Acute and chronic saturated fatty acid treatment as a key instigator of the TLR-mediated inflammatory response in human adipose tissue, in vitro

Abstract

A post-prandial increase in saturated fatty acids (SFAs) and glucose (Glc) activates an inflammatory response, which may be prolonged following restoration of physiological SFAs and Glc levels--a finding referred to as 'metabolic memory'. This study examined chronic and oscillating SFAs and Glc on the inflammatory signalling pathway in human adipose tissue (AT) and adipocytes (Ads) and determined whether Ads are subject to "metabolic memory." Abdominal (Abd) subcutaneous (Sc) explants and Ads were treated with chronic low glucose (L-Glc): 5.6 mM and high glucose (H-Glc): 17.5 mM, with low (0.2 mM) and high (2 mM) SFA for 48 h. Abd Sc explants and Ads were also exposed to the aforementioned treatment regimen for 12-h periods, with alternating rest periods of 12 h in L-Glc. Chronic treatment with L-Glc and high SFAs, H-Glc and high SFAs up-regulated key factors of the nuclear factor-κB (NFκB) pathway in Abd Sc AT and Ads (TLR4, NFκB; P<.05), whilst down-regulating MyD88. Oscillating Glc and SFA concentrations increased TLR4, NFκB, IKKβ (P<.05) in explants and Ads and up-regulated MyD88 expression (P<.05). Both tumor necrosis factor α and interleukin 6 (P<.05) secretion were markedly increased in chronically treated Abd Sc explants and Ads whilst, with oscillating treatments, a sustained inflammatory effect was noted in absence of treatment. Therefore, SFAs may act as key instigators of the inflammatory response in human AT via NFκB activation, which suggests that short-term exposure of cells to uncontrolled levels of SFAs and Glc leads to a longer-term inflammatory insult within the Ad, which may have important implications for patients with obesity and Type 2 diabetes.

Fig. 1

(A–F) The mean relative protein expression (±S.E.M.) of TLR4, MYD88 and TRAF-6 in explants and isolated, mature adipocytes compared with their respective controls (explant tissue and adipocyte cells maintained in L-Glc (5.6 mM), with representative Western blots shown above. Statistical analysis compared expression of the proteins in explants and cells chronically treated with L-Glc: 5.6 mM or H-Glc: 17.5 mM in combination with low (0.2 mM) and high (2 mM) doses of a palmitate: SFA, as well as high glucose alone 17.5 mM, for 48 h (n=6; ⁎P<.05, ⁎⁎P<.01, ⁎⁎⁎P<.001).

Fig. 2

A–D The mean relative protein expression of IKKβ and NFκB (±S.E.M.) in explants and mature adipocytes compared with their respective controls, with representative Western blots shown above. The explants and cells were chronically treated with L-Glc: 5.6 mM or H-Glc: 17.5 mM in combination with low (0.2 mM) and high (2 mM) doses of SFAs, as well as H-Glc alone, for 48 h (n=6; ⁎P<.05, ⁎⁎P<.01, ⁎⁎⁎P<.001).

Fig. 3

(A–D) IL-6 and TNFα fold change in secretion levels (mean±S.E.M.) from control (L-Glc: 5.6 mM) and treated explants and adipocytes. Statistical analysis compared the effect of L-Glc: 5.6 mM or H-Glc: 17.5 mM in combination with low (0.2 mM) and high (2 mM) doses of SFAs, as well as H-Glc alone, on secretion of IL-6 and TNFα, respectively, for 48 h (n=6; ⁎P<.05, ⁎⁎P<.01, ⁎⁎⁎P<.001). (a and b) IL-6 control (mean±S.E.M.) Explants (Exp): 1212.0±299.22 pg/ml; Adipocytes (Ad): 12798.0±5954.16 pg/ml. (c and d) TNFα control (mean±S.E.M.) Exp: 0.30±0.14 pg/ml; Ad: 0.93±0.16 pg/ml.

Fig. 4

(A–F) The mean relative protein expression (±S.E.M.) of TLR-4, MyD88 and TRAF6 in explants and isolated adipocytes intermittently treated with L-Glc: 5.6 mM or H-Glc: 17.5 mM in combination with low (0.2 mM) and high (2 mM) doses of SFAs, as well as high glucose alone 17.5 mM, for 48 h, compared with controls (n=6; ⁎P<.05; ⁎⁎P<.01; ⁎⁎⁎P<.001).

Fig. 5

(A–D) The mean relative protein expression (±S.E.M.) of IKKβ and NFκB in explants and isolated adipocytes intermittently treated with L-Glc: 5.6 mM or H-Glc: 17.5 mM in combination with low (0.2 mM) and high (2 mM) doses of SFAs, as well as high glucose alone 17.5 mM, for 48 h, compared with controls (n=6; ⁎P<.05; ⁎⁎P<.01; ⁎⁎⁎P<.001).

