Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Nov;18(11):1665-72.
doi: 10.1038/nm.2962. Epub 2012 Oct 28.

Immunomodulatory Glycan LNFPIII Alleviates Hepatosteatosis and Insulin Resistance Through Direct and Indirect Control of Metabolic Pathways

Affiliations
Free PMC article

Immunomodulatory Glycan LNFPIII Alleviates Hepatosteatosis and Insulin Resistance Through Direct and Indirect Control of Metabolic Pathways

Prerna Bhargava et al. Nat Med. .
Free PMC article

Abstract

Parasitic worms express host-like glycans to attenuate the immune response of human hosts. The therapeutic potential of this immunomodulatory mechanism in controlling the metabolic dysfunction that is associated with chronic inflammation remains unexplored. We demonstrate here that administration of lacto-N-fucopentaose III (LNFPIII), a Lewis(X)-containing immunomodulatory glycan found in human milk and on parasitic helminths, improves glucose tolerance and insulin sensitivity in diet-induced obese mice. This effect is mediated partly through increased interleukin-10 (Il-10) production by LNFPIII-activated macrophages and dendritic cells, which reduces white adipose tissue inflammation and sensitizes the insulin response of adipocytes. Concurrently, LNFPIII treatment upregulates nuclear receptor subfamily 1, group H, member 4 (Fxr-α, also known as Nr1h4) to suppress lipogenesis in the liver, conferring protection against hepatosteatosis. At the signaling level, the extracellular signal-regulated kinase (Erk)-activator protein 1 (Ap1) pathway seems to mediate the effects of LNFPIII on both inflammatory and metabolic pathways. Our results suggest that LNFPIII may provide new therapeutic approaches to treat metabolic diseases.

