Clodronate liposomes effectively deplete KCs and VATMs of DIO mice.

<p>Wild type mice were fed a HFD for 13 weeks and injected with 2 doses of clodronate liposomes (250 mg/kg) or PBS liposomes i.p. over 6 days with a 3-day interval. We observed no changes in the total animal body weight of PBS liposome-treated mice (42.4±1.8 to 41.6±1.9) or clodronate-treated mice (44.01±2.2 to 41.5±2.6). VAT SVF was isolated and FACS analysis was performed. <i>A</i>.) Upper panels, PBS liposome-treated mice. Lower panels, clodronate-treated mice. Left panels, SiglecF-negative, CD11b- and F4/80-positive cells. Right panels, CD11c-positive cells. Panels are representative of 6 animals per group. <i>B</i>.) Percentage of the total number of cells counted for both CD11b- and F4/80-positive cells and CD11c-positive cells in the VAT of DIO mice. RNA was extracted and quantitative RT-PCR was performed for standard macrophage markers (F480, CD11c) in <i>C.</i>) VAT and <i>D</i>.) liver of DIO mice. Values represent the mean ± SEM. An unpaired student’s t<i>-</i>test was used for comparisons between groups. *P≤0.05, **P≤0.01.</p

Pharmacological intervention via inhibition of IL-1 signaling improves the glucose tolerance in DIO mice.

<p>Wild type mice were fed a HFD for 13 weeks. At 32 days prior to sacrifice, mice were started on daily injections of IL-1Ra (Anakinra; 32 mg/kg) or saline by i.p. administration. Data are representative of 10 mice per group. At the end of 13 weeks, Anakinra-treated mice displayed no changes in <i>A.</i>) total animal body weight and were more glucose tolerant as demonstrated by a reduction in <i>B</i>.) area under the curve (AUC) for the intraperitoneal GTT and <i>C</i>.) fasting insulin levels. Values represent the mean ± SEM. An unpaired student’s t<i>-</i>test was used for comparisons between groups. <i>*</i>P≤0.05, **P≤0.01.</p

Physiological concentrations of IL-1β elicit a biological response in primary mouse hepatocytes to increase TG accumulation and lipogenic gene expression.

<p>Primary hepatocytes from wild type mice were isolated via perfusion, stimulated with the indicated doses of recombinant mouse IL-1β and evaluated after 24-hours. <i>A</i>.) Cells were fixed in 10% formalin and stained with Oil-Red-O to assess TG accumulation. <i>B</i>.) Fas expression was analyzed in primary hepatocyte cell lysates after a 24-hour stimulation with the indicated doses of recombinant IL-1β and normalized to β-actin. <i>C</i>). Relative densitometry from B. <i>D</i>.) TGs were extracted and measured after a 24-hour stimulation with recombinant IL-1β (10 ng/mL) and normalized to the amount of total protein. The data are representative of 4 experiments. All stimulations were performed in duplicate. Values represent the mean ± SEM. A paired student’s t<i>-</i>test was used for comparisons between groups <i>*</i>P≤0.05, ***P≤0.001.</p

Clodronate liposome-mediated KC and VATM depletion ameliorates hepatic steatosis in DIO mice.

<p>Wild type mice were fed a HFD for 13 weeks and injected with 2 doses of clodronate liposomes (250 mg/kg) or PBS liposomes by i.p. over 6 days with a 3-day interval. <i>A</i>.) Livers of DIO mice were isolated, fixed in 10% formalin, embedded in paraffin, and stained with H&E or frozen in OCT and stained with Oil-Red-O to assess steatosis. Images are representative of 12–16 animals at 10X and 20X magnification. <i>B</i>.) Livers of DIO animals were weighed and data is represented as a percentage of total body weight. <i>C</i>.) Total TGs were extracted and normalized to tissue weight. Values represent the mean ± SEM. An unpaired student’s t<i>-</i>test was used for comparisons between groups. <i>*</i>P≤0.05, <i>**</i>P≤0.01.</p

Glycogen content of livers and muscles after treatment with Genz-112638.

<p>C57BL/6 mice that were fed a normal diet (Lean) or a HFD (DIO) were treated as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011239#pone-0011239-g001" target="_blank">Fig. 1</a>. Data shown as mean ± SEM (n = 6–9 mice per group). <i>P</i>>0.05 between all groups.</p

Effect of Genz-112638 on hepatic steatosis.

<p>DIO mice were treated with Genz-112638 or placebo (water) by daily oral gavage at 75 or 125 mg/kg/day for 16 weeks. A) Representative liver sections stained with hematoxylin and eosin (H&E). B) Fatty change score of the livers (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011239#s4" target="_blank">Methods</a>). C) Liver triglyceride levels. Data shown as mean ± SEM (n = 6–10 mice per group). **<i>P</i><0.01 Genz-112638 DIO vs. Water DIO.</p

Effect of Genz-112638 on A) whole body fat mass, and B) whole body lean mass.

<p>C57BL/6 mice were fed a normal diet (Lean) or a HFD (DIO) for 8 weeks. The mice were then treated with Genz-112638 or placebo (water) by daily oral gavage (125 mg/kg/day) for 12 weeks, while remaining on their respective diets. Fat and lean mass were measured by proton magnetic resonance spectroscopy (¹H-MRS) on awake mice. Data shown as mean ± SEM (n = 10 mice per group). *<i>P</i><0.05 Genz-112638 DIO vs. Water DIO.</p

Sphingolipid levels in the livers of DIO mice after treatment with Genz-112638.

<p>C57BL/6 mice were fed a normal diet (Lean) or a HFD (DIO) for 8 weeks. The DIO mice were then treated with Genz-112638 or placebo (water) by twice daily oral gavage (120 mg/kg/day) for 22 weeks, while remaining on the HFD. Sphingolipid levels were determined by high performance liquid chromatography – tandem mass spectrometry (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011239#s4" target="_blank">Materials and Methods</a>). Data shown as mean ± SEM (n = 6–9 mice per group). *<i>P</i><0.05, **<i>P</i><0.01 Genz-112638 DIO vs. Water DIO.</p

Effect of Genz-112638 on A) food consumption and B) body weight.

<p>C57BL/6 mice were fed a normal diet (Lean) or a HFD (DIO) for 8 weeks. The DIO or Lean mice were then treated with Genz-112638 or placebo (water) by daily oral gavage (125 mg/kg/day) for 12 weeks, while remaining on their respective diets. Data shown as mean ± SEM (n = 7–14 mice per group). *<i>P</i><0.05 Genz-112638 DIO (both doses) vs. Water DIO, weeks 0–9. There was no significant difference in body weights between the Water DIO and drug treated groups.</p

Effect of Genz-112638 on hepatic glucose metabolism.

<p>Hyperinsulinemic-euglycemic clamps were performed in normal diet (Lean) or HFD (DIO) fed mice treated with Genz-112638 or placebo (water) by daily oral gavage (125 mg/kg/day) for 12 weeks. Animals were continuously infused with [³H]glucose throughout the clamps. A) Steady-state glucose infusion rates, B) Basal HGP, C) Clamp HGP, D) Insulin-mediated percent suppression of basal HGP. Data shown as mean ± SEM (n = 10 mice per group). #<i>P</i><0.05 vs. Water Lean, *<i>P</i><0.05 Genz-112638 DIO vs. Water DIO.</p