Indirubin, a small molecular deriving from connectivity map (CMAP) screening, ameliorates obesity-induced metabolic dysfunction by enhancing brown adipose thermogenesis and white adipose browning

Abstract

Background: Obesity occurs when the body's energy intake is constantly greater than its energy consumption and the pharmacological enhancing the activity of brown adipose tissue (BAT) and (or) browning of white adipose tissue (WAT) has been considered promising strategies to treat obesity.

Methods: In this study, we took a multi-pronged approach to screen UCP1 activators, including in silico predictions, in vitro assays, as well as in vivo experiments.

Results: Base on Connectivity MAP (CMAP) screening, we obtained multiple drugs that possess a remarkably correlating gene expression pattern to that of enhancing activity in BAT and (or) sWAT signature. Particularly, we focused on a previously unreported drug-indirubin, a compound obtained from the Indigo plant, which is now mainly used for the treatment of chronic myelogenous leukemia (CML). In the current study, our results shown that indirubin could enhance the BAT activity, as evidenced by up-regulated Ucp1 expression and enhanced mitochondrial respiratory function in vitro cellular model. Furthermore, indirubin treatment restrained high-fat diet (HFD)-induced body weight gain, improved glucose homeostasis and ameliorated hepatic steatosis which were associated with the increase of energy expenditure in the mice model. Moreover, we revealed that indirubin treatment increased BAT activity by promoting thermogenesis and mitochondrial biogenesis in BAT and induced browning of subcutaneous inguinal white adipose tissue (sWAT) of mice under HFD. Besides, our results indicated that indirubin induced UCP1 expression in brown adipocytes, at least in part, via activation of PKA and p38MAPK signaling pathways.

Conclusions: Our results clearly show that as an effective BAT (as well as beige cells) activator, indirubin may have a protective effect on the prevention and treatment of obesity and its complications.

Keywords: Brown adipose tissue; Connectivity MAP; Energy expenditure; Indirubin; Obesity.

Fig. 1

CMAP based screen identifies indirubin as a regulator of UCP1. a Flow chart depicting the process of CMAP screen using CITGeneDB to identify potential small-molecule for inducing UCP1. b, c The chemical structure and the CAS number of indirubin. d Dose-dependent effect of indirubin on Ucp1 expression in differentiated C3H10T1/2 cells on day 6 of brown adipogenesis. e RT-qPCR analysis of genes related to thermogenesis, fatty acid oxidation, mitochondrial biogenesis and common adipogenic marks in differentiated C3H10T1/2 cells (6 days of differentiation) after indirubin (10 μM) treatment. f Oxygen consumption rates (OCR) was measured in differentiated C3H10T1/2 cells (6 days of differentiation) in the presence or absence of indirubin using a Seahorse XF24 analyzer. g Basal oxygen consumption and maximum respiratory capacity from seahorse assay were determined. Data in D-G are presented as mean ± SD of six independent experiments performed in duplicate. ^*p < 0.05, ^**p < 0.01 compared with vehicle

Fig. 2

Indirubin treatment protects from HFD-induced obesity by increasing enhancing energy expenditure. a Body weights of mice were measured weekly during the last 8-week period (n = 6 in each group). Insets for A, representative images of mice at the end of 8-week experiment. (B-C) The total fat mass b and lean mass c were determined using miniSpec NMR instrument after 7-week treatment with either vehicle or indirubin. d-h Metabolic cage analyses of mice after 7-week treatment with either vehicle or indirubin (n = 6 in each group). The VO₂ consumption during a 12-h light: 12-h dark cycle d, energy expenditure (EE), mean VO₂ consumption levels e, the respiratory exchange ratio (RER; VCO₂/VO₂) f, and physical avtivity h, were measured simultaneously. g Energy expenditure values adjusted for body weight using ANCOVA. i Food intake (mean daily food consumption) was measured after 7-week treatment with either vehicle or indirubin by calculating the amount of food consumed at 24-h intervals for 6 days. Data are presented as mean ± SD (n = 6 in each group). ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 compared with NCD + vehicle group; ^*p < 0.05, ^**p < 0.01 compared with HFD + vehicle group

Fig. 3

Indirubin treatment improves glucose tolerance and insulin sensitivity in HFD-induced obese mice. a Blood concentrations of glucose in mice fasted 16 h after 6-week treatment with either vehicle or indirubin. b Blood concentrations of insulin in mice fasted overnight after 8-week treatment with either vehicle or indirubin. c Glucose tolerance tests (GTT) performed in mice (i.p. injection of glucose, 1.5 g /kg) fasted 16 h after 6-week treatment with indirubin or vehicle. (n = 6 for each treatment). d Area under curve (AUC) for glucose based on data in c. e Insulin tolerance tests (ITT) performed in mice (i.p. injection of insulin, 0.75 U /kg) fasted 4 h after 7-week treatment with indirubin or vehicle. f Area under curve (AUC) for glucose based on data in e. Data are presented as mean ± SD (n = 6 in each group). ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 compared with NCD + vehicle group; ^*p < 0.05, ^**p < 0.01, ^***p < 0.01 compared with HFD + vehicle group