Conflict of interest statement

COMPETING FINANCIAL INTERESTS

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1
LNFPIII increases Il-10 production and improves insulin sensitivity. (a) Real-time q-PCR examining the expression of M2 (alternative activation) genes in bone marrow derived macrophages treated with Il-4 (20 ng ml−1) or LNFPIII (20 μg ml−1) for 24 hr. (b) Il-10 concentrations in conditioned medium from LNFPIII or vehicle treated macrophages determined by ELISA. PD98059 (10 μM): Erk inhibitor. (c) Serum Il-10 and Il-4 concentrations in vehicle or LNFPIII treated mice (n = 5–7 per treatment) (d and e) Glucose tolerance test (GTT, d) and insulin tolerance test (ITT, e) in vehicle and LNFPIII treated mice. Inset in (e): homeostasis model of assessment-insulin resistance (HOMA-IR). (f) Western blot analyses to examine insulin stimulated Akt phosphorylation in WAT from vehicle and LNFPIII treated mice (from four individual mice per treatment). p-Akt: phospho-Akt; t-Akt: total Akt. (g) The relative level of p-Akt to t-Akt in WAT ±insulin injection quantified by densitometry based on Western signals in (f) or by ELISA-based assays. Values are expressed as means ± SEM. For in vitro assays, the mean and SEM were determined from 3–4 biological replicates for a representative experiment. Experiments were repeated 3 times. In vivo studies were reproduced in three mouse cohorts (n = 5–7 per treatment). Insulin signaling was examined in two of the three cohorts. *P < 0.05 (LNFPIII or Il-4 versus vehicle control).
Figure 2
Figure 2
Reduced inflammation and enhanced insulin signaling in WAT of LNFPIII treated mice. (a) Left panel: WAT histology showing crown-like structures (CLS, indicated with arrows). Scale bar: left images = 400 μm; right images = 100 μm. Right panel: CLSs quantified in 90 fields from 30 sections (3 fields per section) for each individual animal (n = 4 per group). Y-axis: % fields that contains certain percentage of CLSs; X-axis: % CLS-containing adipocytes in a given field; n.d: not detected. (b) Real-time q-PCR analyses of M1 and M2 gene expression in WAT of vehicle- and LNFPIII-treated mice (n = 5 per treatment). (c) Metabolic gene expression in WAT determined by real-time q-PCR. (d) Western blotting showing C/ebp-α and Irs2 protein levels in WAT. Bottom panel: relative C/ebp-α and Irs2 levels normalized to tubulin. Values are expressed as means ±SEM. Metabolic studies were reproduced in three mouse cohorts (n = 5–7 per treatment). Crown-like structures and expression analyses were examined in one and three of the three cohorts, respectively. *P < 0.05 (LNFPIII versus vehicle).
Figure 3
Figure 3
LNFPIII primed macrophage conditioned medium improves insulin sensitivity in 3T3L1 adipocytes in an Il-10 dependent manner. (a) Top panel: Western blotting showing protein levels of total Akt (t-Akt) and insulin stimulated Akt phosphorylation (p-Akt) in 3T3-L1 adipocytes treated with vehicle, LNFPIII or rIl-10 (representative samples from three experiments each with three biological replicates). Bottom left panel: insulin stimulated glucose uptake determined using radioactive 2-[H3]deoxy-D-glucose. Bottom right panel: cellular triglyceride contents measured at day 6 of 3T3-L1 adipocyte differentiation. Vehicle controls for LNFPIII (20 μg ml−1) and Il-10 (10 ng ml−1) were dextran and PBS, respectively. (b) Top panel: Western blotting showing protein levels of t-Akt and insulin stimulated p-Akt in adipocytes treated with control and conditioned medium (CM) from LNFPIII primed wt and Il-10−/− macrophages. Bottom panel: insulin stimulated glucose uptake in adipocytes. (c) Gene expression in 3T3-L1 adipocytes determined by real-time q-PCR. (d) Insulin tolerance test in vehicle and LNFPIII treated Il-10−/− mice (n = 6 per treatment per genotype). AUC: area under the curve of ITT. (e) Ex vivo glucose uptake assay performed in adipose tissue slices collected before and after portal vein injection of 5 U kg−1 insulin. Values are expressed as means ±SEM. For in vitro assays, the mean and SEM were determined from 3–4 biological replicates for a representative experiment. Experiments were repeated three times. Studies in Il-10−/− and control mice were conducted in one cohort (n = 6). *P < 0.05 (treatment versus control).
Figure 4
Figure 4
LNFPIII protects against high fat diet induced hepatic steatosis. (a) Liver histology and triglyceride content analyses to determine hepatic fat accumulation in vehicle and LNFPIII treated mice. Scale bar = 100 μm. (b) Circulating AST and ALT concentrations to assess liver function. (c) Gene expression analyses in livers from vehicle or LNFPIII treated mice (n = 5) by real-time q-PCR. Values are expressed as means ±SEM. Metabolic studies were reproduced in three mouse cohorts (n = 5–7 per treatment). Histology was examined in one and lipid and expression analyses were examined in three of the three cohorts. *P < 0.05 (LNFPIII versus vehicle control).
Figure 5
Figure 5
LNFPIII suppresses lipid synthesis through Fxr-α. (a) De novo lipogenesis (left panel) and fatty acid β-oxidation (right panel) assays in primary hepatocytes treated with vehicle, LNFPIII (20 μg ml−1) or CM from LNFPIII primed wt macrophages. (b) Gene expression in hepatocytes treated with vehicle or LNFPIII determined by real-time q-PCR. (c) Lipogenic (left panel) and β-oxidation (right panel) assays in hepatocytes isolated from wild type (WT) or Fxr-α−/− mice ±LNFPIII ±PD98059. (d and e) Hepatic triglyceride content and serum AST and ALT concentrations in WT and Fxr-α−/− mice ±LNFPIII treatment (n = 6). Values are expressed as means ±SEM. For in vitro assays, the mean and SEM were determined from 3–4 biological replicates for a representative experiment. Experiments were repeated 3 times. Studies for Fxr-α−/− and control mice were conducted in one cohort (n = 6 per treatment per genotype). *P < 0.05 (LNFPIII versus vehicle control).
Figure 6
Figure 6
Induction of Fxr-α activity by LNFPIII is mediated by Erk-Ap1 signaling. (a) Top panel: genomic structure showing alternative promoter usage by human FXR-α1/α2 (promoter 1) and FXR-α3/α4 (promoter 2). Bottom left: Relative activities (RLU) of luciferase reporters driven by human FXR-αpromoter 1 (~2 kb) or human FXR-αpromoter 2 (~0.13 kb) in HepG2 cells ±LNFPIII (20 μg ml−1) or SEA (2 μg ml−1). Bottom right: Western blotting showing Fxr-α protein levels in livers from vehicle or LNFPIII treated mice. (b) Top panel: diagram demonstrating FXR-α promoter 2 (p2–0.13kb) or mutant (p2–0.13kb–M) reporter constructs. The two overlapping AP1 binding sites and the mutation are shown. Bottom panel: Relative luciferase activities of p2–0.13kb and p2–0.13kb–M ± LNFPIII ± PD98059 (c) Erk phosphorylation (p-Erk) in livers of vehicle and LNFPIII treated mice. Bottom panel: Quantification of the Western signal. (d) Western blot analyses showing Erk phosphorylation in hepatocytes ± LNFPIII±PD98059 (representative samples from three experiments each with three biological replicates). Bottom panel: Quantification of the Western signal. Values are expressed as means ± SEM. For in vitro assays, the mean and SEM were determined from 3–4 biological replicates from one of three repeats. Hepatic p-Erk was determined from one of three metabolic study cohorts (n = 5, showing 3 representative samples). *P < 0.05 (LNFPIII or SEA versus vehicle control). (e) Model for direct and indirect regulation of metabolic pathways by LNFPIII. DC: dendritic cells.

Similar articles

See all similar articles

Cited by 35 articles

See all "Cited by" articles

References

    1. Gregor MF, Hotamisligil GS. Inflammatory mechanisms in obesity. Annu Rev Immunol. 29:415–445. - PubMed
    1. Arkan MC, et al. IKK-beta links inflammation to obesity-induced insulin resistance. Nat Med. 2005;11:191–198. - PubMed
    1. Cai D, et al. Local and systemic insulin resistance resulting from hepatic activation of IKK-beta and NF-kappaB. Nat Med. 2005;11:183–190. - PMC - PubMed
    1. Chiang SH, et al. The protein kinase IKKepsilon regulates energy balance in obese mice. Cell. 2009;138:961–975. - PMC - PubMed
    1. Nakamura T, et al. Double-stranded RNA-dependent protein kinase links pathogen sensing with stress and metabolic homeostasis. Cell. 2010;140:338–348. - PMC - PubMed

Publication types

MeSH terms

Feedback