MLK3 promotes metabolic dysfunction induced by saturated fatty acid-enriched diet

Abstract

Saturated fatty acids activate the c-Jun NH₂-terminal kinase (JNK) pathway, resulting in chronic low-grade inflammation and the development of insulin resistance. Mixed-lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase (MAP3K) that mediates JNK activation in response to saturated fatty acids in vitro; however, the exact mechanism for diet-induced JNK activation in vivo is not known. Here, we have used MLK3-deficient mice to examine the role of MLK3 in a saturated-fat diet model of obesity. MLK3-KO mice fed a high-fat diet enriched in medium-chain saturated fatty acids for 16 wk had decreased body fat compared with wild-type (WT) mice due to increased energy expenditure independently of food consumption and physical activity. Moreover, MLK3 deficiency attenuated palmitate-induced JNK activation and M1 polarization in bone marrow-derived macrophages in vitro, and obesity induced JNK activation, macrophage infiltration into adipose tissue, and expression of proinflammatory cytokines in vivo. In addition, loss of MLK3 improved insulin resistance and decreased hepatic steatosis. Together, these data demonstrate that MLK3 promotes saturated fatty acid-induced JNK activation in vivo and diet-induced metabolic dysfunction.

Keywords: inflammation; insulin resistance; mixed-lineage kinase 3; saturated fatty acids.

Fig. 1.

Loss of mixed-lineage kinase 3 (MLK3) reduces diet-induced body weight gain and adiposity. A: wild-type (WT) and MLK3-knockout (KO) mice were maintained on chow (CD) or high-fat diet (HFD) for 16 wk. Body weight was measured monthly. B: after 16 wk on diet, lean and fat mass were determined by MRI. Data are means ± SE; n = 8. *P < 0.05. C–F: CD and HFD mice were monitored for 3 days to measure food consumption (C), physical activity (D), energy expenditure (E), and respiratory quotient (F). Data are means ± SE; n = 8. G: body temperature of WT and MLK3-KO mice is presented. Data are means ± SE; n = 8. H: plasma noradrenaline and adrenaline concentrations were measured on HFD. Data are means ± SE; n = 8. *P < 0.05; ***P < 0.001. NS, not significant.

Fig. 2.

Loss of MLK3 improves diet-induced insulin resistance. A: blood glucose concentration of WT and MLK3-KO mice on CD and HFD were measured after an overnight fast. B: glucose tolerance tests were performed by measuring blood glucose concentrations following intraperitoneal injection of glucose (1.5 g/kg). Area under the curve (AUC) was calculated (right). C: insulin concentration of WT and MLK3-KO mice on CD and HFD were measured after an overnight fast. Data are means ± SE; n = 8. *P < 0.05.

Fig. 3.

MLK3 deficiency decreases secretion of inflammatory markers. A: plasma concentrations of leptin, monocyte chemoattractant protein-1 (MCP-1), and adiponectin were measured in CD and HFD mice. Data are means ± SE; n = 8. B: IL-6 and MCP-1 in conditioned medium were collected for 24 h from visceral fat explants. Data are means ± SE (n = 4) from 2 different experiments. C: phosphorylation and expression of JNK in epididymal fat pads was examined by immunoblot analysis. Quantification of immunoblot data is shown at right. *P < 0.05; **P < 0.01.

Fig. 4.

MLK3 deficiency reduces macrophage infiltration in visceral adipose tissue. A: representative images of hematoxylin and eosin-stained epididymal fat sections from WT and MLK3-KO mice. B: no. of crown-like structures (CLS) in epididymal fat sections from HFD-fed mice was calculated as %total cells. More than 1,000 cells/section were counted. Data are means ± SD; n = 8 mice. C: expression of F4/80, MCP-1, and TNFα was measured by quantitative RT-PCR analysis normalized to the expression of cyclophilin. Data are means ± SE; n = 8. **P < 0.01; *P < 0.05.

Fig. 5.

MLK3 deficiency alters macrophage polarization. A: bone marrow-derived macrophages (BMDM) were left untreated (UT) or were treated with 10 ng/ml lipopolysaccharide (LPS) for 24 h. Inducible nitric oxide synthase (iNOS) expression was measured by quantitative RT-PCR analysis normalized to the expression of cyclophilin. B: BMDM were left UT or were treated with 50 ng/ml IL-4 for 24 h. Arginase expression was measured by quantitative RT-PCR analysis normalized to the expression of cyclophilin. C: BMDM were treated with BSA or 1 mM palmitate (palm) for 6 h. A representative immunoblot of JNK phosphorylation (JNK-P) and expression from 3 independent experiments is shown. D–F: BMDM were treated with 1 mM palmitate for 24 h, and expression of TNFα, iNOS, and arginase was measured by quantitative RT-PCR analysis normalized to the expression of cyclophilin. Data are means ± SE from 3 independent experiments. *P < 0.05; **P < 0.01.

Fig. 6.

MLK3 deficiency improves hepatic steatosis. A: hematoxylin and eosin stain of representative sections of WT and MLK3-KO livers fed HFD for 16 wk. B–D: expression of SCD1, PPARγ, MCP1, carnitine palmitoyltransferase I (CPT I), and fibroblast growth factor 21 (FGF21) was measured by quantitative RT-PCR analysis normalized to the expression of cyclophilin. Data are means ± SE; n = 8. *P < 0.05.