miR-30 promotes thermogenesis and the development of beige fat by targeting RIP140

Abstract

Members of the microRNA (miR)-30 family have been reported to promote adipogenesis and inhibit osteogenesis, yet their role in the regulation of thermogenesis remains unknown. In this study, we show that miR-30b/c concentrations are greatly increased during adipocyte differentiation and are stimulated by cold exposure or the β-adrenergic receptor activator. Overexpression and knockdown of miR-30b and -30c induced and suppressed, respectively, the expression of thermogenic genes such as UCP1 and Cidea in brown adipocytes. Forced expression of miR-30b/c also significantly increased thermogenic gene expression and mitochondrial respiration in primary adipocytes derived from subcutaneous white adipose tissue, demonstrating a promoting effect of miRNAs on the development of beige fat. In addition, knockdown of miR-30b/c repressed UCP1 expression in brown adipose tissue in vivo. miR-30b/c targets the 3'-untranslated region of the receptor-interacting protein 140 (RIP140), and overexpression of miR-30b/c significantly reduced RIP140 expression. Consistent with RIP140 as a target of miR-30b/c in regulating thermogenic gene expression, overexpression of RIP140 greatly suppressed the promoting effect of miR-30b/c on the expression of UCP1 and Cidea in brown adipocytes. Taken together, the data from our study identify miR-30b/c as a key regulator of thermogenesis and uncover a new mechanism underlying the regulation of brown adipose tissue function and the development of beige fat.

Figure 1

Expression profiles of miR-30 family members in brown adipocytes and fat tissues. A: Relative expression levels of miR-30b and -30c during brown adipocyte differentiation (n = 3 independent experiments; *P < 0.05; **P < 0.01; ***P < 0.001). B: Relative UCP1 mRNA levels during brown adipocyte differentiation (n = 3; **P < 0.01; ***P < 0.001 vs. day 0). C: Relative expression levels of miR-30b and -30c during the differentiation of SVF-derived brown adipocytes (n = 3; **P < 0.01; ***P < 0.001). D: Relative expression levels of miR-30b and -30c in fat tissues of C57BL/6 mice (n = 4; *P < 0.05; **P < 0.01). E: Expression levels of miR-30b and -30c in BAT of ob/ob and WT mice (n = 6; ***P < 0.001 vs. WT mice). vWAT, visceral white adipose tissue.

Figure 2

miR-30b/c modulates the expression of thermogenic genes and mitochondrial respiration in brown adipocytes. A: Brown preadipocytes were transfected with miR-30b and -30c mimics (miR-30b/c) or nonspecific controls (miR-NC) for 48 h, followed by induction of differentiation for 3 days. At day 4 after induction, cells were harvested and the levels of miR-30b and miR-30c, as well as mRNA levels of UCP1 and Cidea, were determined by real-time RT-PCR (n = 3; *P < 0.05; ***P < 0.001). B: Brown preadipocytes were transfected with miR-30b and -30c inhibitors (anti-miR-30b/c) or nonspecific controls (anti-miR-NC) for 48 h and then induced to differentiation. The relative levels of miR-30b and miR-30c, as well as mRNA levels of UCP1 and Cidea, were analyzed by RT-PCR (n = 3; **P < 0.01; ***P < 0.001). C: Brown preadipocytes were transfected with miR-30b and -30c inhibitors or controls for 48 h. The basal levels of cell OCRs and levels in the presence of ATP synthase inhibitor oligomycin, uncoupler FCCP, or rotenone/antimycin A were determined using a Seahorse Bioscience XF24 respirometry analyzer (n = 4; *P < 0.05; **P < 0.01; ***P < 0.001).

Figure 3

Overexpression of miR-30b and -30c induces thermogenic gene expression and mitochondrial respiration in white adipocytes. A: Relative expression levels of miR-30b and -30c during the differentiation of SVF-derived white adipocytes (n = 3; *P < 0.05; **P < 0.01). B: SVF-derived preadipocytes were transfected with miR-30b and -30c mimics (miR-30b/c) or nonspecific controls (miR-NC) for 48 h and then subjected to differentiation. At day 4 after the induction of differentiation, cells were harvested and relative levels of miRNA (miR-30b/c) and mRNA (UCP1, Cidea) were determined by real-time PCR (n = 3; *P < 0.05; ***P < 0.001). C: SVF-derived preadipocytes were transfected with miR-30b/c or miR-NC for 48 h. The basal levels of cell OCRs and levels in the presence of ATP synthase inhibitor oligomycin, uncoupler FCCP, or rotenone/antimycin A were determined using a Seahorse Bioscience XF24 respirometry analyzer (n = 4; **P < 0.01 vs. miR-NC).