Fig. 4

Indirubin treatment decreases adipose tissue mass and adipocyte cell size in HFD-induced obese mice. a Mean tissue weights of BAT, iWAT, and eWAT of mice fed NCD or HFD after 8-week treatment with either vehicle or indirubin. b Representive photographs of BAT, iWAT, and eWAT in HFD-induced obese mice after 8-week treatment with either vehicle or indirubin. c Representative hematoxylin and eosin (H&E) staining images of BAT, iWAT, and eWAT of mice fed NCD or HFD after 8-week treatment with either vehicle or indirubin. Scale bar = 50 μm. d Mean adipocyte size of adipose tissues quantified from H&E-stained section in c, six fields per mouse, using Metaxpress software. Data in a, d, e and f are presented as mean ± SD (n = 6 in each group). ^#p < 0.05, ^###p < 0.001 compared with NCD + vehicle group; ^*p < 0.05, ^**p < 0.01, compared with HFD + vehicle group

Fig. 5

Indirubin treatment ameliorated hepatic steatosis in HFD-induced obese mice. a Representative H&E staining (upper) and Oil-red staining (lower) images of liver tissue of mice fed NCD or HFD after 8-week treatment with either vehicle or indirubin. Scale bar = 50 μm. b Representive photographs of liver tissue in HFD-induced obese mice after 8-week treatment with either vehicle or indirubin. c Mean tissue weights of liver were measured in mice fed NCD or HFD after 8-week treatment with either vehicle or indirubin. d-e Liver TG d and TC e content, serum ALT g and AST h were measured in mice fed NCD or HFD after 8-week treatment with either vehicle or indirubin. f RT- qPCR analysis of genes related to adipogenesis and inflammation in livers of mice fed NCD or HFD after 8-week treatment with either vehicle or indirubin. g-h Serum ALT g and AST h were measured in mice fed NCD or HFD after 8-week treatment with either vehicle or indirubin. Data in c-h are presented as mean ± SD (n = 6 in each group). ^##p < 0.01, ^###p < 0.001 compared with NCD + vehicle group; ^*p < 0.05, ^**p < 0.01, ^***p < 0.01 compared with HFD + vehicle group

Fig. 6

Indirubin increases BAT activity by promoting thermogenesis and mitochondrial biogenesis in HFD-induced obese mice. a-e RT-qPCR analysis of genes related to thermogenesis a, fatty acid oxidation b, lipolysis c, common adipogenic marks d and mitochondrial biogenesis in BAT of mice fed NCD or HFD after 8-week treatment with either vehicle or indirubin. f Measurement of mtDMA copy number in BAT of mice fed NCD or HFD after 8-week treatment with either vehicle or indirubin. g Immunohistochemistry (IHC) staining with a UCP1-specific antibody in BAT of mice fed NCD or HFD after 8-week treatment with either vehicle or indirubin. Scale bar = 50 μm. h-i Western blot analysis of proteins levels of UCP1, PGC1α, VDAC1, and OXPHOS in BAT of mice fed NCD h or HFD i after 8-week treatment with either vehicle or indirubin. β-tubulin serves as a loading control. Data in A-F are presented as mean ± SD (n = 6 in each group). ^*p < 0.05, ^**p < 0.01 compared with HFD + vehicle group

Fig. 7

Indirubin promotes browning of sWAT by increasing BAT-specific markers and mitochondrial biogenesis in HFD-induced obese mice. a-f RT-qPCR analysis of genes related to thermogenesis a, fatty acid oxidation b, lipolysis c, common adipogenic marks d, beige cell markers e and mitochondrial biogenesis f in sWAT of mice fed NCD or HFD after 8-week treatment with either vehicle or indirubin. g Measurement of mtDMA copy number in sWAT of mice fed NCD or HFD after 8-week treatment with either vehicle or indirubin. h Immunohistochemistry (IHC) staining with a UCP1-specific antibody in BAT of mice fed NCD or HFD after 8-week treatment with either vehicle or indirubin. Scale bar = 50 μm. i-j Western blot analysis of proteins levels of UCP1, PGC1α, VDAC1, and OXPHOS in BAT of mice fed NCD (H) or HFD i after 8-week treatment with either vehicle or indirubin. β-tubulin serves as a loading control. Data in A-F are presented as mean ± SD (n = 6 in each group). ^#p < 0.05 compared with NCD + vehicle group;^*p < 0.05, ^**p < 0.01 compared with HFD + vehicle group

Fig. 8

Indirubin enhances BAT activity and induces browning of WAT relying on PKA and p38MAPK signaling pathways. a Western blot analysis performed with the indicated antibodies (Phospho-PKA substrate, UCP1, PGC1α, p-CREB) in differentiated C3H10T1/2 cells on day 6 of brown adipogenesis in present or absent of indirubin and (or) PKA inhibitor. β-tubulin serves as a loading control. b Western blot analysis performed with the indicated antibodies (UCP1, PGC1α) in differentiated C3H10T1/2 cells on day 6 of brown adipogenesis in present or absent of indirubin and (or) p38 MAPK inhibitor. β-tubulin serves as a loading control. c Semi-quantitative analysis of p-p38/p38 ratio from (B). d Proposed model for the role of indirubin in activating BAT and browning of WAT, thus having benefit effects on ameliorating obesity and its associated metabolic dysfunction