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AMPK Activation by A-769662 Controls IL-6 Expression in Inflammatory Arthritis.

Guma M, Wang Y, Viollet B, Liu-Bryan R - PLoS ONE (2015)

Bottom Line: Many studies have suggested that AMPK activation exert significant anti-inflammatory and immunosuppressive effects.The effect of A-769662 on bone marrow derived macrophage (BMDM) response to stimulation with TLR2 and TLR4 agonists was tested in vitro.IL-6 expression at both mRNA and protein levels, phosphorylation of p65 NF-κB and MAPK phosphorylation were inhibited by A-769662 in BMDMs stimulated with either TLR2 or TLR4 agonists.

View Article: PubMed Central - PubMed

Affiliation: Division of Rheumatology, Allergy and Immunology, UC San Diego School of Medicine, La Jolla, California, United States of America.

ABSTRACT

Objective: AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase critically involved in the regulation of cellular energy homeostasis. It is a central regulator of both lipid and glucose metabolism. Many studies have suggested that AMPK activation exert significant anti-inflammatory and immunosuppressive effects. In this study, we assessed whether targeted activation of AMPK inhibits inflammatory arthritis in vivo.

Methods: We tested the effect of A-769662, a specific AMPK agonist (60mg/kg/bid) in mouse models of antigen-induced arthritis (AIA) and passive K/BxN serum-induced arthritis. The passive K/BxN serum-induced arthritis model was also applied to AMPKα1-deficient mice. Joints were harvested and subjected to histological analysis. IL-6 expression was measured in both joint tissues and sera by ELISA. The effect of A-769662 on bone marrow derived macrophage (BMDM) response to stimulation with TLR2 and TLR4 agonists was tested in vitro.

Results: AMPK activation by A-769662 reduced inflammatory infiltration and joint damage in both mouse models. IL-6 expression in serum and arthritic joints was significantly decreased in A-769662-treated mice. AMPKα1 deficient mice mildly elicited an increase of clinical arthritis. IL-6 expression at both mRNA and protein levels, phosphorylation of p65 NF-κB and MAPK phosphorylation were inhibited by A-769662 in BMDMs stimulated with either TLR2 or TLR4 agonists.

Conclusions: AMPK activation by specific AMPK agonist A-769662 suppressed inflammatory arthritis in mice as well as IL-6 expression in serum and arthritic joints. These data suggest that targeted activation of AMPK has a potential to be an effective therapeutic strategy for IL-6 dependent inflammatory arthritis.

No MeSH data available.


Related in: MedlinePlus

A-769662 treatment activated AMPK in joints and diminished clinical arthritis in passive K/BxN arthritis.WT mice were injected with 150μl serum from adult K/BxN mice on day 0 to induce arthritis. A-769662 was administrated twice a day from day 0. (A) Proteins of joint tissues from naive and arthritic mice injected with control vehicle (DMSO) and A-769662 (60mg/kg) were extracted and analyzed for phospho-AMPKα, phosphor-ACC, total AMPKα and total ACC by WB. D as DMSO and A as A-796662. Semi-quantitative densitometry analysis of Western blots (arbitrary densitometry units) for phosphorylation of AMPKα and ACC normalized to total AMPKα and ACC respectively is shown. (B) Clinical score at day 7 in mice treated with vehicle, A-769662 at high (60mg/kg) and low dose (30mg/kg) (n = 5 mice per group). (C) Ankle thickness in DMSO-treated animals (black circles, n = 9) and high dose A-769662-treated animals (black squares, n = 5) injected with 150 μl of K/BxN serum on day 0. Values are means ± SEM. * = p<0.05; ** = p<0.01.
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pone.0140452.g003: A-769662 treatment activated AMPK in joints and diminished clinical arthritis in passive K/BxN arthritis.WT mice were injected with 150μl serum from adult K/BxN mice on day 0 to induce arthritis. A-769662 was administrated twice a day from day 0. (A) Proteins of joint tissues from naive and arthritic mice injected with control vehicle (DMSO) and A-769662 (60mg/kg) were extracted and analyzed for phospho-AMPKα, phosphor-ACC, total AMPKα and total ACC by WB. D as DMSO and A as A-796662. Semi-quantitative densitometry analysis of Western blots (arbitrary densitometry units) for phosphorylation of AMPKα and ACC normalized to total AMPKα and ACC respectively is shown. (B) Clinical score at day 7 in mice treated with vehicle, A-769662 at high (60mg/kg) and low dose (30mg/kg) (n = 5 mice per group). (C) Ankle thickness in DMSO-treated animals (black circles, n = 9) and high dose A-769662-treated animals (black squares, n = 5) injected with 150 μl of K/BxN serum on day 0. Values are means ± SEM. * = p<0.05; ** = p<0.01.

