ETC-1002 regulates immune response, leukocyte homing, and adipose tissue inflammation via LKB1-dependent activation of macrophage AMPK

Abstract

ETC-1002 is an investigational drug currently in Phase 2 development for treatment of dyslipidemia and other cardiometabolic risk factors. In dyslipidemic subjects, ETC-1002 not only reduces plasma LDL cholesterol but also significantly attenuates levels of hsCRP, a clinical biomarker of inflammation. Anti-inflammatory properties of ETC-1002 were further investigated in primary human monocyte-derived macrophages and in in vivo models of inflammation. In cells treated with ETC-1002, increased levels of AMP-activated protein kinase (AMPK) phosphorylation coincided with reduced activity of MAP kinases and decreased production of proinflammatory cytokines and chemokines. AMPK phosphorylation and inhibitory effects of ETC-1002 on soluble mediators of inflammation were significantly abrogated by siRNA-mediated silencing of macrophage liver kinase B1 (LKB1), indicating that ETC-1002 activates AMPK and exerts its anti-inflammatory effects via an LKB1-dependent mechanism. In vivo, ETC-1002 suppressed thioglycollate-induced homing of leukocytes into mouse peritoneal cavity. Similarly, in a mouse model of diet-induced obesity, ETC-1002 restored adipose AMPK activity, reduced JNK phosphorylation, and diminished expression of macrophage-specific marker 4F/80. These data were consistent with decreased epididymal fat-pad mass and interleukin (IL)-6 release by inflamed adipose tissue. Thus, ETC-1002 may provide further clinical benefits for patients with cardiometabolic risk factors by reducing systemic inflammation linked to insulin resistance and vascular complications of metabolic syndrome.

Keywords: AMP-activated protein kinase; adipose tissue; cardiometabolic risk factors; cytokines; drug therapy; hypolipidemic drugs; liver kinase B1; macrophages/monocytes; mitogen-activated protein kinases.

Figures

Fig. 1.

ETC-1002 activates AMPK in primary human MDMs. (A) Primary rat hepatocytes and differentiated human MDMs were treated with indicated concentrations of ETC-1002 for 12 h, and then ETC-1002-CoA thioester formation was assessed in cell extracts by HPLC-UV analysis. (B) Primary human MDMs were differentiated in autologous serum for five days, followed by stimulation with 100 ng/ml of LPS in either the absence or presence of ETC-1002 (n = 4 for each experimental condition). Proteins in cell lysates were separated on SDS-PAGE, transferred to PVDF membrane, and blotted with antibodies raised against phosphorylated AMPKα (T172) and ACC (S79) as well as total AMPK and ACC. Density of AMPK- and ACC-specific immunoreactive bands was captured and quantitated with a Kodak 4000MM Image Station. Data are presented as ratio of phosphorylated to total proteins (mean ± SEM). Comparisons between groups were performed by one-way ANOVA and Bonferroni's post hoc multiple comparison tests, *P < 0.05. BDL, below detection limit.

Fig. 2.

ETC-1002 attenuates expression of acute-phase cytokines by LPS-stimulated human MDMs. Macrophages differentiated in autologous serum for five days were stimulated with 100 ng/ml of LPS in either the absence or presence of different concentrations of ETC-1002. Levels of proinflammatory cytokines in the media conditioned by MDMs for 12 h were determined with cytokine arrays. Net signal intensity for each analyte was expressed as percentage of the internal reference standard for each individual array membrane (mean ± SEM). Comparisons between groups were performed by one-way ANOVA and Bonferroni's post hoc multiple comparison tests, *P < 0.05.

Fig. 3.

ETC-1002 inhibits LPS-mediated release of CC- and CXC- chemokine family members by human MDMs. Macrophages differentiated in autologous serum for 5 days were stimulated with 100 ng/ml of LPS in the either absence or presence of different concentrations of ETC-1002. Release of CC- and CXC-chemokine family members in the media conditioned by MDMs for 12 h was measured with cytokine arrays. Net signal intensity for each analyte was expressed as percentage of the internal reference standard for each individual array membrane (mean ± SEM). Comparisons between groups were performed by one-way ANOVA and Bonferroni's post hoc multiple comparison tests, *P < 0.05.

Fig. 4.

ETC-1002 regulates the expression of adhesion molecules and mediators of vascular inflammation by activated MDMs. Macrophages differentiated in autologous serum for five days were stimulated with 100 ng/ml of LPS in either the absence or presence of different concentrations of ETC-1002. Expression of the adhesion molecules and mediators of vascular inflammation in the media conditioned by MDMs for 12 h were determined with (A) cytokine and (B) matrix metalloproteinase arrays. Net signal intensity for each analyte was expressed as percentage of the internal reference standard for each individual array membrane (mean ± SEM). Comparisons between groups were performed by one-way ANOVA and Bonferroni's post hoc multiple comparison tests, *P < 0.05.

Fig. 5.

