Nrf2 induces Ucp1 expression in adipocytes in response to β3-AR stimulation and enhances oxygen consumption in high-fat diet-fed obese mice

Abstract

Cold-induced norepinephrine activates β3-adrenergic receptors (β3-AR) to stimulate the kinase cascade and cAMP-response element-binding protein, leading to the induction of thermogenic gene expression including uncoupling protein 1 (Ucp1). Here, we showed that stimulation of the β3-AR by its agonists isoproterenol and CL316,243 in adipocytes increased the expression of Ucp1 and Heme Oxygenase 1 (Hmox1), the principal Nrf2 target gene, suggesting the functional interaction of Nrf2 with β3-AR signaling. The activation of Nrf2 by tert-butylhydroquinone and reactive oxygen species (ROS) production by glucose oxidase induced both Ucp1 and Hmox1 expression. The increased expression of Ucp1 and Hmox1 was significantly reduced in the presence of a Nrf2 chemical inhibitor or in Nrf2-deleted (knockout) adipocytes. Furthermore, Nrf2 directly activated the Ucp1 promoter, and this required DNA regions located at -3.7 and -2.0 kb of the transcription start site. The CL316,243- induced Ucp1 expression in adipocytes and oxygen consumption in obese mice were partly compromised in the absence of Nrf2 expression. These data provide additional insight into the role of Nrf2 in β3-AR-mediated Ucp1 expression and energy expenditure, further highlighting the utility of Nrf2-mediated thermogenic stimulation as a therapeutic approach to diet-induced obesity. [BMB Reports 2021; 54(8): 419-424].

Fig. 1

β3-AR activation induces Ucp1 and Hmox1 expression in adipocytes. (A) Time-dependent increases in Ucp1 and Hmox1 mRNA levels by treatment with CL316,243 (5 μM) expressed as fold-increases relative to levels in dimethyl sulfoxide (DMSO)-treated C3H10T1/2 adipocytes. (B) Treatment of primary adipocytes isolated from inguinal adipose tissues with CL316,243 (5 μM) for 6 h. (C) Treatment of C3H10T1/2 adipocytes with isoproterenol at 10 nM for 6 h. (D) Differentiated C3H10T1/2 adipocytes were exposed to 37°C or 30°C and the expression of Ucp1 and Hmox1 was measured. The data represent means ± s.e.m. and are representative of three independent experiments. Statistical significance was determined relative to controls using the Student’s t-test (*P < 0.05).

Fig. 2

Nrf2 activation increases Ucp1 expression in adipocytes. (A) tert-Butylhydroquinone (tBHQ) at 25 and 50 μM increases Ucp1 and Hmox1 expression in C3H10T1/2 adipocytes. (B) Time-dependent increases in Ucp1 and Hmox1 mRNA levels by treatment with 50 μM tBHQ expressed as foldincreases relative to the levels in DMSO-treated C3H10T1/2 adipocytes. (C) Treatment with glucose oxidase (2.5 U or 5 U/ml) increases Ucp1 and Hmox1 expression in C3H10T1/2 adipocytes. (D) Time-dependent increases in Ucp1 and Hmox1 mRNA levels by 5 U/ml glucose oxidase expressed as foldincreases relative to levels in DMSO-treated C3H10T1/2 adipocytes. (E) Nrf2 activates the Ucp1 promoter in the reporter assay. 293FT cells were transiently transfected with Ucp1 promoter-luciferase with or without the Nrf2-expressing vector. The proximal 3.7 kb, 2 kb, or 1 kb of the human Ucp1 promoter sequences were fused to express the Ucp1 promoter-driven luciferase reporter. The data represent averages +/− s.e.m. of triplicates and are expressed as fold-increases relative to empty vector-transfected cells. Statistical significance was determined relative to controls using the Student’s t-test (*P < 0.05).

Fig. 3

Nrf2 is essential for tert-butylhydroquinone (tBHQ)-mediated Ucp1 induction in adipocytes. (A) C3H10T1/2 adipocytes were treated with tBHQ (50 μM) and NAC (1 mM) for 12 h and the expression of Ucp1, Hmox1, and Nqo1 was measured. (B) Primary adipocytes isolated from the inguinal fat pads of Nrf2 wild-type (Nrf2 WT) and Nrf2 knockout mice (Nrf2 KO) were treated with tBHQ (50 μM) for 12 h and the expression levels of Ucp1 and Nrf2 target genes (Hmox1, Nqo1, Srxn1, and Gclc) were measured. The data represent means ± s.e.m. and are representative of three independent experiments. Statistical significance was determined relative to controls using the Student’s t-test (*P < 0.05).

Fig. 4

β3-AR-stimulated Ucp1 induction and oxygen consumption are partly dependent upon Nrf2. (A) Primary adipocytes isolated from the inguinal fat pads of Nrf2 WT and Nrf2 KO mice were treated with CL316,243 (5 μM) for 12 or 24 h and the expression levels of Ucp1 and Nrf2 target genes (Hmox1, Nqo1, Maff, Gclc, and Srxn1) were measured. (B) Fourteen-month-old Nrf2 WT and Nrf2 KO female mice were stimulated with β3-AR agonist (CL316,243, 2 mg/kg) and the O₂ consumption rates were measured. (C) The O₂ consumption rates of Nrf2 WT and Nrf2 KO obese male mice fed high-fat diet for 16 weeks were measured. (D) Rectal temperature was measured for 24 h in WT and Nrf2 KO obese mice. The data represent mean ± s.e.m. and statistically significant differences between WT and Nrf2 KO mice were determined by the Student’s t-test *P < 0.05).