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Identification of Creb3l4 as an essential negative regulator of adipogenesis.

Kim TH, Jo SH, Choi H, Park JM, Kim MY, Nojima H, Kim JW, Ahn YH - Cell Death Dis (2014)

Bottom Line: Constitutive overexpression of CREB3L4 resulted in the inhibition of adipocyte differentiation, whereas knockdown of Creb3l4 expression caused differentiation of preadipocytes into mature adipocytes, bypassing the mitotic clonal expansion step.In 3T3-L1 preadipocytes, Creb3l4 knockdown resulted in increased expression of peroxisome proliferator-activated receptor γ (PPARγ2) and CCAAT/enhancer binding protein (C/EBPα), either by increasing the protein stability of C/EBPβ or by decreasing the expression of GATA3, a negative regulator of PPARγ2 expression.Moreover, adipocytes of Creb3l4-knockout mice showed hyperplasia caused by increased adipogenesis, and exhibited improved glucose tolerance and insulin sensitivity, as compared with littermate wild-type mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Yonsei University College of Medicine, Seoul 120-752, Republic of Korea.

ABSTRACT
Understanding the molecular networks that regulate adipogenesis is crucial for combating obesity. However, the identity and molecular actions of negative regulators that regulate the early development of adipocytes remain poorly understood. In this study, we investigated the role of CREB3L4, a member of the CREB3-like family, in the regulation of adiposity. Constitutive overexpression of CREB3L4 resulted in the inhibition of adipocyte differentiation, whereas knockdown of Creb3l4 expression caused differentiation of preadipocytes into mature adipocytes, bypassing the mitotic clonal expansion step. In 3T3-L1 preadipocytes, Creb3l4 knockdown resulted in increased expression of peroxisome proliferator-activated receptor γ (PPARγ2) and CCAAT/enhancer binding protein (C/EBPα), either by increasing the protein stability of C/EBPβ or by decreasing the expression of GATA3, a negative regulator of PPARγ2 expression. Consequently, increased PPARγ2 and C/EBPα levels induced adipocyte differentiation, even in the presence of minimal hormonal inducer. Thus, it can be speculated that CREB3L4 has a role as gatekeeper, inhibiting adipogenesis in 3T3-L1 preadipocytes. Moreover, adipocytes of Creb3l4-knockout mice showed hyperplasia caused by increased adipogenesis, and exhibited improved glucose tolerance and insulin sensitivity, as compared with littermate wild-type mice. These results raise the possibility that Creb3l4 could be a useful therapeutic target in the fight against obesity and metabolic syndrome.

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Creb3l4-KO mice showed adipocyte hyperplasia, lead to improved metabolic parameters. (a) Adipogenic potential of mouse embryonic fibroblasts (MEFs) derived from wild-type (WT) or Creb3l4-knockout (KO) mice. Cells were stained with oil-red O after treatment with insulin (10 μg/ml) and rosiglitazone (2 μM) for 6 days, followed by insulin (1 μg/ml) for 16 days. Microscopic ( × 20) views representative of three independent experiments are shown. (b) Hematoxylin and eosin (H&E) staining of epididymal fat. Scale bar: 100 μm. (c) Adipocyte diameter was quantified with Image J (n=3). (d) Oral glucose tolerance test (GTT), (e) insulin tolerance test (ITT), performed after 14 and 15 weeks, respectively, of feeding HFD (n=12–19). (f) Insulin level from mice was measured by ELISA ( n=9–14 ). Leptin, resistin, and PAI-1 levels from mice fed HFD for 16 weeks were measured by MAGPIX, using a MILLIPLEX MAP mouse magnetic bead panel (n=6–12). Values are expressed as mean±S.E.M., *P<0.05, **P<0.01
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fig9: Creb3l4-KO mice showed adipocyte hyperplasia, lead to improved metabolic parameters. (a) Adipogenic potential of mouse embryonic fibroblasts (MEFs) derived from wild-type (WT) or Creb3l4-knockout (KO) mice. Cells were stained with oil-red O after treatment with insulin (10 μg/ml) and rosiglitazone (2 μM) for 6 days, followed by insulin (1 μg/ml) for 16 days. Microscopic ( × 20) views representative of three independent experiments are shown. (b) Hematoxylin and eosin (H&E) staining of epididymal fat. Scale bar: 100 μm. (c) Adipocyte diameter was quantified with Image J (n=3). (d) Oral glucose tolerance test (GTT), (e) insulin tolerance test (ITT), performed after 14 and 15 weeks, respectively, of feeding HFD (n=12–19). (f) Insulin level from mice was measured by ELISA ( n=9–14 ). Leptin, resistin, and PAI-1 levels from mice fed HFD for 16 weeks were measured by MAGPIX, using a MILLIPLEX MAP mouse magnetic bead panel (n=6–12). Values are expressed as mean±S.E.M., *P<0.05, **P<0.01

