A sustained increase in plasma NEFA upregulates the Toll-like receptor network in human muscle

Abstract

Aims/hypothesis: Insulin-sensitive tissues (muscle, liver) of individuals with obesity and type 2 diabetes mellitus are in a state of low-grade inflammation, characterised by increased Toll-like receptor (TLR) expression and TLR-driven signalling. However, the cause of this mild inflammatory state is unclear. We tested the hypothesis that a prolonged mild increase in plasma NEFA will increase TLR expression and TLR-driven signalling (nuclear factor κB [NFκB] and mitogen-activated kinase [MAPK]) and impair insulin action in muscle of lean healthy individuals.

Methods: Twelve lean, normal-glucose-tolerant participants were randomised to receive a 48 h infusion (30 ml/h) of saline or Intralipid followed by a euglycaemic-hyperinsulinaemic clamp. Vastus lateralis muscle biopsies were performed before and during the clamp.

Results: Lipid infusion impaired insulin-stimulated IRS-1 tyrosine phosphorylation and reduced peripheral insulin sensitivity (p < 0.01). The elevation in circulating NEFA increased expression of TLR3, TLR4 and TLR5, and several MAPK (MAPK8, MAP4K4, MAP2K3) and inhibitor of κB kinase-NFκB (CHUK [IKKA], c-REL [REL] and p65 [RELA, NFKB3, p65]) signalling genes (p < 0.05). The lipid infusion also increased extracellular signal-regulated kinase (ERK) phosphorylation (p < 0.05) and tended to reduce the content of inhibitor of kappa Bα (p = 0.09). The muscle content of most diacylglycerol, ceramide and acylcarnitine species was unaffected. In summary, insulin resistance induced by prolonged low-dose lipid infusion occurs together with increased TLR-driven inflammatory signalling and impaired insulin-stimulated IRS-1 tyrosine phosphorylation.

Conclusions/interpretation: A sustained, mild elevation in plasma NEFA is sufficient to increase TLR expression and TLR-driven signalling (NFκB and MAPK) in lean individuals. The activation of this pathway by NEFA may be involved in the pathogenesis of insulin resistance in humans.

Trial registration: ClinicalTrials.gov NCT01740817.

Fig. 1

Effect of lipid infusion on insulin-stimulated glucose metabolism (M) (a) and HOMA-IR (b). Results are mean ± SE (n = 12). *p < 0.05 vs saline infusion

Fig. 2

Gene expression of TLRs (a), TLR adaptors, interactors and effectors (b) and genes involved in the NFκB, MAPK, IRF and cytokine-mediated signalling pathways (c) following saline (white bars) and lipid infusion (black bars). Only differentially expressed genes are shown. All genes measured are shown in ESM Table 1. Results are means ± SE (n = 10), fold change relative to saline infusion. *p < 0.05 and **p < 0.01 vs saline infusion

Fig. 3

Effect of lipid infusion on phospho-ERK (a), phospho-p38 (b), phospho-JNK (c) and IκBα (d). Data are expressed as the ratio of phosphorylated to total protein, where appropriate, in arbitrary units as fold change compared with saline infusion. Representative immunoblots are shown. Results are means ± SE (n = 12). *p < 0.05 vs saline infusion

Fig. 4

Effect of lipid infusion on total myocellular concentrations of ceramide and DAG (a) (n = 12) and of ceramide species (b), DAG species (c) (n = 7) and acylcarnitine species (d) (n=10). Results are means ± SE. *p < 0.05 and **p < 0.01 vs saline infusion

Fig. 5

Effect of lipid infusion on plasma TNF-α (a) , IL-6 (b) and fetuin-A concentrations (c) before (white bars) and after (black bars) saline and lipid infusion. Results are means ± SE (n = 12). **p < 0.01 vs pre-infusion

Fig. 6

Effect of lipid infusion on phospho-IRS-1^tyr612 (a), phospho-Akt (b), phospho-GSK3α (c), phospho-GSK3β (d) and phospho-AS160 (e). Data are expressed as a ratio of phosphorylated to total protein, in arbitrary units, as insulin-stimulated fold change over basal. Representative immunoblots are shown. Results are means ± SE (n = 9). *p < 0.05 vs saline infusion