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. 2012;7(10):e47358.
doi: 10.1371/journal.pone.0047358. Epub 2012 Oct 15.

The Essential Role of Mbd5 in the Regulation of Somatic Growth and Glucose Homeostasis in Mice

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Free PMC article

The Essential Role of Mbd5 in the Regulation of Somatic Growth and Glucose Homeostasis in Mice

Yarui Du et al. PLoS One. .
Free PMC article

Abstract

Methyl-CpG binding domain protein 5 (MBD5) belongs to the MBD family proteins, which play central roles in transcriptional regulation and development. The significance of MBD5 function is highlighted by recent studies implicating it as a candidate gene involved in human 2q23.1 microdeletion syndrome. To investigate the physiological role of Mbd5, we generated knockout mice. The Mbd5-deficient mice showed growth retardation, wasting and pre-weaning lethality. The observed growth retardation was associated with the impairment of GH/IGF-1 axis in Mbd5-null pups. Conditional knockout of Mbd5 in the brain resulted in the similar phenotypes as whole body deletion, indicating that Mbd5 functions in the nervous system to regulate postnatal growth. Moreover, the mutant mice also displayed enhanced glucose tolerance and elevated insulin sensitivity as a result of increased insulin signaling, ultimately resulting in disturbed glucose homeostasis and hypoglycemia. These results indicate Mbd5 as an essential factor for mouse postnatal growth and maintenance of glucose homeostasis.

Conflict of interest statement

Competing Interests: Co-author Guoliang Xu is a PLOS ONE Editorial Board member, and this does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Targeted disruption of Mbd5 in mice.
(A) The strategy for the generation of a targeted Mbd5 allele. Numbered black boxes represent the coding exons and open boxes represent the 5′ untranslated region (UTR). The LoxP and Frt sites are shown as black and gray triangles, respectively. The floxed region contains exon 1, which encodes the majority of the MBD domain. The probes used in the Southern blot analysis to correctly identify targeted ES cells are indicated by the horizontal bars. The restriction sites used to digest ES genomic DNA were BamHI (B) and EcoRV (E). The PCR primers used for genotyping are indicated with arrows P1, P2 and P3. (B) Identification of targeted ES clones by Southern blot analysis using two different probes. (C) Verification of the mutant allele in homozygous and heterozygous mice by genomic PCR with primers P1 and P2. (D) Loss of Mbd5 mRNA in knockout mice. RNA samples from the brain, pancreas and liver were examined by RT-PCR. The forward primer used was located in the targeted genomic region. A targeted allele following Cre-deletion generated no PCR product, and the floxed allele generated a 305-bp product.
Figure 2
Figure 2. Postnatal growth retardation and pre-weaning lethality of Mbd5−/− mice.
(A) Gross morphology of typical wild-type (+/+), heterozygous (+/−) and homozygous (−/−) mice at postnatal day 14 (P14). The Mbd5-knockout mice had significantly smaller body size than their littermates, and the reduction in body weight was accompanied by a reduction in body length. (B) Survival curve of Mbd5−/− pups (n = 48) and their littermate controls. (C) Growth curve of Mbd5−/− pups (n = 4–5 for each gender) and their littermate controls (For WT, n = 4–5 for each gender, for heterozygotes, n = 9–10 for each gender). The offspring generated from heterozygous intercrosses of Mbd5+/− mice were weighed at 3-day intervals as indicated. (D) Growth gain curve of Mbd5−/− mice (n = 9) and their littermate controls (8 wild-type and 19 heterozygotes). The growth rates shown were calculated by subtracting the value of cumulative weight at a particular day from that of three days before, i.e., each point represents the weight gain of the three preceding days. (E) Reduction of subcutaneous fat (white arrows) and perigonadal fat (black arrows) in Mbd5-knockout mouse at P14.
Figure 3
Figure 3. Reduced somatotropic signaling in Mbd5-knockout mice.
(A) Pituitary expression of GH in control and knockout mice. Gapdh was used for normalization. Gene expression levels were determined by real-time PCR in P14 mice. At least 5 pairs of matched female mice were used for the comparison. (B–D) RNA levels of Ghr, Als and Igf-1 in the liver of control and knockout mice at P14. β-actin was used for normalization. n = 3 per group. (E) Serum IGF-1 concentrations in P14 mice. For each group, n = 5–6. (F) Pituitary from KO and wild-type littermates at P14. (G, H) Pituitary GH stores (G) and GH content normalized to pituitary protein level (H) in 2-week-old Mbd5 KO mice and their littermate controls. n = 4 per group. *, P<0.05, **, P<0.01; ***, P<0.001.
Figure 4
Figure 4. Specific deletion of Mbd5 in brain results in similar phenotypes as the whole body deletion.
(A) Genotype confirmation of Mbd5 brain-specific knockout mice by PCR with primers indicated. (B) Mbd5 mRNA level in different tissues of Mbd5 BSKO mice normalized to the control levels. Gapdh was used for normalization. At least 3 pairs of matched mice were used for comparison. (C) Body weight of Mbd5 BSKO mice and their littermates controls at P7. n = 4 per genotype. (D) Serum GH concentrations in 2-week-old BSKO and littermate controls. n = 4 per genotype. (E) Expression levels of GH/IGF-1 axis associated genes in Mbd5 BSKO mice and littermate controls at P14 as measured by real-time PCR. β-actin was used for normalization. n = 3 per group. (F) Serum IGF-1 concentrations in P14 mice. At least 6 pairs of matched mice were used for comparison. *, P<0.05; ***P<0.001.
Figure 5
Figure 5. Disturbed glucose homeostasis with elevated insulin sensitivity in Mbd5−/− mice.
(A) Blood glucose levels in WT, Mbd5+/− and Mbd5−/− mice in the fed state at the indicated ages. The numbers in brackets indicate the number of animals studied. (B) Blood glucose and serum insulin levels of 2-week-old mice in the fed state. For each group, n = 5. (C) The OGTT of control and Mbd5-knockout male mice at P14. For each group, n = 5. (D) The ITT of control and Mbd5-knockout male mice at P14. For each group, n = 5. *, P<0.05; **, P<0.01; ***, P<0.001. (E) Akt activation in the liver of control and Mbd5-knockout mice at P14. The protein levels were analyzed by western blotting with the antibodies indicated. α-tubulin was used as a loading control. (F) Alteration in the mRNA levels of glycolytic genes in the livers of Mbd5-knockout mice at P14. The expression of each gene was normalized to β-actin. At least 5 pairs of matched mutant and wild-type mice were used for comparison. (G) Lactate levels in the livers of Mbd5-knockout mice and controls at P14. n = 3 per genotype *, P<0.05; **, P<0.01; ***, P<0.001.

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Grant support

This work was supported by grants from the Ministry of Science and Technology China (2007CB947503 and 2009CB941101), and National Science Foundation of China (30730059 to G.X., 31028012 to X.S. and 31000641 to Y.D.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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