Mentions: To further confirm the effects of CREB3L4 in adipogenesis, adipocyte differentiation in Creb3l4 KO and WT mouse embryonic fibroblasts (MEFs) was compared. Primary Creb3l4 KO MEFs showed an increase in lipid accumulation and morphological differentiation compared with WT MEFs at D22, even in the insulin-containing medium (Figure 9a). To elucidate the physiological role of Creb3l4 in adipogenesis, both WT and Creb3l4-KO mice were fed with a 45% high-fat diet (HFD) for up to 16 weeks. There are no significant differences in body weight and fat mass (data not shown). However, the size of adipocyte fat cells in Creb3l4-KO mice was smaller than that in WT mice (Figures 9b and c). The average of epididymal adipocyte diameter in WT mice was 184 μm, in the Creb3l4-KO mice, average of adipocyte size was smaller (151 μm) (Figure 9c). In addition, Creb3l4-KO mice exhibited an increased number of these small adipocytes (hyperplasia), which may contribute to improved glucose tolerance and insulin sensitivity (Figures 9d and e). HFD-fed Creb3l4-KO mice showed decreased fasting glucose levels (Figure 9d) and were resistant to diet-induced hyperinsulinemia (Figure 9f). However, plasma lipid profiles, including triglyceride, total cholesterol, and non-esterified free fatty acid levels, were not significantly different between these groups (Supplementary Figure S7a). Because adipose tissue can affect systemic glucose homeostasis by secreting adipokines, we measured adipokine levels. We found lower levels of leptin, resistin, and PAI-1 in Creb3l4-KO mice than in WT mice, suggesting that deletion of Creb3l4 led to an altered adipokine profile that may contribute to improved insulin sensitivity (Figure 9f). In addition, insulin-stimulated Akt phosphorylation (S473) was augmented in WAT and liver tissues, but not in muscle, of the Creb3l4-KO mice (Supplementary Figure S7b). These results indicate that adipocyte hyperplasia due to increased adipogenesis in HFD-fed Creb3l4-KO mice could be an adaptation to overcome glucose intolerance and insulin resistance.


Identification of Creb3l4 as an essential negative regulator of adipogenesis.

Kim TH, Jo SH, Choi H, Park JM, Kim MY, Nojima H, Kim JW, Ahn YH - Cell Death Dis (2014)

Creb3l4-KO mice showed adipocyte hyperplasia, lead to improved metabolic parameters. (a) Adipogenic potential of mouse embryonic fibroblasts (MEFs) derived from wild-type (WT) or Creb3l4-knockout (KO) mice. Cells were stained with oil-red O after treatment with insulin (10 μg/ml) and rosiglitazone (2 μM) for 6 days, followed by insulin (1 μg/ml) for 16 days. Microscopic ( × 20) views representative of three independent experiments are shown. (b) Hematoxylin and eosin (H&E) staining of epididymal fat. Scale bar: 100 μm. (c) Adipocyte diameter was quantified with Image J (n=3). (d) Oral glucose tolerance test (GTT), (e) insulin tolerance test (ITT), performed after 14 and 15 weeks, respectively, of feeding HFD (n=12–19). (f) Insulin level from mice was measured by ELISA ( n=9–14 ). Leptin, resistin, and PAI-1 levels from mice fed HFD for 16 weeks were measured by MAGPIX, using a MILLIPLEX MAP mouse magnetic bead panel (n=6–12). Values are expressed as mean±S.E.M., *P<0.05, **P<0.01
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4260748&req=5

