Sodium butyrate alleviates adipocyte inflammation by inhibiting NLRP3 pathway

Xukai Wang, Gang He, Yan Peng, Weitian Zhong, Yan Wang, Bo Zhang, Xukai Wang, Gang He, Yan Peng, Weitian Zhong, Yan Wang, Bo Zhang

Abstract

Insulin resistance (IR) is a common feature of Type II diabetes, metabolic disorders, hypertension and other vascular diseases. Recent studies showed that obesity-induced inflammation may be critical for IR. To investigate the anti-inflammatory effect of sodium butyrate (NaB) on obesity-induced inflammation, the db/db mice were intraperitoneally injected with NaB for 6 weeks. Glucose control was evaluated by glucose tolerance test (GTT) and insulin tolerance test (ITT). Adipose tissue was harvested for gene expression analysis. 3T3-L1 adipocytes were treated with Tnf-α to mimic the inflammatory state and gene expression was detected by realtime PCR and Western blotting. Our results showed that NaB treatment improved glucose control in db/db mice as determined by GTT and ITT tests. Gene expression analysis showed that NaB inhibited cytokines and immunological markers including CD68, Interferon-γ and Mcp in adipose tissues in db/db mice. Moreover, NaB inhibited cytokine releasing in 3T3-L1 adipocytes treated with TNF-α. Further analysis of inflammation pathway showed that NLRP3 was activated in db/db mice, which was efficiently inhibited by NaB treatment. Our data suggest that inhibition of obesity-induced inflammation alleviates IR, and NaB might be a potential anti-inflammatory agent for obesity.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1. NaB improved glucose control in…
Figure 1. NaB improved glucose control in db/db mice.
(A) NaB treatment significantly decreased the body weight of db/db mice. Intraperitoneal glucose tolerance test (B), insulin tolerance test (C) and fasting glucose (D) were detected at the end of treatment as described in Materials and Methods. Data are the means ± SE. *P < 0.05, by Student’s t test (N vs NaB). N: PBS-treated controls; NaB: NaB-treated group.
Figure 2. NaB inhibited lymphocyte infiltration into…
Figure 2. NaB inhibited lymphocyte infiltration into adipose tissues.
(A–B) Epididymal adipose tissues (EAT) and subcutaneous adipose tissue (SAT) were harvested from db/db mice treated with or without NaB for six weeks. Total RNA was isolated for realtime RT-PCR. Data are expressed in ΔCt values normalized against the mean Ct of GAPDH. Fold changes were calculated as 2^(ΔΔCtN-NaB). *P < 0.05, #P < 0.01 by Student’s t test. (C) Immunohistochemical staining of macrophage marker F4/80 in mouse EAT. N: Sham-treated group; NaB: NaB-treated group.
Figure 3. NaB inhibited cytokine expression in…
Figure 3. NaB inhibited cytokine expression in db/db mice adipose tissues.
Epididymal adipose tissues (A) and subcutaneous adipose tissue (B) were harvested from db/db mice treated with or without NaB for six weeks. Total RNA was isolated for realtime RT-PCR. Data are expressed in ΔCt values normalized against the mean Ct of GAPDH. Fold changes were calculated as 2^(ΔΔCtN-NaB). *P < 0.05, #P < 0.01 by Student’s t-test. N: PBS-treated group; NaB: NaB-treated group.
Figure 4. HDACi inhibited the cytokine expression…
Figure 4. HDACi inhibited the cytokine expression in 3T3-L1 adipocytes.
3T3-L1 cells were treated with TNF-α (50 ng/mL) combined with or without NaB(1 mM) or TSA(0.5 μM) for 8 hr. Total RNA was isolated for realtime RT-PCR to detect the gene expression of IL-1β and IL-6 (A). Supernatants of cell culture were harvested for ELISA assay to detect IL-1 and IL-6 in the culture medium (B). N: Normal control. *P < 0.05 by t-test (TNF vs NaB or TSA).
Figure 5. HDACi inhibited NLRP3 inflammasome pathway…
Figure 5. HDACi inhibited NLRP3 inflammasome pathway in 3T3-L1 cells.
3T3-L1 cells were treated with TNF-α combined without or with NaB (1 mM) and TSA (0.5 μM) for 8 hr. (A) Total RNA was isolated for realtime RT-PCR. mRNA expression was normalized to that of GAPDH. (B) Total proteins were isolated from treated 3T3-L1 cells for Western blots to detect the protein level of NLRP3 and IL-1. GAPDH was used as controls for equal loading. N: Normal control. (C) Densitometry measurement of Western blots. The experiments were repeated at least three times. *P < 0.05, #P < 0.01 by t-test (TNF vs NaB or TSA).
Figure 6. NaB inhibited LRP3 inflammasome pathway…
Figure 6. NaB inhibited LRP3 inflammasome pathway in db/db mice.
Epididymal adipose tissues (EAT) and subcutaneous adipose tissue (SAT) were harvested from db/db mice treated with or without NaB for six weeks. Total RNA was isolated for realtime RT-PCR (A). Data are expressed in ΔCt values normalized against the mean Ct of GAPDH. Fold changes were calculated as 2^(ΔΔCtN-NaB). Total proteins were isolated for Western-blot (B). The bar graph of the densitometry measurement showed the decreased expression of NLRP3 and active IL-1 in db/db adipose tissues treated without or with NaB (C). *P < 0.05, #P < 0.01 by Student’s t test.

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