12/15-lipoxygenase is required for the early onset of high fat diet-induced adipose tissue inflammation and insulin resistance in mice - PubMed (original) (raw)

12/15-lipoxygenase is required for the early onset of high fat diet-induced adipose tissue inflammation and insulin resistance in mice

Dorothy D Sears et al. PLoS One. 2009.

Abstract

Background: Recent understanding that insulin resistance is an inflammatory condition necessitates searching for genes that regulate inflammation in insulin sensitive tissues. 12/15-lipoxygenase (12/15LO) regulates the expression of proinflammatory cytokines and chemokines and is implicated in the early development of diet-induced atherosclerosis. Thus, we tested the hypothesis that 12/15LO is involved in the onset of high fat diet (HFD)-induced insulin resistance.

Methodology/principal findings: Cells over-expressing 12/15LO secreted two potent chemokines, MCP-1 and osteopontin, implicated in the development of insulin resistance. We assessed adipose tissue inflammation and whole body insulin resistance in wild type (WT) and 12/15LO knockout (KO) mice after 2-4 weeks on HFD. In adipose tissue from WT mice, HFD resulted in recruitment of CD11b(+), F4/80(+) macrophages and elevated protein levels of the inflammatory markers IL-1beta, IL-6, IL-10, IL-12, IFNgamma, Cxcl1 and TNFalpha. Remarkably, adipose tissue from HFD-fed 12/15LO KO mice was not infiltrated by macrophages and did not display any increase in the inflammatory markers compared to adipose tissue from normal chow-fed mice. WT mice developed severe whole body (hepatic and skeletal muscle) insulin resistance after HFD, as measured by hyperinsulinemic euglycemic clamp. In contrast, 12/15LO KO mice exhibited no HFD-induced change in insulin-stimulated glucose disposal rate or hepatic glucose output during clamp studies. Insulin-stimulated Akt phosphorylation in muscle tissue from HFD-fed mice was significantly greater in 12/15LO KO mice than in WT mice.

Conclusions: These results demonstrate that 12/15LO mediates early stages of adipose tissue inflammation and whole body insulin resistance induced by high fat feeding.

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Conflict of interest statement

Competing Interests: Support for this research was provided, in part, by the University of California Discovery Program Project #bio03-10383 with matching grant funds provided by Pfizer, Inc (DDS, JMO). JC is an employee of Pfizer and participated as a collaborator in sample and data analysis for the project. The terms of this arrangement have been reviewed and approved by the University of California, San Diego in accordance with its conflict of interest policies. JC participated as described above, no other person or entity of Pfizer, Inc. had a role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. There are no other competing interests (as listed in the PLoS Competing Interests Policy) known to us.

Figures

Figure 1

Figure 1. Proinflammatory chemokine production by 15LO-expressing cells.

A. Fold difference in OPN and MCP-1 mRNA expression in murine fibroblasts constitutively expressing human 15LO or LacZ. OPN and MCP-1 mRNA levels were measured by qPCR of total RNA and were normalized to GAPDH mRNA. B. Levels of MCP-1 protein in conditioned media from 15LO and LacZ cells, as assayed by ELISA. C. Autoradiograph of OPN protein in conditioned media from 15LO and LacZ cells, as determined by western blotting.

Figure 2

Figure 2. FACS analysis of macrophage content in adipose tissue SVCs.

A. HFD-induced fold change in the percent of live adipose tissue SVCs expressing surface markers F4/80, CD11b, and both F4/80 and CD11b. SVCs were isolated from eWAT of mice fed NC, two-week HFD, or four-week HFD. *p<0.05 vs WT NC, ˆp<0.05 vs WT two-week HFD, $p<0.05 vs WT four-week HFD.

Figure 3

Figure 3. Adipose tissue cytokine levels.

Cytokine protein levels measured in eWAT lysates from WT (black bars) and 12/15LO KO (gray bars) mice fed NC or two-week HFD. Values are mean±standard error. 8–10 animals per group. *p<0.05 vs diet-matched WT, #p<0.05 vs strain-matched NC.

Figure 4

Figure 4. Characterization of whole body insulin sensitivity.

Hyperinsulinemic euglycemic clamp studies were conducted to calculate (A) Ginf, (B) GDR, (C) HGO, and (D) GDR normalized to fat pad mass as a percent of body weight in WT (black bars) and 12/15LO KO (gray bars) mice fed NC or two-week HFD. Values are mean±standard error. 7–9 animals per group. *p<0.05 vs WT HFD, #p<0.05 vs WT NC.

Figure 5

Figure 5. Basal and insulin-stimulated Akt phosphorylation in muscle of HFD-fed mice.

WT (black bars) and 12/15LO KO (gray bars) mice were fed HFD for two weeks. Mice were then injected intraperitoneally with 0.85 U/kg insulin or vehicle. After 15 min, mice were sacrificed and gastrocnemius muscles were rapidly isolated. Phospho-Ser473 Akt and total Akt levels in muscle lysates were measured by western blotting. A. Representative western blot images. B. Quantitation of all Akt western blot images. Phospho-Ser473 Akt/total Akt ratio values were normalized to the maximal signal observed. Bar graph values are mean±standard error. Insulin-induced fold changes in Akt phosphorylation are shown within the bars. 2–6 animals per group. *p<0.05.

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