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

Tateya S, Rizzo NO, Handa P, Cheng AM, Morgan-Stevenson V, Daum G, Clowes AW, Morton GJ, Schwartz MW, Kim F - Diabetes (2011)

Bottom Line: 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.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.Our findings also identify the NO/VASP pathway as a novel potential target for the treatment of obesity-associated liver insulin resistance.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Washington, Seattle, Washington, USA.

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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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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Figure 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.

Mentions: We next investigated whether VASP signaling is a determinant in hepatic responses to inflammatory stimuli in vitro. VASP was overexpressed in AML12 hepatocytes using retroviral transduction (Fig. 6A). Overexpression of VASP was associated with reduced palmitate-mediated increases in phospho–IκB-α levels (Fig. 6B) and restoration of insulin-mediated p-Akt signaling even in the presence of palmitate (Fig. 6C). Thus, increased VASP signaling in hepatocytes is sufficient to attenuate palmitate-dependent NF-κB activation. Next, primary hepatocytes were isolated from Vasp−/− and littermate control mice. The absence of Vasp was sufficient to increase phospho–IκB-α levels, even in the absence of palmitate (Fig. 6D), and the addition of palmitate did not further increase phospho–IκB-α levels in Vasp-deficient hepatocytes. These results suggest that the absence of Vasp is sufficient in reproducing the inflammatory effects of palmitate. In addition, the anti-inflammatory effects of DETA-NO or 8Br-cGMP were no longer apparent in Vasp-deficient hepatocytes (Fig. 6D).


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

Tateya S, Rizzo NO, Handa P, Cheng AM, Morgan-Stevenson V, Daum G, Clowes AW, Morton GJ, Schwartz MW, Kim F - Diabetes (2011)

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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Figure 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.
Mentions: We next investigated whether VASP signaling is a determinant in hepatic responses to inflammatory stimuli in vitro. VASP was overexpressed in AML12 hepatocytes using retroviral transduction (Fig. 6A). Overexpression of VASP was associated with reduced palmitate-mediated increases in phospho–IκB-α levels (Fig. 6B) and restoration of insulin-mediated p-Akt signaling even in the presence of palmitate (Fig. 6C). Thus, increased VASP signaling in hepatocytes is sufficient to attenuate palmitate-dependent NF-κB activation. Next, primary hepatocytes were isolated from Vasp−/− and littermate control mice. The absence of Vasp was sufficient to increase phospho–IκB-α levels, even in the absence of palmitate (Fig. 6D), and the addition of palmitate did not further increase phospho–IκB-α levels in Vasp-deficient hepatocytes. These results suggest that the absence of Vasp is sufficient in reproducing the inflammatory effects of palmitate. In addition, the anti-inflammatory effects of DETA-NO or 8Br-cGMP were no longer apparent in Vasp-deficient hepatocytes (Fig. 6D).

Bottom Line: 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.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.Our findings also identify the NO/VASP pathway as a novel potential target for the treatment of obesity-associated liver insulin resistance.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Washington, Seattle, Washington, USA.

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.

Show MeSH
Related in: MedlinePlus