Mentions: Next, we tested the effect of A-769662 on inflammatory arthritis in mice in vivo. We first used the K/BxN passive serum transfer model, which is dependent on innate but not adaptive immunity [13]. We observed that phosphorylation of AMPKα and acetyl-CoA carboxylase (ACC), an AMPK downstream target, in arthritic joint tissues decreased during the peak of inflammation at day 5 (Fig 3A and S2 Fig), but was increased in mice treated with A-769662 (Fig 3A). We used two different doses (30mg/kg/bid and 60mg/kg/bid) of A-769662. As shown in Fig 3B and 3C, higher dose of A-769662 significantly lowered clinical score at day 7, and ankle swelling from day 3. Histopathological analysis at day 7 showed reduced inflammatory cell infiltration, joint destruction and cartilage damage in A-769662-treated mice compared with vehicle-treated controls (Fig 4A and 4B). To evaluate the influence of A-769662 on synovial inflammatory mediators, we determined IL-6 and IL-1β production from these mice on day 5. Clinical score at day 5 was 12 ± 0.4 and 5.5 ± 1.3 for DMSO and A-769662-treated mice (0 = 0.002), respectively. Interestingly, amounts of IL-6, but not IL-1β, were significantly lower in A-769662-treated mice (Fig 4C), suggesting an important role of activation of AMPK in suppression of IL-6 expression.


AMPK Activation by A-769662 Controls IL-6 Expression in Inflammatory Arthritis.

Guma M, Wang Y, Viollet B, Liu-Bryan R - PLoS ONE (2015)

A-769662 treatment activated AMPK in joints and diminished clinical arthritis in passive K/BxN arthritis.WT mice were injected with 150μl serum from adult K/BxN mice on day 0 to induce arthritis. A-769662 was administrated twice a day from day 0. (A) Proteins of joint tissues from naive and arthritic mice injected with control vehicle (DMSO) and A-769662 (60mg/kg) were extracted and analyzed for phospho-AMPKα, phosphor-ACC, total AMPKα and total ACC by WB. D as DMSO and A as A-796662. Semi-quantitative densitometry analysis of Western blots (arbitrary densitometry units) for phosphorylation of AMPKα and ACC normalized to total AMPKα and ACC respectively is shown. (B) Clinical score at day 7 in mice treated with vehicle, A-769662 at high (60mg/kg) and low dose (30mg/kg) (n = 5 mice per group). (C) Ankle thickness in DMSO-treated animals (black circles, n = 9) and high dose A-769662-treated animals (black squares, n = 5) injected with 150 μl of K/BxN serum on day 0. Values are means ± SEM. * = p<0.05; ** = p<0.01.
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pone.0140452.g003: A-769662 treatment activated AMPK in joints and diminished clinical arthritis in passive K/BxN arthritis.WT mice were injected with 150μl serum from adult K/BxN mice on day 0 to induce arthritis. A-769662 was administrated twice a day from day 0. (A) Proteins of joint tissues from naive and arthritic mice injected with control vehicle (DMSO) and A-769662 (60mg/kg) were extracted and analyzed for phospho-AMPKα, phosphor-ACC, total AMPKα and total ACC by WB. D as DMSO and A as A-796662. Semi-quantitative densitometry analysis of Western blots (arbitrary densitometry units) for phosphorylation of AMPKα and ACC normalized to total AMPKα and ACC respectively is shown. (B) Clinical score at day 7 in mice treated with vehicle, A-769662 at high (60mg/kg) and low dose (30mg/kg) (n = 5 mice per group). (C) Ankle thickness in DMSO-treated animals (black circles, n = 9) and high dose A-769662-treated animals (black squares, n = 5) injected with 150 μl of K/BxN serum on day 0. Values are means ± SEM. * = p<0.05; ** = p<0.01.
Mentions: Next, we tested the effect of A-769662 on inflammatory arthritis in mice in vivo. We first used the K/BxN passive serum transfer model, which is dependent on innate but not adaptive immunity [13]. We observed that phosphorylation of AMPKα and acetyl-CoA carboxylase (ACC), an AMPK downstream target, in arthritic joint tissues decreased during the peak of inflammation at day 5 (Fig 3A and S2 Fig), but was increased in mice treated with A-769662 (Fig 3A). We used two different doses (30mg/kg/bid and 60mg/kg/bid) of A-769662. As shown in Fig 3B and 3C, higher dose of A-769662 significantly lowered clinical score at day 7, and ankle swelling from day 3. Histopathological analysis at day 7 showed reduced inflammatory cell infiltration, joint destruction and cartilage damage in A-769662-treated mice compared with vehicle-treated controls (Fig 4A and 4B). To evaluate the influence of A-769662 on synovial inflammatory mediators, we determined IL-6 and IL-1β production from these mice on day 5. Clinical score at day 5 was 12 ± 0.4 and 5.5 ± 1.3 for DMSO and A-769662-treated mice (0 = 0.002), respectively. Interestingly, amounts of IL-6, but not IL-1β, were significantly lower in A-769662-treated mice (Fig 4C), suggesting an important role of activation of AMPK in suppression of IL-6 expression.

