要約のみTohoku University盧尚建課

So  Kyoung-ha

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Tohoku University博士(農学)博士学位論文 (Thesis(doctor))要約のみthesi

So, Kyoung-ha

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Study on the Roles of Adipogenin on FatAccumulation in Mice and Cattle著者 So  Kyoung-ha学位授与機関 Tohoku University学位授与番号 11301甲第16457号URL http://hdl.handle.net/10097/60398   Study on the Roles of Adipogenin on Fat Accumulation  in Mice and Cattle (マウスとウシの脂肪蓄積における Adipogeninの 役割に関する研究)    2015     Kyoung-ha So   Introduction  Adipose tissue plays a critical role in the regulation of metabolic homeostasis and energy balance. It also serves an important function as a secretory/endocrine organ that mediates numerous physiological and pathological processes. The global obesity rate nearly doubled between the 1980s and 2014. Obesity is a known risk factor for numerous health problems, including hypertension, high cholesterol, diabetes, cardiovascular disease, respiratory problems (asthma), musculoskeletal diseases (arthritis), and cancer (OECD Factbook, 2014). Adipose tissue growth involves both expansion of existing adipocytes and development of new adipocytes from precursor cells (preadipocytes or adipoblasts). The increase in adipocyte size is primarily due to lipid accumulation within the cells. The increase in adipose tissue mass is caused by an increase in the abundance of mature cells. For decades, the functional mechanisms of adipocyte differentiation have been extensively investigated using preadipocyte culture systems. Furthermore, the importance of factors related to adipose tissue growth and adipogenesis has been demonstrated using knockout mouse models. The relationship of several biological factors to adipogenesis and the development of adipose tissue have already been established. Leptin, a hormone secreted by adipocytes, serves to regulate body weight and energy expenditure (Friedman, 1998; Zhang et al., 1994). Peroxisome proliferator-activated receptor γ2 (PPAR-γ2) and the CCAAT/enhancer-binding protein (C/EBP) family of transcription factors are crucial factors in maintaining the differentiated state of adipocytes (Hu et al., 1996; Spiegelman and Flier, 1996). The blood level of adiponectin, an adipocyte-derived hormone, was lower in obese and high-fat diet-fed mice (Kadowaki and Yamauchi, 2005; Yamauchi et al., 2001). In addition, several cytokines (TNF-α, resistin, and chemerin) are involved in insulin resistance and glucose metabolism (Bozaoglu et al., 2007; Goralski et al., 2007; Hotamisligil et al., 1996; Peraldi et al., 1996; Roh et al., 2007; Steppan et al., 2001). Japanese Black cattle produces high-quality beef, which is known for its extremely high marbling, which itself is dependent on several factors including breed, age at slaughter, aging process and packaging method (Busboom et al., 1993; Matsuhashi et al., 2011). Many studies have investigated the genetic effects on meat quality; hence, the SNPs of many candidate genes have been tested (Li et al., 2013). The fatty acid composition of adipose tissue of cattle has become an important production parameter for the meat industry, because higher concentrations of MUFA causes a lower fat-melting point, which affects the softness of fat and the beef flavor and ultimately influences meat quality (Melton et al., 1982). Japanese Black cattle has a greater genetic predisposition for producing carcass fat with higher concentrations of MUFAs than Holstein, Japanese Brown, and Charolais (Zembayashi et al., 1995). Thus, SCD, SREBP-1, and FASN are considered determinants of the fatty acid composition in Japanese Black cattle (Abe et al., 2009; Hoashi et al., 2007; Roy et al., 2005; Taniguchi et al., 2004). Our group reported the possibility that adipogenin (ADIG) regulates adipogenesis and fat accumulation in mice and bovine species (Hong et al., 2005; Hong