Indomethacin treatment prevents high fat diet-induced obesity and insulin resistance but not glucose intolerance in C57BL/6J mice

Abstract

Chronic low grade inflammation is closely linked to obesity-associated insulin resistance. To examine how administration of the anti-inflammatory compound indomethacin, a general cyclooxygenase inhibitor, affected obesity development and insulin sensitivity, we fed obesity-prone male C57BL/6J mice a high fat/high sucrose (HF/HS) diet or a regular diet supplemented or not with indomethacin (±INDO) for 7 weeks. Development of obesity, insulin resistance, and glucose intolerance was monitored, and the effect of indomethacin on glucose-stimulated insulin secretion (GSIS) was measured in vivo and in vitro using MIN6 β-cells. We found that supplementation with indomethacin prevented HF/HS-induced obesity and diet-induced changes in systemic insulin sensitivity. Thus, HF/HS+INDO-fed mice remained insulin-sensitive. However, mice fed HF/HS+INDO exhibited pronounced glucose intolerance. Hepatic glucose output was significantly increased. Indomethacin had no effect on adipose tissue mass, glucose tolerance, or GSIS when included in a regular diet. Indomethacin administration to obese mice did not reduce adipose tissue mass, and the compensatory increase in GSIS observed in obese mice was not affected by treatment with indomethacin. We demonstrate that indomethacin did not inhibit GSIS per se, but activation of GPR40 in the presence of indomethacin inhibited glucose-dependent insulin secretion in MIN6 cells. We conclude that constitutive high hepatic glucose output combined with impaired GSIS in response to activation of GPR40-dependent signaling in the HF/HS+INDO-fed mice contributed to the impaired glucose clearance during a glucose challenge and that the resulting lower levels of plasma insulin prevented the obesogenic action of the HF/HS diet.

Keywords: Adipose Tissue; Cyclooxygenase (COX); Cyclooxygenase Inhibitors; G Protein-coupled Receptor (GPCR); Glucose Intolerance; Insulin Resistance; Insulin Secretion.

© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Figures

FIGURE 1.

Effect of indomethacin supplementation on body weight gain and adipose tissue mass. Mice were fed RD, HF/HS, or HF/HS+INDO for 7 weeks. The mice were killed, and liver and adipose tissue depots were dissected out and weighed. A, body weight development during 7 weeks of feeding. B, mean total body weight gain after 7 weeks of feeding. C, mean weight of eWAT, retroperitoneal white adipose tissue (rWAT), and iWAT. D, mean liver weight. E, mean weight of iBAT. F, total energy intake calculated from the amount of food eaten during the experiment. G, energy efficiency calculated as weight gain relative to megajoules (kJ). H, feces were collected for 48 h, and the fat content was measured to estimate percentage of fat absorption. I, total percentage of weight loss after 12-h starvation in mice treated with the respective diets. All results are presented as mean ± S.E. (error bars) (n = 8–9). Statistical significances are denoted with asterisks as follows: *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.

FIGURE 2.

The effect of indomethacin on metabolic performance, expression of markers of brown and brown-like adipocytes, and plasma levels of glycerol, NEFA, alanine aminotransferase (ALT), and aspartate transaminase (AST) after 7 weeks of feeding. Mice fed RD, HF/HS, and HF/HS+INDO were placed in an open circuit chamber for 24 h for measurements of O2 consumption and CO2 production. A and B, O2 uptake and production of CO2 expressed as ml/h/kg. C, calculation of respiration exchange ratio (RER). D–F, mRNA levels of markers of brown and brown-like adipocytes in iBAT, eWAT, and iWAT. Ucp1, uncoupling protein 1; Dio2, deiodinase, iodothyronine, type II; Ppargc1a, peroxisome proliferator-activated receptor γ, coactivator 1 α. G, IHC staining with UCP1 antibody for detection of multilocular cells in representative sections from eWAT and iWAT. H, plasma levels of ALT measured in the fed state and aspartate transaminase measured in the fasted state (12 h). I, plasma glycerol levels in fed and fasted mice. J, plasma levels of NEFA in the fed and the fasted state. The results are presented as mean ± S.E. (error bars) (n = 8–9). Statistical significances are denoted with asterisks as follows: *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.

FIGURE 3.

Effects of indomethacin supplementation on adipocyte morphology and inflammation.A, morphology of eWAT and iWAT from mice fed RD, HF/HS, and HF/HS+INDO for 7 weeks (n = 3). The tissues were stained with hematoxylin-eosin. Micrographs from one representative mouse in each group are shown. B–I, quantitative real-time RT-PCR analysis of markers of macrophage infiltration and inflammation in eWAT (B–E) and iWAT. F–I, Cd68, cluster of differentiation 68; Emr1, Egf-like module containing, mucin-like, hormone receptor-like 1; Serpine1, serpin peptidase inhibitor, clade E, member 1; Ccl2, chemokine ligand 2. J, IHC staining with F4/80-antibody for detection of macrophages and crownlike structures in representative sections from eWAT and iWAT. The results are presented as mean ± S.E. (error bars) (n = 8–9). Statistical significances are denoted with asterisks as follows: **, p ≤ 0.01; ***, p ≤ 0.001.

