Anti-Inflammatory Mechanisms of Curcumin and Its Metabolites in White Adipose Tissue and Cultured Adipocytes

Abstract

The plant-derived polyphenol curcumin alleviates the inflammatory and metabolic effects of obesity, in part, by reducing adipose tissue inflammation. We hypothesized that the benefits of curcumin supplementation on diet-induced obesity and systemic inflammation in mice occur through downregulation of white adipose tissue (WAT) inflammation. The hypothesis was tested in adipose tissue from high-fat diet-induced obese mice supplemented with or without curcumin and in 3T3-L1 adipocytes treated with or without curcumin. Male B6 mice were fed a high-fat diet (HFD, 45% kcal fat) with or without 0.4% (w/w) curcumin supplementation (HFC). Metabolic changes in these mice have been previously reported. Here, we determined the serum levels of the curcumin metabolites tetrahydrocurcumin (THC) and curcumin-O-glucuronide (COG) using mass spectrometry. Moreover, we determined interleukin 6 (IL-6) levels and proteomic changes in LPS-stimulated 3T3-L1 adipocytes treated with or without curcumin by using immunoassays and mass spectrometry, respectively, to gain further insight into any altered processes. We detected both curcumin metabolites, THC and COG, in serum samples from the curcumin-fed mice. Both curcumin and its metabolites reduced LPS-induced adipocyte IL-6 secretion and mRNA levels. Proteomic analyses indicated that curcumin upregulated EIF2 and mTOR signaling pathways. Overall, curcumin exerted anti-inflammatory effects in adipocytes, in part by reducing IL-6, and these effects may be linked to the upregulation of the mTOR signaling pathway, warranting additional mechanistic studies on the effects of curcumin and its metabolites on metabolic health.

Keywords: 3T3-L1 adipocytes; C57BL/6J (B6) mice; curcumin; curcumin-O-glucuronide; high-fat diet; inflammation; obesity; tetrahydrocurcumin; white adipose tissue.

Figure 1

Detection of curcumin metabolites in mouse serum. We used the mass spectrometry technique to detect curcumin metabolites in the serum samples of the HF and HFC group mice. Tetrahydrocurcumin (THC) and Curcumin-O-glucuronide (COG) were detected in the serum samples of the HFC-fed mice. Means without a common letter differ (p-value < 0.05), n = 3.

Figure 2

Curcumin dose–response and cell viability experiments: Curcumin reduced LPS-induced inflammatory cytokine IL-6 secretion in the cultured adipocytes. (A) In the non-differentiated preadipocytes, curcumin dose-dependently reduced IL-6 secretion at all doses (1–20 µM). (B) However, in the differentiated adipocytes, curcumin doses between 5–15 µM were more effective in reducing IL-6 secretion induced by LPS (200 ng/mL). (C) Up to 10 µM, curcumin did not induce any cell toxicity. In the differentiated adipocytes, a 5 µM dose of curcumin was the lowest dose that reduced IL-6 secretion without any toxicity and had similar effects compared to 10 µM. Thus, this dose was applied in the subsequent cell culture experiments. The MTT assay results were averaged from 2 biological replicate experiments, where 4 to 5 wells per treatment were used for each treatment group per experiment. (D,E) A curcumin dose of 5 µM reduced IL-6 secretion in LPS (200 ng/mL)-induced mature 3T3-L1 cells. However, MCP-1 secretion was not affected by curcumin at the dose used. Means without a common letter differ (p-value < 0.05), n = 3; 4–6 wells per treatment group in each experiment.

Figure 3

Dose–response effects of curcumin metabolites (COG and THC) on cell viability and IL-6 secretion. (A,B) The MTT assay showed that none of the doses (1–10 µM) of curcumin metabolites tested reduced cell viability in the 3T3-L1 adipocytes. (C) In the differentiated adipocytes, only 1 µM of COG reduced IL 6 secretion; (D) THC dose-dependently reduced IL-6 secretion with significant effects at 5 and 10 µM of THC. Means without a common letter differ (p-value < 0.05); duplicate experiments with 6–12 wells in each MTT test and 4–6 wells in each ELISA test were carried out.

Figure 4

Proteomic analyses of curcumin effects in 3T3-L1 adipocytes. A total of 2106 proteins were differentially expressed, of which 209 proteins were significantly up- or downregulated by curcumin (0.2-fold change cut-off; n = 3, control = 200 ng/mL of LPS and treatment = 200 ng/mL of LPS + 1 µM curcumin).

Figure 5

Curcumin-modulated upstream regulators (A) and pathways (B) (ranked based on statistical significance). The adipocytes’ proteomics data were analyzed with IPA pathway analysis application. The EIF2 and mTOR pathways were upregulated (z-score: 3 and 1.342, respectively, p-value < 0.05), which was supported by the inhibition of the upstream regulator of Rictor and Sirolimus (z-score of −2.132 and −2.693, respectively, p-value < 0.05). GraphPad software was used for this visualization.