et al., 2006). ADIG is highly expressed in the white adipose tissue in mice; ADIG level was significantly elevated during adipocyte differentiation of 3T3-L1 cells. siRNA-induced inhibition of ADIG reduced 3T3-L1 adipogenic differentiation and adipogenesis. Transfection of 3T3-L1 cells with EGFP-ADIG fusion protein confirmed that ADIG was located exclusively in the membranes of 3T3-L1 cells. Furthermore, ADIG was identified in cattle, pigs, and humans, that is, species in which mRNA and proteins are approximately 66–86% conserved. These reports suggested that ADIG might be an important factor in adipocyte development and adipogenesis. However, the functional role of ADIG in the metabolic processes of fat accumulation and adipogenesis in vivo and in vitro remain unknown.  Hence, the aim of the study was as follows;  1. To investigate phenotypic characteristics of ADIG gene-deficient mice 2. To investigate the regulation of ADIG gene expression in 3T3-L1 cells and mouse embryonic fibroblasts 3. To investigate the effects of single-nucleotide polymorphisms (SNP) of the bovine ADIG gene in Japanese Black cattle   Chapter 1. Adipose tissue metabolism in ADIG-deficient (ADIG-KO) mice  This study aimed to investigate the phenotypic changes as well as adipose tissue metabolism in ADIG-KO mice in vivo.  To determine the impact of ADIG on adipocyte lipid metabolism, body weight, and glucose metabolism, we investigated the phenotypic changes in ADIG-KO mice fed normal-fat diet including the body weight, fat accumulation and levels of gene expression.  These results shown that the body weight of ADIG-KO mice was significantly lower than that of ADIG wild-type (ADIG-WT) mice and the deposition of white adipose tissue was also significantly reduced (Figure 1).  In the adipose tissue, the expression levels of PPAR-γ2 gene was reduced in ADIG-KO mice than in ADIG-WT mice (data not shown).  The phenotypic analysis of ADIG-KO mice suggests that ADIG regulates lipid and glucose metabolism in adipose tissue through impaired insulin secretion, which results in growth retardation.  Chapter 2. Regulation of ADIG gene expression in 3T3-L1 cells and mouse embryonic fibroblasts (MEFs)  This study aimed to investigate the regulation of ADIG gene expression during adipocyte differentiation in 3T3-L1 preadipocytes and MEFs derived from ADIG-KO mice. 3T3-L1 preadipocytes were differentiated with adipogenic mixtures (insulin, dexamethasone, and IBMX) for 10 days, after which the expression levels of ADIG and lipid accumulations were measured. The expression of ADIG gene was up-regulated after adipocyte differentiation of 3T3-L1 cells and lipid accumulation was also significantly increased after adipocyte differentiation (Figure 2). To further examine the contribution of ADIG to adipogenesis, primary MEFs were isolated and differentiated from the embryos of ADIG-KO and ADIG-WT mice at 13.5 days postcoitum. In the stage of matured adipocytes, the MEFs of ADIG-KO mice showed lower lipid accumulation than those of ADIG-WT (data not shown). These results suggest that the ADIG expression was induced after adipocyte differentiation in vitro.  Chapter 3. The SNP G.7302 G>A of the bovine gene ADIG is sex-dependently associated with fatty acid composition in Japanese Black cattle.  Carcass traits and fatty acid composition have become important production parameters in the beef industry. It has been reported that bovine ADIG plays a crucial role in adipocyte differentiation and fat deposition (Hong et al., 2005). The purpose of this study was to investigate the association of SNPs in ADIG with fatty acid composition and carcass traits in Japanese Black cattle (n = 338). As shown in Figure 3, we identified six SNPs: g.7050 A>G, g.7302 G>A, g.9852 C>T, g.9873 C>G, g.10089 A>G, and g.10142 T>C. Steers with g.7302 G>A exhibited higher carcass weight and larger rib eye area and thickness. Steers with the genotype GG showed higher levels of C18:1 and monounsaturated fatty acids (MUFA), but lower levels of C16:0 and saturated fatty acids (SFA) than steers with the genotype GA (P < 0.05). Heifers had higher percentages of C18:1 and MUFA, but lower percentages of C14:0, C16:0, C18:0, and SFA, compared to steers (Table 1). These results suggest that g.7302 G>A is related to fatty acid composition in a sex-dependent manner in Japanese Black cattle.  