FIGURE 4.

Effects of indomethacin supplementation on lipid accumulation in liver and muscle and insulin sensitivity. Quantitative analyses of lipids in the liver and muscle of mice fed RD, HF/HS, and HF/HS+INDO for 7 weeks were performed (n = 8–9). TAG (A) and diacylglycerol (DAG) (B) in liver are shown. TAG (C) and DAG (D) in tibialis anterior muscle are shown. E, HOMA-IR calculated from fasting plasma (12 h) levels of insulin and glucose. Shown are QUICKI (F) and ITT (G) after 6 weeks on the respective diets. Blood glucose from RD-fed mice was significantly different from the level in mice given a HF/HS diet at time points 15, 30, 45, and 60 after injection, but is not shown in the figure. All results are presented as mean ± S.E. (error bars) (n = 8–9). Statistical significances are denoted with asterisks as follows: *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.

FIGURE 5.

Effects of indomethacin supplementation on glucose tolerance, insulin sensitivity, hepatic glucose production, and glucose-stimulated insulin secretion.A, plasma blood glucose levels in fed and 12-h fasted animals after 7 weeks of feeding. B, intraperitoneal glucose tolerance test (2 g/kg) performed on mice fasted for 6 h. C, GSIS during GTT after 7 weeks of treatment with the respective diets. D, pyruvate tolerance test after 6 weeks of feeding with an RD, HF/HS, and HF/HS+INDO diet. E–F, mRNA levels of glucose-6-phosphatase (G6pc) and phosphoenolpyruvate carboxykinase 1 (Pck1) in liver in the fed state. The results are presented as mean ± S.E. (error bars) (n = 8–9). G and H, GTT and GSIS after 1 week on the respective diets. I and J, GTT and GSIS after 2 weeks. K and L, GTT and GSIS after 3 weeks. Statistical significances are denoted with asterisks as follows: *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.

FIGURE 6.

Effects of acute COX inhibition with indomethacin, indomethacin combined in a balanced RD diet, and indomethacin supplementation to already obese animals.A–C, acute effects of indomethacin on GTT and GSIS in C57BL/6J mice given an RD diet for 1 week. The mice were given indomethacin (2.5 mg/kg, body weight) orally 1 h before a glucose tolerance test during which GSIS was also evaluated. D–F, acute effects of indomethacin on glucose tolerance and GSIS in mice fed an HF/HS diet for 10 weeks. G, weight of eWAT, rWAT, and iWAT in mice fed RD and RD+INDO for 7 weeks. H and I, GTT and GSIS after 6 weeks of feeding with RD supplemented with indomethacin. J–O, mice were fed an RD or HF/HS diet for 10 weeks and then continued on either an HF/HS diet with or without indomethacin supplementation or the RD diet for an additional 8 weeks. Body weight (J), feed efficiency (K), lean body mass (L), and fat mass (M) are shown for 6 weeks after changing the diet. N and O, GTT and GSIS on the mice shown in J–M. The results are presented as mean ± S.E. (error bars) (n = 8–9). Statistical significances are denoted with asterisks as follows: *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.

FIGURE 7.

Effects of indomethacin and the GPR40 activation on glucose-stimulated insulin secretion.A, plasma insulin levels in fed and 12 h-fasted animals after 8 weeks of feeding RD, HF/HS, or HF/HS+INDO. B, pancreatic islet and section sizes were analyzed, and islet size as a percentage of total pancreas size was calculated (n = 5). C, first phase insulin secretion and glucose-stimulated insulin secretion were measured after 3 h of fasting. D, MIN6 cells treated with indomethacin at concentrations of 0.1, 1, or 10 μm. E, MIN6 cells treated with vehicle, indomethacin (1 μm), TUG469 (10 μm), or TUG469 (10 μm) + INDO (1 μm) at both low (2.8 mm) and high (20 mm) glucose concentration. After 1 h of incubation, insulin release was measured. F, effects of TUG469 and indomethacin on β-arrestin-2 recruitment to mouse GPR40. Bioluminescence resonance energy transfer signals normalized to the maximal TUG469 response obtained following treatment with varying concentrations of TUG469 or INDO on their own or to varying concentrations of INDO in the presence of 100 nm TUG469. The results are presented as mean ± S.E. (error bars) (n = 8–9). Statistical significances are denoted with asterisks as follows: *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001.