A low dose lipid infusion is sufficient to induce insulin resistance and a pro-inflammatory response in human subjects

Abstract

Objective: The root cause behind the low-grade inflammatory state seen in insulin resistant (obesity and type 2 diabetes) states is unclear. Insulin resistant subjects have elevations in plasma free fatty acids (FFA), which are ligands for the pro-inflammatory toll-like receptor (TLR)4 pathway. We tested the hypothesis that an experimental elevation in plasma FFA (within physiological levels) in lean individuals would upregulate TLR4 and activate downstream pathways (e.g., MAPK) in circulating monocytes.

Research design and methods: Twelve lean, normal glucose-tolerant subjects received a low dose (30 ml/h) 48 h lipid or saline infusion on two different occasions. Monocyte TLR4 protein level, MAPK phosphorylation, and expression of genes in the TLR pathway were determined before and after each infusion.

Results: The lipid infusion significantly increased monocyte TLR4 protein and phosphorylation of JNK and p38 MAPK. Lipid-mediated increases in TLR4 and p38 phosphorylation directly correlated with reduced peripheral insulin sensitivity (M value). Lipid increased levels of multiple genes linked to inflammation, including several TLRs, CD180, MAP3K7, and CXCL10. Monocytes exposed in vivo to lipid infusion exhibited enhanced in vitro basal and LPS-stimulated IL-1β secretion.

Conclusions: In lean subjects, a small increase in plasma FFA (as seen in insulin resistant subjects) is sufficient to upregulate TLR4 and stimulate inflammatory pathways (MAPK) in monocytes. Moreover, lipids prime monocytes to endotoxin. We provide proof-of-concept data in humans indicating that the low-grade inflammatory state characteristic of obesity and type 2 diabetes could be caused (at least partially) by pro-inflammatory monocytes activated by excess lipids present in these individuals.

Fig 1. Effect of lipid infusion on TLR cell surface expression and intracellular MAPK phosphorylation in peripheral blood monocytes.

Cell surface protein levels of TLR4 (A), CD14 (B) and TLR2 (C) and basal (D) and LPS-stimulated (E) MAPK phosphorylation were determined by flow cytometry as described under ‘Materials and methods’. All values are the mean ± SEM of data obtained from 10–12 subjects. *P<0.05.

Fig 2. Effects of lipid infusion on expression of genes in the TLR signaling pathway in peripheral blood monocytes.

Expression of 84 genes central to TLR-mediated signal transduction was determined using a human TLR Signaling Pathway RT² Profiler PCR Array as described under ‘Materials and methods’. All values are mean ± SEM of data obtained from 12 subjects. *P<0.05 pre- vs. post-infusion within group; #P<0.05 post-saline vs. post-lipid.

Fig 3. Effects of lipid infusion on cytokine production in cultured monocytes.

Monocytes were isolated using a Dynabeads® Untouched™ Human Monocytes Kit. Two hundred thousand cells were either left untreated or treated with 1 μg/ml LPS for 18 h. Basal (A) and LPS-stimulated (B) cytokine production was determined by multiplex assay as described under ‘Materials and methods’. All values are mean ± SEM of data obtained from 12 subjects. *P<0.05.