Bottom Line: Many studies have suggested that AMPK activation exert significant anti-inflammatory and immunosuppressive effects.The effect of A-769662 on bone marrow derived macrophage (BMDM) response to stimulation with TLR2 and TLR4 agonists was tested in vitro.IL-6 expression at both mRNA and protein levels, phosphorylation of p65 NF-κB and MAPK phosphorylation were inhibited by A-769662 in BMDMs stimulated with either TLR2 or TLR4 agonists.

View Article: PubMed Central - PubMed

Affiliation: Division of Rheumatology, Allergy and Immunology, UC San Diego School of Medicine, La Jolla, California, United States of America.

ABSTRACT

Objective: AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase critically involved in the regulation of cellular energy homeostasis. It is a central regulator of both lipid and glucose metabolism. Many studies have suggested that AMPK activation exert significant anti-inflammatory and immunosuppressive effects. In this study, we assessed whether targeted activation of AMPK inhibits inflammatory arthritis in vivo.

Methods: We tested the effect of A-769662, a specific AMPK agonist (60mg/kg/bid) in mouse models of antigen-induced arthritis (AIA) and passive K/BxN serum-induced arthritis. The passive K/BxN serum-induced arthritis model was also applied to AMPKα1-deficient mice. Joints were harvested and subjected to histological analysis. IL-6 expression was measured in both joint tissues and sera by ELISA. The effect of A-769662 on bone marrow derived macrophage (BMDM) response to stimulation with TLR2 and TLR4 agonists was tested in vitro.

Results: AMPK activation by A-769662 reduced inflammatory infiltration and joint damage in both mouse models. IL-6 expression in serum and arthritic joints was significantly decreased in A-769662-treated mice. AMPKα1 deficient mice mildly elicited an increase of clinical arthritis. IL-6 expression at both mRNA and protein levels, phosphorylation of p65 NF-κB and MAPK phosphorylation were inhibited by A-769662 in BMDMs stimulated with either TLR2 or TLR4 agonists.

Conclusions: AMPK activation by specific AMPK agonist A-769662 suppressed inflammatory arthritis in mice as well as IL-6 expression in serum and arthritic joints. These data suggest that targeted activation of AMPK has a potential to be an effective therapeutic strategy for IL-6 dependent inflammatory arthritis.

No MeSH data available.


Related in: MedlinePlus