Figure 4

Upregulation of miR-30b and -30c by cold exposure and by β-adrenergic receptor signaling. C57BL/6 mice were maintained at room temperature (25°C) or exposed to cold (4°C) for 1 week. The relative levels of miRNAs and UCP1 in BAT (A) and sWAT (B) of the mice were analyzed (n = 4–5; *P < 0.05; **P < 0.01, ***P < 0.001 vs. 25°C). C: SVF-derived preadipocytes were cultured and induced to differentiation. On day 4 after induction of differentiation, cells were treated with the β3-adrenergic receptor activator CL-316,243, the nonselective β-adrenergic receptor activator isoproterenol, the cAMP inducer Forskolin, or a vehicle control for 24 h. Relative miR-30b and -30c levels were determined by RT-PCR (n = 6; *P < 0.05; **P < 0.01). SVF-derived preadipocytes were transfected with miR-30b and -30c mimics (miR-30b/c) or nonspecific controls (miR-NC) for 48 h and then subjected to differentiation. At day 4 after induction, cells were treated with β-adrenergic receptor activator CL-316,243 or vehicle control for 24 h, and relative mRNA levels of UCP1 (D) and Cidea (E) were determined by real-time PCR (n = 6; *P < 0.05; ***P < 0.001).

Figure 5

miR-30b and -30c target RIP140. A: The 3′ UTR sequence of the RIP140 gene is conserved in vertebrates, including mice, humans, chimpanzees, and rats. The gray block demonstrates the miR-30 targeting sites of the 3′ UTR, and asterisks show the mutant points. B: Expression profiles of RIP140 mRNA (lower graph) and protein (upper blots) during the differentiation of brown adipocytes (n = 3; *P < 0.05; **P < 0.01 vs. day 0). C: HEK293T cells were cotransfected with or without miR-30 mimics, together with WT or mutant (Mut) RIP140 3′ UTR inserted downstream of the firefly luciferase gene construct. After transfection (48 h), cells were collected and relative luciferase activity was determined using a Dual-Glo luciferase assay system (n = 4; **P < 0.01 vs. blank and Mut). D: HEK293T cells were cotransfected with miR-30b/c mimics (miR-30) and blank or 3′ UTR–RIP140 expression construct (WT) or 3′ UTR mutant–RIP140 construct (MUT). After transfection (48 h), cells were collected and RIP140 expression was analyzed by Western blot.

Figure 6

RIP140 mediates the effects of miR-30 on thermogenic gene expression in the brown adipocytes. Preadipocytes were transfected with miR-30 mimics (A and B) or inhibitors (C and D) and stimulated to undergo differentiation. Differentiating adipocytes were collected for either RT-PCR or Western blot analyses to determine expression levels of mRNAs (A and C) or proteins (B and D), respectively (n = 3; *P < 0.05; **P < 0.01 vs. respective controls). E: Brown adipose cells were transfected with blank or an RIP140 expression construct or cotransfected with or without miR-30 mimics. After transfection (48 h), cells were collected and gene expression was analyzed by RT-PCR (n = 4; *P < 0.05; **P < 0.01; ***P < 0.001).

Figure 7

Knockdown of miR-30b/c decreases UCP1 expression and mitochondrial respiration in mouse BAT in vivo. Eight-week-old male C57BJ6 mice received agomir, antagomir-30b/c, or their respective scrambled negative control (NC) through subcutaneous injection. Three days after injection, BATs were collected and the expression of miRNAs was verified by real-time RT-PCR (A and C; n = 6/group; *P < 0.05; **P < 0.01 vs. respective NCs). B: The expression of RIP140 and UCP1 was determined by Western blot (upper blots) and statistical analysis (lower graph) in BAT of mice injected with an miRNA agomir or control (n = 4; *P < 0.05). D: The expression of RIP140 and UCP1 in BAT of mice injected with an miRNA antagomir or control (n = 6; *P < 0.05). E: The basal levels of OCRs in BAT from mice injected with an miRNA antagomir or control were determined using a Seahorse Bioscience XF24 respirometry analyzer (n = 5; *P < 0.05).