Abstract
Chito-oligosaccharide (COS) is a natural bioactive compound, which has been shown to suppress lipid metabolic genes and lipid accumulation in differentiating adipocytes. Leptin has been identified as a key regulator of energy homeostasis and is known to be under epigenetic regulation during adipogenesis. Hence, the first objective of this experiment was to compare leptin gene (LEP) expression and leptin secretion during the different stages of adipogenesis and to investigate the effect of COS on these processes. As COS inhibited LEP expression during adipogenesis, the second aim was to investigate the methylation dynamics of a ‘CpG’ island in the proximal region of the LEP promoter during adipogenesis and to determine the effect of COS on this process. Mouse 3T3-L1 cells were stimulated to differentiate in the absence or presence of COS and the levels of leptin mRNA and protein were evaluated on days 0, 2, 4 and 6 post-induction of differentiation (PID). The extent of de-methylation of six CpG sites was evaluated. LEP mRNA transcript and protein could not be detected on either day 0PID or 2PID. In contrast, both were detected on day 4PID (P<0.05) and 6PID (P<0.001) and both were inhibited by COS (P<0.001). Of the six CpG sites analyzed, CpG_52, CpG_62 and CpG_95 became 11.5, 5.0 and 5.0% de-methylated between day 2PID and 6PID, respectively. COS blocked this de-methylation event at CpG_52 (P<0.001), CpG_62 (P<0.01) and CpG_95 (P<0.01) on day 6PID. These data suggest that COS can have an epigenetic effect on differentiating adipocytes, a novel biological function of COS which has potential applications for the manipulation of leptin gene expression, adipogenesis, and conditions within the metabolic syndrome spectrum.
Highlights
Obesity is rapidly becoming an epidemic in both developed and developing nations of the world [1], [2]
Bisulfite Modification of DNA and Pyrosequencing Based on observations that COS can inhibit the differentiation of pre-adipocytes to mature adipocytes, and de-methylation of the leptin gene (LEP) promoter is necessary for production of leptin by mature adipocytes, we investigated the methylation pattern at the CpG sites present in a 134 bp region of the mouse LEP promoter
LEP gene expression: The expression of the LEP gene was at the minimum limit of detection in pre-adipocytes and early differentiation stage adipocytes (Figure 1A)
Summary
Obesity is rapidly becoming an epidemic in both developed and developing nations of the world [1], [2]. Bisulfite Modification of DNA and Pyrosequencing Based on observations that COS can inhibit the differentiation of pre-adipocytes to mature adipocytes, and de-methylation of the LEP promoter is necessary for production of leptin by mature adipocytes, we investigated the methylation pattern at the CpG sites present in a 134 bp region of the mouse LEP promoter. Effect of COS on Leptin Protein Secretion and LEP Gene Expression during Adipogenesis
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