Transgenic restoration of long-chain n-3 fatty acids in insulin target tissues improves resolution capacity and alleviates obesity-linked inflammation and insulin resistance in high-fat-fed mice

Abstract

Objective: The catabasis of inflammation is an active process directed by n-3 derived pro-resolving lipid mediators. We aimed to determine whether high-fat (HF) diet-induced n-3 deficiency compromises the resolution capacity of obese mice and thereby contributes to obesity-linked inflammation and insulin resistance.

Research design and methods: We used transgenic expression of the fat-1 n-3 fatty acid desaturase from C. elegans to endogenously restore n-3 fatty acids in HF-fed mice. After 8 weeks on HF or chow diets, wild-type and fat-1 transgenic mice were subjected to insulin and glucose tolerance tests and a resolution assay was performed. Metabolic tissues were then harvested for biochemical analyses.

Results: We report that the n-3 docosanoid resolution mediator protectin D1 is lacking in muscle and adipose tissue of HF-fed wild-type mice. Accordingly, HF-fed wild-type mice have an impaired capacity to resolve an acute inflammatory response and display elevated adipose macrophage accrual and chemokine/cytokine expression. This is associated with insulin resistance and higher activation of iNOS and JNK in muscle and liver. These defects are reversed in HF-fed fat-1 mice, in which the biosynthesis of this important n-3 docosanoid resolution mediator is improved. Importantly, transgenic restoration of n-3 fatty acids prevented obesity-linked inflammation and insulin resistance in HF-fed mice without altering food intake, weight gain, or adiposity.

Conclusions: We conclude that inefficient biosynthesis of n-3 resolution mediators in muscle and adipose tissue contributes to the maintenance of chronic inflammation in obesity and that these novel lipids offer exciting potential for the treatment of insulin resistance and diabetes.

FIG. 1.

HF feeding reduces n-3 availability for resolution mediator synthesis. A: 8 weeks of HF feeding raised but fat-1 (F1) transgenesis restored the long-chain n-6:n-3 ratio in membrane phospholipids of muscle, liver, and epididymal adipose tissue. C, standard laboratory chow; AA, arachidonic acid (20:4 n-6); EPA, eicosapentaenoic acid (20:5 n-3); DPA, docosapentaenoic acid (22:5 n-3); DHA, docosahexaenoic acid (22:6 n-3). Data are mean ± SEM (n = 3). **P < 0.01 versus WTC; ***P < 0.001 versus WTC; †P < 0.05 versus WTHF; ††P < 0.01 versus WTHF. B: Comparison of n-3 docosanoid and eicosanoid biosynthetic pathways by LC-MS/MS in muscle, liver, and epididymal adipose tissue of HF-fed mice revealed that the docosanoid biosynthetic pathway has greater flux in metabolic tissues. Above left schematic diagram of docosanoid biosynthetic pathway showing the biosynthetic marker 17-HDoHE and PD1 (10R,17S-dihydroxydocosa-4Z,7Z,11E,13E,15Z,19Z-hexaenoic acid [28]) as well as the immediate PD1 precursor 17-HpDoHE. At right, the eicosanoid pathway showing 18-HEPE and RvE1 (5S,12R,18R-trihydroxy-6Z,8E,10E,14Z,16E-EPA [29]). ND indicates not detected. Data are mean ± SEM (n = 9–14). *P < 0.05, **P < 0.01, ***P < 0.001 versus 17-HDoHE. C: Representative LC-MS/MS spectra for 17-HDoHE, PD1, and 18-HEPE; retention times were 22.6, 18.2, and 20.6 min, respectively. D: Comparison of n-3 docosanoid and eicosanoid biosynthetic pathway activity by LC-MS/MS in muscle, liver, and epididymal adipose tissue of HF-fed wild-type and F1 mice reveals increased levels of docosanoid resolution mediator synthesis in muscle and adipose tissue of F1 mice compared with wild-type mice. Data are mean ± SEM (n = 6–10). *P < 0.05 versus WTHF. (A high-quality color representation of this figure is available in the online issue.)

FIG. 2.

Transgenic restoration of n-3 resolution mediators reestablishes resolution capacity and prevents adipose inflammation in HF-fed mice. A: Clearance of inflammatory PMN infiltrates in dorsal air pouches of HF-fed mice is impaired during the resolution phase of the in vivo resolution assay (n = 5–6). B: Percent infiltrate clearance 6.5 h after TNFα injection was reduced by ∼35% in obese HF-fed wild-type mice; HF-fed F1 transgenic mice displayed normal infiltrate clearance (n = 5–6). C: Representative image of F4/80 immunohistochemistry in epididymal adipose tissue show mass accumulation of macrophage in HF-fed wild-type mice that is prevented in HF-fed transgenic mice. MØ, macrophage. D: Percent F4/80+ cells in epididymal adipose tissue (n = 4–6). E: Macrophages present in adipose HF-fed wild-type mice formed multiple CLS; the formation of these inflammatory macrophage aggregates was greatly reduced in HF-fed F1 mice (number of CLS per 100 adipocytes). F–J: Chemokine and cytokine expression in epididymal adipose tissue was elevated by HF compared with chow feeding in wild-type mice, but these factors were not significantly raised by HF feeding in F1 mice (n = 5–10). All data are mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 versus WTC; †P < 0.05, ††P < 0.01, †††P < 0.001 versus WTHF.

FIG. 3.

Transgenic restoration of long-chain n-3 PUFA protects against obesity-linked insulin resistance and glucose intolerance. A: HF-diet–induced elevation of fasting plasma insulin was prevented by transgenic restoration of n-3 derived resolution mediators (n = 4–9). B: Glycemic excursion from 1.5 U/kg i.p. ITT was normalized in HF-fed F1 mice (n = 8–12). C: Glycemic excursion expressed as percent basal glycemia. D: Percent basal glycemia at T = 15 min after insulin injection. E: Glycemic excursion from 1g/kg i.p. GTT expressed as percent basal glycemia (n = 7–11), and (F) area under the curve from GTT show that HF-fed F1 mice are partially protected from glucose intolerance. G: HF-fed F1 mice develop similar obesity to wild-type mice. Weight gain (n = 16–20). H: Epididymal fat pad weight (n = 9–14). I: Liver weight (n = 9–14). J: Representative hematoxylin and eosin stained liver sections showing similar accumulation of fat vesicles in both WTHF and F1HF mice. All data are mean ± SEM, ND not detected, *P < 0.05, **P < 0.01, ***P < 0.001 versus respective chow-fed control; †P < 0.05 versus WTHF. (A high-quality color representation of this figure is available in the online issue.)

FIG. 4.

n-3 resolution mediators improve insulin signaling by blunting JNK and iNOS in muscle and liver. Transgenic restoration of n-3 resolution mediators improves insulin signaling to Akt in muscle and liver and blunts activation of JNK and iNOS in these tissues. A–E: Immunoblots for pAKTser473, total AKT, pJNKthr183/tyr185, and total JNK in gastrocnemius muscle and liver, and iNOS in muscle (n = 5–9). Quantification of densitometry analyses are shown below the representative gels. Lanes were run on the same gel but were noncontiguous. All data are mean ± SEM, ND not detected, *P < 0.05, **P < 0.01, ***P < 0.001 versus respective chow-fed control; †tP < 0.05 versus WTHF.