Endothelial NO/cGMP/VASP signaling attenuates Kupffer cell activation and hepatic insulin resistance induced by high-fat feeding - PubMed (original) (raw)

. 2011 Nov;60(11):2792-801.

doi: 10.2337/db11-0255. Epub 2011 Sep 12.

Affiliations

Endothelial NO/cGMP/VASP signaling attenuates Kupffer cell activation and hepatic insulin resistance induced by high-fat feeding

Sanshiro Tateya et al. Diabetes. 2011 Nov.

Abstract

Objective: Proinflammatory activation of Kupffer cells is implicated in the effect of high-fat feeding to cause liver insulin resistance. We sought to determine whether reduced endothelial nitric oxide (NO) signaling contributes to the effect of high-fat feeding to increase hepatic inflammatory signaling and if so, whether this effect 1) involves activation of Kupffer cells and 2) is ameliorated by increased NO signaling.

Research design and methods: Effect of NO/cGMP signaling on hepatic inflammation and on isolated Kupffer cells was examined in C57BL/6 mice, eNos(-/-) mice, and Vasp(-/-) mice fed a low-fat or high-fat diet.

Results: We show that high-fat feeding induces proinflammatory activation of Kupffer cells in wild-type mice coincident with reduced liver endothelial nitric oxide synthase activity and NO content while, conversely, enhancement of signaling downstream of endogenous NO by phosphodiesterase-5 inhibition protects against high fat-induced inflammation in Kupffer cells. Furthermore, proinflammatory activation of Kupffer cells is evident in eNos(-/-) mice even on a low-fat diet. Targeted deletion of vasodilator-stimulated phosphoprotein (VASP), a key downstream target of endothelially derived NO, similarly predisposes to hepatic and Kupffer cell inflammation and abrogates the protective effect of NO signaling in both macrophages and hepatocytes studied in a cell culture model.

Conclusions: These results collectively imply a physiological role for endothelial NO to limit obesity-associated inflammation and insulin resistance in hepatocytes and support a model in which Kupffer cell activation during high-fat feeding is dependent on reduced NO signaling. Our findings also identify the NO/VASP pathway as a novel potential target for the treatment of obesity-associated liver insulin resistance.

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Figures

FIG. 1.

FIG. 1.

The effect of high-fat (HF) feeding on liver NO, hepatic inflammation, and Kupffer cell activation. Wild-type (WT) mice were fed either a low-fat (LF) or HF diet for 2, 4, or 8 weeks. A: Hepatic NO content during HF and LF feeding as measured by electron spin resonance spectroscopy (n = 9). B: Liver eNOS phosphorylation of Ser 1177 as measured by Western blot (n = 9). C: Liver phospho–IκB-α as measured by Western blot, normalized to total GAPDH levels (n = 9). D: Liver IL-6. E: TNF-α mRNA (n = 9). F: ICAM mRNA levels (n = 9). G: Relative mRNA levels from isolated Kupffer cells as measured by RT-PCR after 2, 4, and 8 weeks of LF or HF diet (n = 5 mice). H: In parallel experiments in WT mice, hepatic insulin signaling was assessed following intraperitoneal injection of insulin or saline vehicle (n = 5 per group). Liver protein lysates were analyzed for IRS-1 tyrosine phosphorylation (_p_–IRS-1), IRS-1, pAkt, and Akt levels by enzyme-linked immunosorbent assay. *P < 0.05.

FIG. 2.

FIG. 2.

Hepatic inflammation and insulin resistance in eNos−/− mice. eNos−/− mice and littermate control mice were fed a low-fat (LF) or a high-fat (HF) diet for 4 weeks from age 8 weeks. A and B: Liver lysates were analyzed for IκB-α phosphorylation by Western blot and IL-6 mRNA expression by RT-PCR (n = 7). *P < 0.05. C: Inflammatory markers from isolated Kupffer cells by RT-PCR (n = 5). *P < 0.05. D: Hepatic insulin (Ins) signaling at the level of Akt phosphorylation (n = 5). *P < 0.05. E: Hepatic triglyceride (TG) content (n = 7). *P < 0.05. Veh, vehicle; WT, wild type. IB, immunoblot; kD, kilodalton.

FIG. 3.

FIG. 3.

