TIMP3 overexpression in macrophages protects from insulin resistance, adipose inflammation, and nonalcoholic fatty liver disease in mice

Abstract

The tissue inhibitor of metalloproteinase (TIMP)3, a stromal protein that restrains the activity of proteases and receptors, is reduced in inflammatory metabolic disorders such as type 2 diabetes mellitus (T2DM) and atherosclerosis. We overexpressed Timp3 in mouse macrophages (MacT3) to analyze its potential antidiabetic and antiatherosclerotic effects. Transgenic mice with myeloid cells targeting overexpression of TIMP3 were generated and fed a high-fat diet for 20 weeks. Physical and metabolic phenotypes were determined. Inflammatory markers, lipid accumulation, and insulin sensitivity were measured in white adipose tissue (WAT), liver, and skeletal muscle. In a model of insulin resistance, MacT3 mice were more glucose tolerant and insulin sensitive than wild-type mice in both in vitro and in vivo tests. Molecular and biochemical analyses revealed that increased expression of TIMP3 restrained metabolic inflammation and stress-related pathways, including Jun NH2-terminal kinase and p38 kinase activation, in WAT and liver. TIMP3 overexpression in macrophages resulted in reduced activation of oxidative stress signals related to lipid peroxidation, protein carbonylation, and nitration in WAT and liver. Our data show that macrophage-specific overexpression of TIMP3 protects from metabolic inflammation and related metabolic disorders such as insulin resistance, glucose intolerance, and nonalcoholic steatohepatitis.

FIG. 1.

MacT3 mice are protected against HFD-induced insulin resistance. WT and MacT3 mice were fed an HFD for 20 weeks. A: Body weight curve during HFD (n = 9 per group). B: Fasting and fed glucose and insulin levels (n = 9 per group; *P < 0.05, Student t test). C: Intraperitoneal glucose tolerance test and intraperitoneal insulin tolerance test (n = 9 per group; *P < 0.05, one-way ANOVA). D: Adiponectin, leptin, TNF-α, IL-6, and transaminase levels (n = 9 per group; *P < 0.05, Student t test. Data are means ± SD). E: Vo₂ in free-fed MacT3 mice over a period of 24 h (line chart) compared with Vo₂ in WT mice after 20 weeks of HFD. F: Mean 24-h values for Vo₂, Vco₂, respiratory quotient (RER), locomotor activity, and food intake. (E and F: n = 6 per group. Student t test, *P < 0.05 for MacT3 HFD vs. WT HFD mice. Data are means ± SD.)

FIG. 2.

MacT3 mice show improved insulin-stimulated glucose transport in vivo. A: Anterior images from the FDG PET scans of WT and MacT3 mice fed an HFD for 20 weeks. Arrows indicate sites with different glucose uptake in forelimbs and hind limbs. Maximum standardized uptake values (SUV_max) indicating an increased glucose uptake in MacT3 vs. WT mice. (n = 5 per group; *P < 0.05, Student t test. Data are means ± SD.) B: Skeletal muscle tissue from overnight-fasted WT and MacT3 mice fed an HFD for 20 weeks was dissected 5 min after insulin injection, and Akt phosphorylation (p) was analyzed by Western blot. (n = 8 per group; **P < 0.01, Student t test. Data are means ± SD.) A representative image of four mice per group is shown. a.u., arbitrary unit. (A high-quality color representation of this figure is available in the online issue.)

FIG. 3.

MacT3 mice are protected against HFD-induced changes in adipose tissue. A: Representative computed tomography scan section indicating a decreased visceral adipose tissue fraction in MacT3 vs. WT mice fed an HFD for 20 weeks, confirmed by visceral adipose tissue quantification. (n = 4 per group; *P < 0.05, Student t test. Data are means ± SD.) B: Epididymal fat pads were harvested and weighed. (n = 5 per group; *P < 0.05, Student t test. Data are means ± SD.) C: TIMP3 mRNA and protein levels were increased and ADAM17 activity was reduced in adipose tissue of MacT3 mice fed an HFD compared with WT mice. A representative image of four mice per group is shown. (n = 8 per group; *P < 0.05, Student t test. Data are means ± SD.) D: TIMP3 and adiponectin (considered adipocyte markers) and mRNA expression in mature adipocytes (AFs) and SVF. (n = 5 per group; **P < 0.01 and ***P < 0.001, Student t test. Data are means ± SD.) E: Representative sections of adipose tissue stained with hematoxylin-eosin (H/E) showing an increased adipocyte density and a reduction in mean adipocyte area in MacT3 mice vs. WT mice. (n = 5 per group; **P < 0.01 and *P < 0.05, Student t test. Data are means ± SD.) F: WAT expression of leptin and adiponectin mRNA. Expression of mRNA was determined by real-time PCR and normalized to 18S RNA. (n = 5 per group; *P < 0.05, Student t test. Data are means ± SD.) a.u., arbitrary unit. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 4.