Conclusion  In summary, these studies highlight the functional roles of ADIG in the regulation of fat accumulation and adipogenesis as follows:  1. In ADIG-KO mice fed a normal-fat diet, the body and adipose tissue weights were reduced than ADIG-WT mice. 2. ADIG expression was up-regulated after adipocyte differentiation. 3. In the study of SNPs of bovine ADIG, we found 6 SNPs. With the SNP g.7302 G>A, steers with the genotype GG showed higher levels of C18:1 and MUFA and lower levels of C16:0 and SFA than those with the genotype GA (P < 0.05). These results suggest that g.7302 G>A is related to fatty acid composition in a sex-dependent manner in Japanese Black cattle.       Figure 1. Body weight curves (A) and Adipose tissue weight (B) between ADIG-KO and WT male mice fed normal chow diet. Data presented are the mean ± SEM values (n = 10–11). ** P < 0.01, * P < 0.05 versus WT.        Figure 2. The expression level of ADIG gene (A) and lipid accumulation (B) during adipocyte differentiation of 3T3-L1 cells. Results are the mean ± SEM values (n = 6). * P < 0.05 versus day 5. N.D means not differentiation.   1015202530354 5 6 7 8 9 10 11 12 13 14 15Body weight (g)WeeksWT (n=11)KO (n=10)*** **** ** **** ** ** **** **A)0123456WT KOTotal fat (g)p = 0.06B)A) B)024681012140 5 10AIDG / 18STime (day)*0 5 10Day of differentiation N.D    Figure 3. Structure of the bovine ADIG gene and location of the polymorphic sites.      Exon3① ②③④ ⑤⑥Exon1 Exon2ATG TGA5’ 3’Table 1. Effect of SNPg.7302 G>A in the ADIG gene on carcass traits and fatty acid composition in Japanese Black cattle (n = 338)   CW = Carcass weight; REA = Rib eye area; RT = Rib thickness; SFT = Subcutaneous fat thickness; YE = Yield estimate; BMG = Beef marbling grade; BCG = Beef color grade; BTG = Beef texture grade; C14:0 = Myristic acid; C16:0 = Palmitic acid; C16:1 = Palmitoleic acid; C18:0 = Stearic acid; C18:1 = Oleic acid; C18:2 = Linoleic acid; SFA = Saturated fatty acids (C14:0 + C16:0 + C18:0); MUFA = Monounsaturated fatty acids (C16:1 + C18:1). Data presented are the mean ± SEM values.  Steers (n = 235) Heifers (n = 103)Traits GG (n = 186) GA (n = 49) P value GG (n = 90) GA (n = 13) P valueCW (kg) 483.48±51.41 488.93 ±53.56 0.91 407.08±44.65 407.35±33.87 0.98 REA (cm²) 62.37±9.08 62.82±7.22 0.68 59.52±9.24 59.38±8.89 0.96 RT (cm) 8.23±0.86 8.12±0.80 0.91 7.90±0.91 7.97±0.67 0.79 SFT (cm) 2.50±0.78 2.44±0.63 0.60 2.58±0.70 2.74±0.90 0.47 YE (%) 74.74±1.59 74.71±1.24 0.45 75.06±1.72 74.91±1.39 0.76 BMG 6.81±2.20 6.53±1.89 0.42 6.86±2.33 6.62±2.43 0.59 BCG 4.22±0.84 4.12±0.81 0.42 4.22±0.88 4.08±0.86 0.44 BTG 4.33±0.74 4.37±0.67 0.89 4.37±0.79 4.23±0.73 0.43 C14:0 2.47±0.48 2.62±0.52 0.06 2.43±0.45 2.35±0.32 0.50 C16:0 25.69±1.79 26.55±1.67 0.003 25.25±1.90 25.14±1.67 0.28 C16:1 4.30±0.83 4.27±0.87 0.70 4.48±0.71 4.51±0.69 0.59 C18:0 11.03±1.58 11.07±1.71 0.77 10.93±1.48 10.85±1.60 0.86 C18:1 53.81±2.63 52.96±2.26 0.04 54.48±2.56 54.94±2.47 0.55 C18:2 2.71±0.53 2.53±0.51 0.73 2.43±0.55 2.22±0.35 0.50 SFA 39.18±2.78 40.24±2.42 0.02 38.61±2.74 38.33±2.66 0.74 MUFA 58.11±2.69 57.23±2.30 0.04 58.96±2.62 59.45±2.62 0.54 References  Abe, T., J. 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Study on the Roles of Adipogenin on Fat Accumulation in Mice and Cattle

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マウスとウシの脂肪蓄積におけるAdipogeninの役割に関する研究

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