Fig. 6

(A–H) The fold change in TNFα secretion levels (mean±S.E.M.) from control (L-Glc:5.6 mM) and explants and adipocytes that went through an alternating treatment regime that consisted of L-Glc for 12 h, then L-Glc: 5.6 mM or H-Glc: 17.5 mM in combination with low (0.2 mM) and high (2 mM) doses of SFAs, as well as H-Glc alone, for a period of 48 h in total (n=6; ⁎P<.05). (a) 0–12 h control (mean±S.E.M.) Exp: 0.39±0.18 pg/ml. (b) 12–24 h Control Exp: 0.32±0.13 pg/ml. (c) 24–36 h Control Exp: 0.29±0.15 pg/ml. (d) 36–48 h Control Exp: 0.28±0.13 pg/ml. (e) 0–12 h Control (mean±S.E.M.) Ad: 6.13±1.18 pg/ml. (f) 12–24 h Control Ad: 0.90±0.20 pg/ml. (g) 24–36 h Control Ad: 0.57±0.20 pg/ml. (h) 36–48 h Control Ad: 0.66±0.23 pg/ml.

Fig. 6

(A–H) The fold change in TNFα secretion levels (mean±S.E.M.) from control (L-Glc:5.6 mM) and explants and adipocytes that went through an alternating treatment regime that consisted of L-Glc for 12 h, then L-Glc: 5.6 mM or H-Glc: 17.5 mM in combination with low (0.2 mM) and high (2 mM) doses of SFAs, as well as H-Glc alone, for a period of 48 h in total (n=6; ⁎P<.05). (a) 0–12 h control (mean±S.E.M.) Exp: 0.39±0.18 pg/ml. (b) 12–24 h Control Exp: 0.32±0.13 pg/ml. (c) 24–36 h Control Exp: 0.29±0.15 pg/ml. (d) 36–48 h Control Exp: 0.28±0.13 pg/ml. (e) 0–12 h Control (mean±S.E.M.) Ad: 6.13±1.18 pg/ml. (f) 12–24 h Control Ad: 0.90±0.20 pg/ml. (g) 24–36 h Control Ad: 0.57±0.20 pg/ml. (h) 36–48 h Control Ad: 0.66±0.23 pg/ml.

Fig. 7

A–H. The fold change in IL-6 secretion levels (mean±S.E.M.) from control (L-Glc:5.6 mM) and explants and adipocytes that went through alternating treatments of L-Glc for 12 h, then L-Glc: 5.6 mM or H-Glc: 17.5 mM in combination with low (0.2 mM) and high (2 mM) doses of SFAs, as well as H-Glc alone, for a period of 48 h in total (n=6, p-values: P<.05⁎). (a) 0–12 h Control (mean±S.E.M.) Exp: 447.9±160.90 pg/ml. (b) 12–24 h Control (mean±S.E.M.) Exp: 240.22±53.85 pg/ml. (c) 24–36 h Control (mean±S.E.M.) Exp: 193.04±45.16 pg/ml. (d) 36–48 h Control (mean±S.E.M.) Exp: 479.24±247.40 pg/ml. (e) 0–12 h Control (mean±S.E.M.) Ad: 1863.7±95.90 pg/ml. (f) 12–24 h Control Ad: 860.60±272.93 pg/ml. (g) 24–36 h Control Ad: 739.18±221.14 pg/ml. (h) 36–48 h Control Ad: 593.46±193.86 pg/ml.

Fig. 7

A–H. The fold change in IL-6 secretion levels (mean±S.E.M.) from control (L-Glc:5.6 mM) and explants and adipocytes that went through alternating treatments of L-Glc for 12 h, then L-Glc: 5.6 mM or H-Glc: 17.5 mM in combination with low (0.2 mM) and high (2 mM) doses of SFAs, as well as H-Glc alone, for a period of 48 h in total (n=6, p-values: P<.05⁎). (a) 0–12 h Control (mean±S.E.M.) Exp: 447.9±160.90 pg/ml. (b) 12–24 h Control (mean±S.E.M.) Exp: 240.22±53.85 pg/ml. (c) 24–36 h Control (mean±S.E.M.) Exp: 193.04±45.16 pg/ml. (d) 36–48 h Control (mean±S.E.M.) Exp: 479.24±247.40 pg/ml. (e) 0–12 h Control (mean±S.E.M.) Ad: 1863.7±95.90 pg/ml. (f) 12–24 h Control Ad: 860.60±272.93 pg/ml. (g) 24–36 h Control Ad: 739.18±221.14 pg/ml. (h) 36–48 h Control Ad: 593.46±193.86 pg/ml.