Hematological and acute-phase responses to diet-induced obesity in IL-6 KO mice

Obesity is associated with chronic inflammation and elevated levels of IL-6. The role of IL-6 in induction of acute-phase proteins and modulation of haematological responses has been demonstrated in models of inflammation and aging, but not in obesity. We hypothesized that IL-6 is necessary to regulate the acute-phase response and hematological changes associated with diet-induced obesity (DIO) in mice. Feeding a 60% kcal/fat diet for 13 weeks to C57BL6 WT male mice induced a significant increase in IL-6 expression in visceral adipose tissue (VAT), but not liver, compared to mice fed chow diet. Significantly elevated IL-6 levels were present in the peritoneal lavage fluid, but not plasma, of DIO compared to lean mice. A comparable degree of obesity, hepatomegaly, hyperleptinemia, VAT inflammation and insulin resistance was observed in DIO WT and IL-6 KO mice compared to WT and KO mice fed chow diet. Significant leukocytosis was observed in DIO WT but not DIO KO mice compared to lean groups. A significant reduction in platelet counts, without alterations in platelet size, percentage of circulating reticulated platelets and number of bone marrow megakaryocytes, was present in DIO KO mice compared to each other group. Hepatic expression of thrombopoietin was comparable in each group, with DIO WT and KO mice having reduced VAT expression compared to lean mice. Lean KO mice had significantly elevated plasma levels of thrombopoietin compared to each other group, whereas liver-associated thrombopoietin levels were comparable in each group. Deficiency of IL-6 resulted in blunted hepatic induction of the acute-phase protein serum amyloid A-1, whereas expression of hepcidin-1 and -2, LPS-binding protein, ceruloplasmin, plasminogen activator inhibitor-1 and thrombospondin-1 was IL-6- independent. In conclusion, in the absence of overt metabolic alterations, IL-6 modulates leukocytosis,thrombopoiesis induction of SAA-1, but not other acute-phase proteins in obese mice

Deficiency of ANXA1 modulates adiposity in female Balb/c mice.

<p><u>Panel A</u>: Body weight in grams, (n = 14 – 20). <u>Panel B</u>: Fat mass in grams measured by DXA. <u>Panel C</u>: Percentage of fat mass to BW (n = 14 – 19). <u>Panel D</u>: Plasma leptin levels (n = 13 – 17). <u>Panel E</u>: Leptin mRNA expression in VAT (n = 4 – 5). <u>Panel F</u>: Plasma adiponectin levels (n = 13 – 17). <u>Panel G</u>: Adiponectin mRNA expression in VAT (n = 5). <u>Panel H</u>: PPARγ mRNA expression in VAT (n = 4 – 5). <u>Panel I</u>: Lean mass in grams measured by DXA. <u>Panel J</u>: Median adipocyte size in VAT and SAT (n = 4 – 5), H&E-stained slides for VAT and SAT magnified to 10X. *p<.05 vs. respective diet-matched WT group, p<.05 vs. WT-Chow, #p<.05 vs. KO-Chow. Data are mean ± SEM.</p

Corticosterone and 11βHSD1 levels in WT and ANXA1 KO mice.

<p><u>Panel A</u>: Plasma corticosterone levels in fed mice (n = 5 – 13). <u>Panel B</u>: Expression of 11βHSD1 mRNA in VAT of fed mice (n = 4 – 5). *p<.05 vs. respective diet-matched WT group, p<.05 vs. WT-Chow, #p<.05 vs. KO-Chow. Data are mean ± SEM.</p

Modulation of ANXA1 expression in adipose tissue.

<p><u>Panel A</u>: Time course of ANXA1 mRNA expression in VAT and SAT of male C57BL6 mice fed chow or HFD for 5, 9 or 13 weeks. Data are expressed as fold difference vs. the respective tissue in chow mice at 5 weeks. ***p<0.001 vs. respective tissue in chow mice. <u>Panel B</u>: Expression of ANXA1 mRNA in VAT SVF and adipocytes of male C57BL6 mice fed chow or HFD for 13 weeks (n = 9). *p<0.05, ***p<0.001 vs. respective fraction in chow mice. <u>Panels C and D</u>: Number (Panel C) and Mean Fluorescence Intensity (Panel D) of ANXA1⁺ cells per mg of tissue in the F4/80^- and F4/80⁺ populations from VAT of male C57BL6 mice fed chow or HFD for 13 weeks evaluated by flow cytometry (n = 5). ***p<0.001 vs. respective cell population in chow mice. <u>Panel E</u>: Expression of ANXA1 mRNA at eight weeks of age in VAT of male WT and <i>ob/ob</i> mice fed chow diet (n = 3). <u>Panel F</u>: Expression of ANXA1 mRNA in VAT of male WT and IL-6 KO mice fed chow or HFD for 13 weeks (n = 3 – 5). **p<.005 vs. WT-HFD, p<.05 vs. WT-Chow. Data are mean ± SEM.</p

Markers of VAT inflammation in WT and ANXA1 KO mice.

