (left) and quantified using measurements of the OD at 510 nm (right). The oil red O staining quantified results were normalized to control cells. *, P<0.05; **, P<0.01. The results shown here are representative of three independent experiments. Error bars, S. D.

Fig 5. C/EBPα activates the transcription of Ccny during adipogenesis.

(A) C/EBPα knockdown reduces the expression of Ccny. 3T3-L1 cells were stably transfected with C/EBPα-shRNA (Ri) and NC as control. The CCNY protein levels of the cells were detected on day 0 and day 8 after the MDI induction. Hsp90 is the loading control. The results shown here are representative of three independent experiments. (B) 293T cells were co-transfected with the pGL3-Ccny promoter and a C/EBPβ, C/EBPα or control vector. The results are expressed as the firefly luciferase activity and normalized to the Renilla luminescence. *, P<0.05 C/EBPα compared with control. Error bars, S. D. (C) C/EBPα binds to the Ccny promoter in 3T3-L1 cells. The MDI-induced 3T3-L1 cells at the indicated time points (days 0, 4 and 8) were subjected to ChIP with an anti-C/EBPα or IgG antibody. C/EBPα bound to the Ccny promoter (primer pair c) but not the primer pair d; the binding of the PPARγ2 promoter (primer pair a), but not the <-2,000 bp region (primer pair b), was detected as the control.

Fig 6. Ccny deficiency inhibits the activation of insulin signaling.

Stable Ccny knockdown HepG2 cells, differentiated stable Ccny knockdown 3T3-L1 cells and primary hepatocytes of Ccny KO mice were stimulated with insulin for 15 min (HepG2, 100 nmol; 3T3-L1, 100 nmol; Hepatocytes, 10 nmol). (A) Western blot analysis of the phosphorylation of insulin receptor β (IRβ), AKT, and GSK3β. (B) The relative ratios of phosphorylated AKT and GSK3β were quantified (HepG2, from three independent experiments; 3T3-L1, from three independent experiments; Hepatocytes, n = 3). (C) Ccny KO mice and littermate controls (n = 2) of age 16 weeks were fasted overnight, IP injection of insulin (5U/kg body weight). The mice were humanely destroyed after 5 minutes and the liver, WAT and muscle were excised and used in western blotting analysis. (D) The relative ratios of phosphorylated AKT and GSK3β. *, P<0.05; **, P<0.01 WT vs Ccny KO, or con vs Ccny-Ri. Error bars, S. E.

Fig 7. Ccny deficiency blocks the insulin-stimulated expression of target genes.

(A) Western blot analysis of SREBP1 and FASN in stable Ccny knockdown (Ccny-Ri) HepG2 cells, hepatocytes and liver tissue from Ccny KO mice and WT controls. (B) The relative levels of SREBP1 and FASN were quantified (HepG2, from three independent experiments; Hepatocytes, n = 3; Liver tissue, n = 3). (C) Real-time PCR analysis of the mRNA levels of lipogenic genes in the hepatocytes and liver of WT and Ccny KO mice. *, P<0.05; **, P<0.01 WT vs Ccny KO, or con vs Ccny-Ri. Error bars, S. E.