ETC-1002-mediated modulation of AMPK and MAPK signaling pathways. MDMs were differentiated in autologous serum for five days, and ETC-1002 at various concentrations was added to the media 1 h prior to LPS stimulation (100 ng/ml). Cell lysates were collected 3 h after LPS challenge (n = 3 for each experimental condition). (A) Proteins in cell lysates were separated on SDS-PAGE, transferred to PVDF membrane blotted with antibodies specific for phosphorylated and total AMPK, ACC, p38, and JNK1/2. (B) Phosphorylation status of AMPK, ACC, p38, and JNK1/2 in MDM cell lysates was quantitated by densitometry of the respective immunoreactive bands. Data are presented as ratio of phosphorylated to total proteins (mean ± SEM). Comparisons between groups were performed by one-way ANOVA and Bonferroni's post hoc multiple comparison tests, *P < 0.05.

Fig. 6.

ETC-1002 activates macrophage AMPK and inhibits immune response via LKB1-dependent mechanism. Primary human MDMs differentiated for five days in autologous serum were transfected with negative control “mock” or LKB1 siRNA for 5 h followed by 24 h recovery period. Unstimulated macrophages or cells induced with 100 ng/ml of LPS cells were then treated with vehicle or 100 µM ETC-1002. Proteins in cell lysates were separated on SDS-PAGE, transferred to PVDF membrane, and blotted with (A) anti-LKB1 and anti-β-Actin antibodies or (B) antibodies specific for phosphorylated and total AMPK. (C) Media conditioned by macrophages was collected, and levels of IL-6 and CCL2/MCP-1were determined by ELISA. LKB1 expression and phosphorylation status of AMPK in MDM cell lysates were quantitated by densitometry of the respective immunoreactive bands. For LKB1, data are presented as ratio of total LKB1 to total β-actin (mean ± SEM). For AMPK, data are presented as ratio of phosphorylated to total AMPK protein. Levels of IL-6 and CCL2/MCP-1 in macrophage-conditioned media are expressed as picograms per milliliter (mean ± SEM). Comparisons between groups performed by two-tailed nonpaired Student t-test, *P < 0.05, or by one-way ANOVA and Bonferroni's post hoc multiple comparison tests, *P < 0.05.

Fig. 7.

ETC-1002 inhibits homing of leukocytes into inflammatory site in vivo. C57BL/6 male mice were injected intraperitoneally with 1 ml of 4% sterile thioglycollate, followed by administration of either vehicle or 30 mg/kg of ETC-1002 via oral gavage. (A) Neutrophil and macrophage transmigration into peritoneal cavity was evaluated 24 h and 72 h after intraperitoneal thioglycollate challenge, respectfully. (B) Expression of matrix metalloproteinase in the media conditioned by MDMs for 12 h was determined with MMP array. Cell migration into peritoneal cavity was estimated by number of leukocytes in the peritoneal lavage fluid and expressed as mean ± SEM. Group comparison performed with nonpaired two-tailed Student t-test, *P < 0.05. For MMP array, net signal intensity for each analyte was expressed as percentage of the internal reference standard for each individual array membrane (mean + SEM). Comparisons between groups were performed by one-way ANOVA and Bonferroni's post hoc multiple comparison tests, *P < 0.05.

Fig. 8.

ETC-1002 reduces epididymal fat-pad mass and adipose tissue inflammation in a mouse model of diet-induced obesity. C57BL/6 mice were fed with chow (ChD) or 60% high-fat diet (HFD) for nine weeks. HFD groups (n = 10/group) were administered with vehicle alone or ETC-1002 at 30 mg/kg/day via oral gavage. (A) Macrograph of epididymal fat pads from the ChD- and HFD-fed animals administered with either vehicle (HFD vehicle) or 30 mg/kg of ETC-1002 (HFD ETC-1002). Left graph: Mass of epididymal fat pads obtained from the mice in different experimental groups. Right graph: IL-6 release into the media conditioned by adipose tissue explant ex vivo. Fragments of epididymal fat pads were maintained ex vivo in serum-free RPMI, and IL-6 levels were measured in a media conditioned by tissue explants for 12 h. (B) Phosphorylation of AMPK, p38, and JNK1/2 and expression levels of F4/80 in adipose tissue lysates were quantitated by densitometry of the respective immunoreactive bands. For AMPK, p38, and JNK1/2, data are presented as ratio of phosphorylated to total proteins (mean ± SEM). For F4/80 expression, data are normalized to β-actin (mean ± SEM). Comparisons between groups were performed by one-way ANOVA and Bonferroni's post hoc multiple comparison test, *P < 0.05. Data are expressed as mean ± SEM.

Fig. 9.

Mechanism of anti-inflammatory effects of ETC-1002 and its therapeutic implications. In primary human MDMs, activation of AMPK by ETC-1002 offsets the LPS-induced immune response. Upregulation of AMPK (blue) and inhibition of MAPK (red) activities attenuate release of proinflammatory cytokines, chemokines and adhesion molecules by stimulated MDMs. Mitigated proinflammatory signaling networks prevent leukocyte homing and activation in subendothelial space or visceral adipose tissues. By quenching inflammation in target organs, ETC-1002 may offer an additional clinical benefit for patients with cardiometabolic risk factors, potentially improving insulin sensitivity and reducing CAD. ACS, acute coronary syndrome; IR, insulin resistance.