J Biochem. 2019 Jan 1;165(1):47-55.

CREG1 promotes uncoupling protein 1 expression and brown adipogenesis in vitro

Tatsuya Kusudo,1,2 Michihiro Hashimoto,1 Naoya Kataoka,1,3 Yongxue Li,1 Aya Nozaki,1 and Hitoshi Yamashita1

1Department of Biomedical Sciences, College of Life and Health Sciences, Chubu University, Kasugai 487-8501, Japan.

2Faculty of Human Sciences, Tezukayama Gakuin University, Sakai 590-0113, Japan.

3Department of Integrative Physiology, Nagoya University School of Medicine, Nagoya 466-8550, Japan.

Correspondence should be addressed to Hitoshi Yamashita, Department of Biomedical Sciences, College of Life and Health Sciences, Chubu University, Kasugai 487-8501, Japan, E-mail: hyamashi@isc.chubu.ac.jp

 

Abstract

Brown adipocytes play a critical role for adaptive thermogenesis to regulate body temperature in cold or to circumvent diet-induced obesity. In this study, we investigated the role of cellular repressor of E1A-stimulated genes 1 (CREG1) on brown adipogenesis and uncoupling protein 1 (UCP1) expression by using in vitro culture models. In murine mesenchymal stem cell line C3H10T1/2, Creg1 mRNA expression significantly increased in a time-dependent manner along with Ucp1 mRNA induction in brown adipogenesis. Creg1 gene overexpression upregulated the expression of brown fat-related genes including Ucp1 but its suppression downregulated these gene expression in C3H10T1/2 cells. Unlike the brown adipogenesis, Creg1 mRNA expression decreased significantly after differentiation stimulation in white adipogenesis of 3T3-L1 cells. Either Creg1 gene overexpression or suppression hardly affected white adipogenesis. In addition, CREG1 protein stimulated brown adipogenesis and rescued the adipogenesis in the absence of thyroid hormone in C3H10T1/2 cells. In reporter assay, CREG1 induction stimulated Ucp1 promoter activity, which was enhanced by co-expression with thyroid hormone receptors. The effect of CREG1 on Ucp1 promoter activity was also stimulated by retinoic acid. These results strongly suggest that CREG1 plays an important role on the regulation of UCP1 expression and brown adipogenesis.

PMID: 30295852

 

Supplement:

Active BAT is detected in adult human, whereas its amount is reduced with age and correlated inversely to the degree of adiposity. Discovery of inducible brown-like adipocytes, so-called beige adipocytes, in white adipose tissue (WAT) has accelerated basic and clinical studies on the stimulation of brown fat formation and activity as a potential therapeutic target against obesity and metabolic syndrome. CREG1 is a secreted glycoprotein in mouse and human that inhibits cell growth and enhances differentiation of human embryonic teratocarcinoma NTERA-2 cells. However, involvement of CREG1 into adipocyte differentiation and, if any, the role of this protein on brown adipogenesis was totally unknown. Here we demonstrate for the first time that CREG1 stimulates Ucp1 gene expression and promotes brown fat formation in vitro and in vivo.

 

CREG1 stimulates brown adipogenesis and compensates T3 effect for the differentiation in vitro

To clarify the contribution of CREG1 to brown adipogenesis, we examined the role of CREG1 on the differentiation of C3H10T1/2 cells, which is an established model of brown adipogenesis. Creg1 gene overexpression in C3H10T1/2 cells infected with Ad-Creg1 stimulated adipogenesis and significantly increased the mRNA levels of Ucp1 compared to those in the control cells (Figure 1A). Conversely, Creg1 gene silencing attenuated adipogenesis and the gene expression pattern was like a mirror image of that in the Creg1 gene overexpression experiment (Figure 1B). In addition, subtraction analysis of common stimulators including 3,5,3’-triiodothyronine (T3) and insulin revealed that CREG1 substitutes T3 function for brown adipogenesis in the culture (Figure 1C). Interestingly, CREG1 significantly increased the mRNA level of fibroblast growth factor 21 (fgf21), which is a regulator of WAT browning, in the control cells without T3. These results suggest that the secreted CREG1 can promote brown adipocyte differentiation by autocrine/paracrine mechanism.