Effect of daily sildenafil during high-fat (HF) feeding on liver NF-κB and hepatic insulin signaling. Six-week-old male C57BL/6 mice were maintained on either a low-fat (LF; 10% saturated fat) or HF (60% saturated fat) diet for 8 weeks, and for the last 2 weeks of the diet, study mice received 30 mg/kg per day oral sildenafil (n = 8) or vehicle (n = 8). A: IκB-α phosphorylation in liver lysates. B: IL-6 mRNA levels. C: Inflammatory markers from isolated Kupffer cells by quantitative RT-PCR (n = 5). *P < 0.05. D: Hepatic insulin (Ins) signaling at the level of Akt phosphorylation (n = 5). *P < 0.05. E: Hepatic triglyceride (TG) content (n = 5). *P < 0.05. ctl, control; veh, vehicle. IB, immunoblot; kD, kilodalton.

FIG. 4.

FIG. 4.

Effect of VASP deficiency on hepatic and Kupffer cell inflammation and hepatic insulin signaling. A: Hepatic PKG expression and phosphorylation of VASP Ser 239 from wild-type (WT) mice fed either a low-fat (LF) or high-fat (HF) diet for 4 weeks from age 8 weeks (n = 5). B_–_E: Vasp−/− mice and littermate control mice were fed an LF diet for 4 weeks. B: Body weight (BW) (g). C: Body composition (%). Liver lysates were analyzed for IκB-α phosphorylation by Western blot (D), and IL-6 mRNA expression was measured by RT-PCR (E) (n = 8). *P < 0.05. F: Kupffer cells were isolated from Vasp−/− mice or WT control mice on an LF diet for 4 weeks. Inflammatory markers as measured by RT-PCR (n = 5). *P < 0.05. G and H: In parallel experiments in Vasp−/− (n = 7) and littermate control (n = 9) mice, hepatic insulin (Ins) signaling was assessed following intraperitoneal injection of insulin. IRS-1 and IRS-2 tyrosine phosphorylation and Akt serine phosphorylation. *P < 0.05. I: Hepatic triglyceride (TG) from Vasp−/− and control (n = 5). *P < 0.05. Veh, vehicle. IB, immunoblot; kD, kilodalton.

FIG. 5.

FIG. 5.

The effect of VASP signaling on RAW cell inflammatory responses to LPS. RAW cells were transduced with VASP (VASP-OE) or control (Con) vector. A: VASP Western blot. B: RAW cells were stimulated with LPS (5 ng/mL) and IFN-γ (12 ng/mL) for 4 h and then analyzed for TNF-α, IL-6, and iNOS mRNA expression (n = 3). *P < 0.05. C: Peritoneal macrophages from VASP−/− and wild-type (WT) control mice were stimulated with LPS (5 ng/mL) and IFN-γ (12 ng/mL) for 4 h in the presence or absence of DETA-NO (10 μmol/L for 4 h) or 8Br-cGMP (10 μmol/L for 4 h). *P < 0.05. IB, immunoblot; kD, kilodalton.

FIG. 6.

FIG. 6.

The effect of VASP signaling on hepatocyte inflammatory responses to palmitate. AML12 hepatocytes were transduced with VASP (VASP-OE) or control (Con) vector. A: VASP Western blot. B: Hepatocytes were treated with 100 μmol/L palmitate (Pal) or vehicle for 4 h, and a representative IκB-α phosphorylation Western blot from one of three independent experiments is shown. C: Insulin (Ins)-mediated pAkt signaling (10 nmol/L insulin for 15 min) in transduced hepatocytes, following 4 h palmitate (100 μmol/L). Ser 473 Akt phosphorylation was assessed by Western blot analysis, and fold increase over control condition was calculated (n = 3). *P < 0.05. D: Primary hepatocytes were isolated and cultured from Vasp−/− mice or wild-type (WT) control mice. Isolated hepatocytes were treated with palmitate (100 μmol/L) for 4 h following 4-h pretreatment with 10 μmol/L DETA-NO or 10 μmol/L 8Br-cGMP. IκB-α phosphorylation was assessed by Western blot analysis. *P < 0.05. E: Insulin-mediated pAkt (10 nmol/L for 15 min) following treatment with palmitate (100 μmol/L for 4 h), DETA-NO (10 μmol/L for 4 h), or 8BrcGMP (10 μmol/L for 4 h). Akt Ser 473 phosphorylation assessed by Western blot (n = 3). *P < 0.05. IB, immunoblot; kD, kilodalton.

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