fig9: Creb3l4-KO mice showed adipocyte hyperplasia, lead to improved metabolic parameters. (a) Adipogenic potential of mouse embryonic fibroblasts (MEFs) derived from wild-type (WT) or Creb3l4-knockout (KO) mice. Cells were stained with oil-red O after treatment with insulin (10 μg/ml) and rosiglitazone (2 μM) for 6 days, followed by insulin (1 μg/ml) for 16 days. Microscopic ( × 20) views representative of three independent experiments are shown. (b) Hematoxylin and eosin (H&E) staining of epididymal fat. Scale bar: 100 μm. (c) Adipocyte diameter was quantified with Image J (n=3). (d) Oral glucose tolerance test (GTT), (e) insulin tolerance test (ITT), performed after 14 and 15 weeks, respectively, of feeding HFD (n=12–19). (f) Insulin level from mice was measured by ELISA ( n=9–14 ). Leptin, resistin, and PAI-1 levels from mice fed HFD for 16 weeks were measured by MAGPIX, using a MILLIPLEX MAP mouse magnetic bead panel (n=6–12). Values are expressed as mean±S.E.M., *P<0.05, **P<0.01
Mentions: To further confirm the effects of CREB3L4 in adipogenesis, adipocyte differentiation in Creb3l4 KO and WT mouse embryonic fibroblasts (MEFs) was compared. Primary Creb3l4 KO MEFs showed an increase in lipid accumulation and morphological differentiation compared with WT MEFs at D22, even in the insulin-containing medium (Figure 9a). To elucidate the physiological role of Creb3l4 in adipogenesis, both WT and Creb3l4-KO mice were fed with a 45% high-fat diet (HFD) for up to 16 weeks. There are no significant differences in body weight and fat mass (data not shown). However, the size of adipocyte fat cells in Creb3l4-KO mice was smaller than that in WT mice (Figures 9b and c). The average of epididymal adipocyte diameter in WT mice was 184 μm, in the Creb3l4-KO mice, average of adipocyte size was smaller (151 μm) (Figure 9c). In addition, Creb3l4-KO mice exhibited an increased number of these small adipocytes (hyperplasia), which may contribute to improved glucose tolerance and insulin sensitivity (Figures 9d and e). HFD-fed Creb3l4-KO mice showed decreased fasting glucose levels (Figure 9d) and were resistant to diet-induced hyperinsulinemia (Figure 9f). However, plasma lipid profiles, including triglyceride, total cholesterol, and non-esterified free fatty acid levels, were not significantly different between these groups (Supplementary Figure S7a). Because adipose tissue can affect systemic glucose homeostasis by secreting adipokines, we measured adipokine levels. We found lower levels of leptin, resistin, and PAI-1 in Creb3l4-KO mice than in WT mice, suggesting that deletion of Creb3l4 led to an altered adipokine profile that may contribute to improved insulin sensitivity (Figure 9f). In addition, insulin-stimulated Akt phosphorylation (S473) was augmented in WAT and liver tissues, but not in muscle, of the Creb3l4-KO mice (Supplementary Figure S7b). These results indicate that adipocyte hyperplasia due to increased adipogenesis in HFD-fed Creb3l4-KO mice could be an adaptation to overcome glucose intolerance and insulin resistance.

Bottom Line: Constitutive overexpression of CREB3L4 resulted in the inhibition of adipocyte differentiation, whereas knockdown of Creb3l4 expression caused differentiation of preadipocytes into mature adipocytes, bypassing the mitotic clonal expansion step.In 3T3-L1 preadipocytes, Creb3l4 knockdown resulted in increased expression of peroxisome proliferator-activated receptor γ (PPARγ2) and CCAAT/enhancer binding protein (C/EBPα), either by increasing the protein stability of C/EBPβ or by decreasing the expression of GATA3, a negative regulator of PPARγ2 expression.Moreover, adipocytes of Creb3l4-knockout mice showed hyperplasia caused by increased adipogenesis, and exhibited improved glucose tolerance and insulin sensitivity, as compared with littermate wild-type mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Yonsei University College of Medicine, Seoul 120-752, Republic of Korea.

ABSTRACT
Understanding the molecular networks that regulate adipogenesis is crucial for combating obesity. However, the identity and molecular actions of negative regulators that regulate the early development of adipocytes remain poorly understood. In this study, we investigated the role of CREB3L4, a member of the CREB3-like family, in the regulation of adiposity. Constitutive overexpression of CREB3L4 resulted in the inhibition of adipocyte differentiation, whereas knockdown of Creb3l4 expression caused differentiation of preadipocytes into mature adipocytes, bypassing the mitotic clonal expansion step. In 3T3-L1 preadipocytes, Creb3l4 knockdown resulted in increased expression of peroxisome proliferator-activated receptor γ (PPARγ2) and CCAAT/enhancer binding protein (C/EBPα), either by increasing the protein stability of C/EBPβ or by decreasing the expression of GATA3, a negative regulator of PPARγ2 expression. Consequently, increased PPARγ2 and C/EBPα levels induced adipocyte differentiation, even in the presence of minimal hormonal inducer. Thus, it can be speculated that CREB3L4 has a role as gatekeeper, inhibiting adipogenesis in 3T3-L1 preadipocytes. Moreover, adipocytes of Creb3l4-knockout mice showed hyperplasia caused by increased adipogenesis, and exhibited improved glucose tolerance and insulin sensitivity, as compared with littermate wild-type mice. These results raise the possibility that Creb3l4 could be a useful therapeutic target in the fight against obesity and metabolic syndrome.

Show MeSH
Related in: MedlinePlus