Protective effect of transgenic expression of TIMP3 in macrophages on inflammation in WAT. A: Representative sections of WAT from WT and MacT3 mice fed an HFD for 20 weeks stained with F4/80, CD3, and CD31, revealing a reduction in macrophage and T-cell infiltration and in vessel density. (n = 5 mice per group; *P < 0.05, Student t test. Data are means ± SD.) B: WAT expression of genes involved in inflammation. Expression of mRNA was determined by real-time PCR and normalized to 18S RNA. (n = 5 per group; *P < 0.05 and **P < 0.01, Student t test. Data are means ± SD.) C: WAT from WT and MacT3 mice fed an HFD for 20 weeks was lysed and analyzed for phosphorylation of JNK and p38. D: WAT from WT and MacT3 mice fed an HFD for 20 weeks was lysed and analyzed for phosphorylation of Akt by Western blot (upper panel). Adipose tissue from overnight-fasted WT and MacT3 mice fed an HFD for 20 weeks was dissected 5 min after insulin injection; insulin-stimulated Akt phosphorylation (p) was analyzed by Western blot (lower panel). (C and D: For all Western blots, n = 8 per group; a representative image of four mice per group is shown. *P < 0.05 and **P < 0.01, Student t test. Data are means ± SD.) a.u., arbitrary unit. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 5.

Reduced hepatic steatosis in HFD-fed MacT3 mice. A: Livers of MacT3 and WT mice fed an HFD for 20 weeks were harvested and weighed. Results are expressed in grams. (n = 10 per group; *P < 0.05, Student t test. Data are means ± SD.) B: TIMP3 mRNA. C and D: TIMP3 protein levels (C) and ADAM17 activity (D) in livers of MacT3 mice compared with WT mice. (n = 8 per group; *P < 0.05, Student t test. Data are means ± SD.) E: TIMP3 and albumin (hepatocyte [HEP] marker) mRNA expression in purified hepatocytes and NPCs from MacT3 and WT mouse livers. (n = 5 per group; **P < 0.01 and ***P < 0.001, Student t test. Data are means ± SD). F: Representative sections of liver stained with hematoxylin-eosin (H/E), Oil Red O, F4/80, CML, or nitroTyr (N-Tyr). Arrow indicates a lipogranuloma in WT mice. CV, central vein. (n = 5 per group; **P < 0.01 and *P < 0.05, Student t test. Data are means ± SD.) a.u., arbitrary unit. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 6.

Reduced hepatic oxidative (Ox) stress and improved insulin signaling in HFD-fed MacT3 mice. A: TBARS assay detecting lipid peroxidation products (fold change). (n = 5 per group; *P < 0.05, Student t test. Data are means ± SD.) B: mRNA expression of indicated markers in liver tissue of WT and MacT3 mice was analyzed by real-time PCR, expressed relative to WT, and normalized to expression of 18S. CAT, catalase. (n = 5 per group; *P < 0.05, Student t test. Data are means ± SD). C: Livers from WT and MacT3 mice fed an HFD for 20 weeks were lysed and analyzed for phosphorylation (p) of Akt and JNK by Western blot. For insulin-stimulated Akt phosphorylation, livers from overnight-fasted mice were dissected 5 min after insulin injection and analyzed by Western blot. (n = 8 per group. A representative image of four mice per group is shown. *P < 0.05, Student t test. Data are means ± SD.) a.u., arbitrary unit. (A high-quality color representation of this figure is available in the online issue.)