Animal studies were carried out using wild-type (WT) and Mck–Mcp-1 Tg mice, all on a C57Bl/6J background . The mice were kindly donated by D. Patsouris (Université Claude Bernard Lyon 1, France). Only male mice were used. Mice were kept in temperature controlled rooms (21 ± 1°C) on a 12 h light–dark cycle. For the studies using chow-fed mice, 16 WT and 19 Mcp-1-Tg mice were fed regular chow (RMH-B Arie Blok, Woerden, the Netherlands). At 14–17 weeks of age, eight mice per group were subjected to an intraperitoneal glucose tolerance test and a week later to an intraperitoneal insulin tolerance test. Mice were euthanised at 17–20 weeks of age after a 6 h fast at zeitgeber time (ZT)7–ZT9. Before being euthanised, eight mice of each genotype received an intraperitoneal injection of insulin (2 U/kg body weight in 100 μl saline [154 mmol/l NaCl], n = 4 per genotype) or saline (100 μl, n = 4 per genotype) followed by euthanasia exactly 5 min later by cervical dislocation. For the remaining mice, blood was collected by orbital puncture under isoflurane anaesthesia and mice were euthanised by cervical dislocation. For the high-fat feeding study, 12 WT and 12 Mcp-1-Tg mice were fed regular chow (RMH-B Arie Blok) after weaning. At 12–16 weeks of age, mice were switched to a high-fat diet (HFD) containing 60 energy% fat (D12492; Research Diets Inc., New Brunswick, NJ, USA). Food intake and body weights were registered weekly. After 8 weeks of high-fat feeding, mice were subjected to an intraperitoneal glucose tolerance test and a week later to an intraperitoneal insulin tolerance test. Mice were euthanised after 10 weeks of high-fat feeding after a 6 h fast at ZT7–ZT9. Before being euthanised, four mice of each genotype received an intraperitoneal injection of insulin (2 U/kg body weight in 100 μl saline, n = 2 per genotype) or saline (100 μl, n = 2 per genotype) followed by euthanasia exactly 5 min later by cervical dislocation. For the remainder of the mice, blood was collected by orbital puncture under isoflurane anaesthesia and mice were euthanised by cervical dislocation. Tissues were excised and immediately frozen in liquid nitrogen followed by storage at −80°C. All experiments were blind to group and outcome assessment. All animal experiments were authorised by the animal welfare committee of Wageningen University.
Intraperitoneal glucose tolerance test
After a 6 h fast mice were injected intraperitoneally with glucose (1.2 g/kg body weight for mice on chow, 0.8 g/kg body weight for mice on HFD). Blood was collected by tail bleeding after 0, 15, 30, 60, 90 and 120 min, and glucose was measured using an Accu-Chek compact (Roche Diagnostics, Almere, the Netherlands).
Intraperitoneal insulin tolerance test
After a 6 h fast mice were injected intraperitoneally with insulin (0.75 U/kg body weight). Blood was collected by tail bleeding after 0, 15, 30, 45, 60 and 90 min, and glucose was measured using an Accu-Chek compact (Roche Diagnostics).
Histological analysis of musculus gastrocnemius morphology and macrophage infiltration was done using hematoxylin and eosin (H&E) staining. On the day of euthanasia, samples were frozen in isopentane mixed with dry ice and stored at −80°C. Tissue was embedded in Tissue-Tek O.C.T. Compound (Sakura Finetek, Alpen aan de Rijn, the Netherlands), sectioned at 10 μm in a cryostat chamber, fixed on a Superfrost glassslide, left to dry for 30 min and stored at −20°C. Samples were stained at room temperature in Mayer hematoxylin solution for 10 min and in eosin Y solution for 30 s.
Measurement of plasma variables
Blood was collected into EDTA-coated tubes. Blood samples were placed on ice and centrifuged at 4°C for 10 min at 10,000 g. Plasma was collected and stored at −80°C. To measure plasma MCP-1 concentration a DuoSet ELISA Development kit against mouse MCP-1 was used (R&D Systems, Minneapolis, MN, USA). For insulin measurements an ultra-sensitive mouse insulin ELISA kit was used (Crystal chemicals, Downers Grove, IL, USA). For the other plasma measurements the following kits were used: NEFA Reagent set, Triglycerides Liquicolor, Cholesterol Liquicolor (all Instruchemie, Delfzijl, the Netherlands) and Glucose GOD FS (DiaSys, Holzheim, Germany). All measurements were performed according to the manufacturers’ protocols.
Microarray analysis was performed on RNA of the musculus gastrocnemius as described in the electronic supplementary material (ESM) Methods.
RNA isolation and quantitative real-time PCR
Total RNA was extracted from different mouse tissues using TRIzol reagent (Invitrogen, Bleiswijk, the Netherlands) and a Tissuelyser II (Qiagen, Venlo, the Netherlands). Reverse transcription was performed using Superscript II and oligo(dT) primers (Invitrogen). PCR-amplifications were carried out using iQ SYBR Green Supermix on a CFX384 Touch real-time PCR detection system (Bio-Rad, Hercules, CA, USA).
The sequences of the primers used for quantitative real-time PCR (qPCR) are shown in ESM Table 1. All genes were normalised to 36b4. Primer efficiencies were determined using LinRegPCR v11.1 (http://LinRegPCR.nl) . Relative expression of the transcript levels was calculated as described previously .
Total protein extracts were obtained from musculus gastrocnemius to detect MCP-1. Tissue was homogenised using a Tissuelyser II (Qiagen) in protein extraction buffer without bromophenol blue (62.5 mmol/l Tris HCl, pH 6.8, 2% SDS, 10% glycerol and 10 mmol/l dithiothreitol) and protein concentration was measured using Bradford protein assay. Protein (50 μg) was boiled, subjected to SDS-PAGE and proteins transferred to nitrocellulose membrane (Immobilon; Millipore, Billerica, MA, USA). Membranes were blocked and incubated with anti-MCP-1 (R17; Santa Cruz, Dallas, TX, USA) and anti-Actin (Sigma Aldrich, St Louis, MO, USA).
To obtain total protein extracts from musculus gastrocnemius to detect phospho-Akt, tissue was homogenised using a Tissuelyser II (Qiagen) in protein extraction buffer with phosphatase inhibitors (50 mmol/l Tris HCl, pH 4.7, 1 mmol/l EDTA, 150 mmol/l NaCl, 1% NP40, 0.25% sodium deoxycholate, 2 mmol/l sodium orthovanadate and 5 mmol/l sodium fluoride, containing freshly added protease inhibitor cocktail [Roche Applied Science, Penzberg, Germany]). After being centrifuged at 20,800 g for 10 min at 4°C, supernatant fractions were collected and 50 μg of protein was boiled with Laemmli sample buffer. The protein was subjected to SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Immobilon; Millipore). Membranes were blocked and incubated with anti-Akt (in house), anti-phospho-Akt (Ser473) (Cell Signaling Technology, Danvers, MA, USA) and anti-Actin (Sigma Aldrich). Quantification was carried out using ImageJ 1.49m (http://imagej.nih.gov).
For statistical analysis Student’s t test was used. Data are means ± SEM and p < 0.05 is considered statistically significant. One mouse from the WT group, fed HFD, was excluded from subsequent analysis because it was mistakenly injected with a higher dose of insulin during the insulin tolerance test. Some qPCR data on gene expression were excluded from the results section because the genes were expressed at a low level, with Ct values greater than 32 in both the WT and Mcp-1-Tg mice.