 

 

CREG1 enhances THR-driven Ucp1 transcription with RXRa

To address the role of CREG1 on the molecular mechanism of transcriptional regulation for brown-fat selective genes, we examined the involvement of CREG1 in Ucp1 transcriptional regulation. The Ucp1 promoter activity was increased by Creg1 expression, as well as by thyroid hormone receptor a (Thra) and Thrb, compared to the control in reporter assay, and the activity was synergistically increased by co-expression of Creg1 with these nuclear receptors (Figure 2A). Similar to THRa and retinoid x receptor a (RXRa), which are known to form heterodimer for Ucp1 gene activation, CREG1 was recruited to the enhancer region, but not the promoter, in the differentiated C3H10T1/2 cells (Figure 2B), indicating the involvement of CREG1 in Ucp1 transcriptional machinery. Moreover, inhibition of RXR action resulted in a significant suppression of Ucp1 promoter activity in the absence of T3 and blunted the stimulatory effect of CREG1. On the other hand, the 9-cis retinoic acid (RA)-mediated enhancement of CREG1 effect was completely inhibited by a RXR antagonist (HX531), indicating that the stimulatory effect of CREG1 for Ucp1 transcription greatly depends on RXR action (Figure 2C). We then examined whether CREG1 can directly interact with these nuclear receptors. In our in vitro experiments, RXRa was found to bind CREG1 (Figure 2D). Together, we propose a model for the CREG1 role on Ucp1 transcription (Figure 2E). Namely, THR/RXRα heterodimer binds to the thyroid response element (TRE) and retinoic acid response element (RARE) in BAT-specific enhancer of Ucp1 prompter and stimulates Ucp1 transcription, which activity is strongly enhanced by T3 and RA. CREG1 also enhances this transcriptional activity through binding to RXRα possibly even in the situation lacking T3.

 

 

 

CREG1 stimulates brown fat formation and improves diet-induced obesity in mice

To investigate the role of CREG1 in brown fat formation in adipose tissues and energy metabolism in vivo, we created aP2-Creg1-transgenic (Tg) mice (Hashimoto et al., 2019). Although no phenotypic differences were found in body weight between the wild-type (WT) and Tg mice under a standard chow diet condition, Tg mice were found to be more resistant to diet-induced obesity than WT mice in the absence of a difference in food intake under high-fat (HF) diet condition. As expected, immunohistochemical analysis revealed striking changes in Tg mice, relative to WT mice, in brown adipocyte morphology in BAT and browning of WAT coupled with strong UCP1 immunoreactivity (Figure 3). Fgf21 mRNA level was also drastically elevated in the WAT of Tg mice. These results indicated that an increase in CREG1 in adipose tissues substantially stimulates browning and UCP1 expression in mice. Moreover, HF-diet-fed Tg mice exhibited an improvement of glucose and insulin tolerance as well as obesity-associated fatty liver relative to WT mice. These effects of CREG1 were confirmed in obese mice injected with CREG1 protein via osmotic pump, in which body weight gain, but not food intake, was significantly reduced in the CREG1 group and accompanied with improvement of glucose tolerance and fatty liver, compared with PBS group (our unpublished data).

 

 

 

In the in vivo experiments, we successfully confirmed the effects of CREG1 for induction of brown fat formation and obesity inhibition, indicating a systemic role of CREG1 as an endocrine factor. In particular, the analyses of Creg1-Tg mice indicate that CREG1 induction in adipose tissues stimulates browning and causes the resistant phenotypes against obesity and fatty liver under a HF diet condition in mice. It is important to note that serum CREG1 level is quite high (microgram order per ml) and positively correlates with body weight in normal mice. On the other hand, the serum CREG1 levels in ob/ob and db/db mice were significantly lower than that in normal mice and not associated with body weight (our unpublished data). The data on Creg1-Tg mice also suggest that CREG1 is a novel endogenous regulator of Fgf21 expression. Hypothyroidism in patients or animal models is often associated with hypothermia, weight gain and dyslipidemia. Considering the importance of thyroid hormone regulation of metabolism, CREG1 could make up for T3 action with RXRα not only in adipose tissues but also in other tissue including liver in case of a lowering of thyroid hormone action such as fasting. Future studies are required to clarify the precise mechanism of CREG1 that promotes brown/beige adipogenesis and the therapeutic potential for type 2 diabetes; however, our studies might open up a new perspective in the control of brown fat formation and in the complex mechanism of thyroid system in metabolism.

 

References:

  1. Kusudo T, Hashimoto M, Kataoka N, Li Y, Nozaki A, and Yamashita H. (2019) CREG1 promotes uncoupling protein 1 expression and brown adipogenesis in vitro. J Biochem., 165(1):47-55. doi: 10.1093/jb/mvy083.
  2. Hashimoto M, Kusudo T, Takeuchi T, Kataoka N, Mukai T, Yamashita H. (2019) CREG1 stimulates brown adipocyte formation and ameliorates diet-induced obesity in mice. FASEB J., 33(7):8069-8082. doi: 10.1096/fj.201802147RR.