<p>Expression of CD68, IL-6, IL-1β, CCL2, and IL-10 mRNA in VAT (n = 8 – 10). p<.05 vs. WT-Chow, #p<.05 vs. KO-Chow. Data are mean ± SEM.</p

Expression of markers of lipolysis in VAT.

<p><u>Panel A</u>: Expression of ATGL, HSL, and Galectin-12 mRNA in fasted mice and FAS, ACC, and SREBF1 mRNA in fed mice in VAT (n = 5). <u>Panel B</u>: Ratio of pHSL to HSL protein in fasted mice in VAT by western blot (n = 3). *p<.05 vs. respective diet-matched WT group, p<.05 vs. WT-Chow, #p<.05 vs. KO-Chow. Data are mean ± SEM.</p

Rosiglitazone Improves Survival and Hastens Recovery from Pancreatic Inflammation in Obese Mice

Obesity increases severity of acute pancreatitis (AP) by unclear mechanisms. We investigated the effect of the PPAR-gamma agonist rosiglitazone (RGZ, 0.01% in the diet) on severity of AP induced by administration of IL-12+ IL-18 in male C57BL6 mice fed a low fat (LFD) or high fat diet (HFD), under the hypothesis that RGZ would reduce disease severity in HFD-fed obese animals. In both LFD and HFD mice without AP, RGZ significantly increased body weight and % fat mass, with significant upregulation of adiponectin and suppression of erythropoiesis. In HFD mice with AP, RGZ significantly increased survival and hastened recovery from pancreatic inflammation, as evaluated by significantly improved pancreatic histology, reduced saponification of visceral adipose tissue and less severe suppression of erythropoiesis at Day 7 post-AP. This was associated with significantly lower circulating and pancreas-associated levels of IL-6, Galectin-3, osteopontin and TIMP-1 in HFD + RGZ mice, particularly at Day 7 post-AP. In LFD mice with AP, RGZ significantly worsened the degree of intrapancreatic acinar and fat necrosis as well as visceral fat saponification, without affecting other parameters of disease severity or inflammation. Induction of AP lead to major suppression of adiponectin levels at Day 7 in both HFD and HFD + RGZ mice. In conclusion, RGZ prevents development of severe AP in obese mice even though it significantly increases adiposity, indicating that obesity can be dissociated from AP severity by improving the metabolic and inflammatory milieu. However, RGZ worsens selective parameters of AP severity in LFD mice

Effect of RGZ on hematological parameters in mice with AP.

<p>Mice received two injections of IL -12+ IL-18 and were evaluated at Day 1 and Day 7. Control mice received vehicle. Circulating while blood cells (WBC) (<b>A</b>), % neutrophils (<b>B</b>), % lymphocytes (<b>C</b>) and % monocytes (<b>D</b>) as well as red blood cells (RBC) (<b>E</b> absolute value, <b>F</b> % change from baseline), concentration of hemoglobin (<b>G</b> absolute value, <b>H</b> % change from baseline), and hematocrit (<b>I</b> absolute value, <b>I</b> % change from baseline) were quantified in blood obtained from LFD (green columns), LFD + RGZ (orange columns), HFD (blue columns) or HFD + RGZ (pink columns) groups. Data are mean +/− SEM of 8–12 mice per group.</p

Effect of RGZ on adipokine levels in mice with AP.

<p>Mice in the LFD (green columns), LFD + RGZ (orange columns), HFD (blue columns) or HFD + RGZ (pink columns) groups received two injections of IL-12+ IL-18 and were evaluated at Day 1 and Day 7. Control mice received vehicle. Circulating levels of leptin (<b>A</b>) and adiponectin (APN) (<b>B</b>) were measured by ELISA. Data are mean +/− SEM of 8–12 mice per group.</p

Experimental design.

<p>Timing of LFD and HFD feeding with and without RGZ, administration of vehicle or IL-12+ IL-18 (2 injections, 24 h apart), and termination of the experiment is shown.</p