Glucose metabolism in the Fgl1 null mouse.

<p>A) <i>Fgl1^−/−</i> mice have fasting hyperglycemia (P = 0.005, n = 10 for <i>Fgl1^+/+</i> and n = 8 for <i>Fgl1^−/−</i>). B) Glucose tolerance tests of fasted 3 month old mouse. The graph represents a plot of plasma glucose versus time after i.p. administration of glucose. The superimposed panel represents plots of average area under the curve (AUC) for each mouse. The baseline was set as the mean pre-glucose administration plasma level. The difference is glucose levels as determined from the AUC is significant (P = 0.002, n = 4 per group). C) Insulin tolerance test on 3 month old fasted mouse shows a similar rate of decline of glucose levels between the <i>Fgl1^+/+</i> and <i>Fgl1^−/−</i> mice. AUC calculations after normalizing for baseline glucose for the first 45 min of the test shows no differences between Fgl1 containing and deficient mice (n = 5 per group). D) Insulin levels are not different between <i>Fgl1^+/+</i> and <i>Fgl1^−/−</i> mice. E) HOMA scores are not different for <i>Fgl1^+/+</i> and <i>Fgl1^−/−</i> (n = 5 for <i>Fgl1^+/+</i> and 4 for <i>Fgl1^−/−</i>). F) Graph of glucose levels following administration of pyruvate to fasted mice. Inset is AUC calculation which shows a difference in glucose levels over the duration of the experiment (P = 0.029, n = 5 per group).</p

Length and tissue mass of <i>Fgl1^+/+</i> and <i>Fgl1^−/−</i> mice.

<p>(n = 5 for both cohorts except for liver (*) where n = 9).</p

Structure, content and activity of adipose tissues in the Fgl1 null mouse.

<p>A) Representative H&E stains (40× magnification) of brown adipose tissue. Lipid droplets are larger in <i>Fgl1^−/−</i> mice. B) Quantitation of lipid droplet size show significant difference between <i>Fgl1^+/+</i> and <i>Fgl1^−/−</i> mice (P = 0.011, n = 5 per group). C) Expression of brown adipose genes. Note the paradoxical up regulation of DiO2 and UCP1 (P = 0.002 and 0.0001 respectively. n = 11 per group except for Perilipin and HSL where n = 5 and 6 respectively). D) ¹⁸FDG incorporation into BAT. The % uptake represents the uptake of injected dose per gram of tissue. Note the marked decrease in radioisotope uptake in BAT (P = 0.05, n = 5 per group). E). Representative H&E stains (40× magnification) of white adipose tissue. Lipid droplets are larger in <i>Fgl1^−/−</i> mice. F) Quantitation of number of cells per HPF shows smaller number of white adipose cells in <i>Fgl1^−/−</i> (P = 0.005, n = 5). G) Expression of white adipose genes. Glut4, leptin and perilipin are significantly down regulated (*) with a P<0.04 for each. P for ATGL (#) is 0.06. n = 4–6 mice per group.</p

Food intake and indirect calorimetry.

<p>Scatter plot of average food intake versus average weight for individually housed <i>Fgl1^+/+</i> (blue squares, n = 4) and <i>Fgl1^−/−</i>(red circles, n = 8) taken daily over an 18 day period. Note that <i>Fgl1^−/−</i> remain larger that wild types for the duration of the experiment. B and C) Indirect calorimetric values for VO₂ and VCO₂ respectively and D) RER. The RER is significant irrespective of day and night cycles (P = 0.04 and 0.016 respectively) and over the entire 24 h (P = 0.019). E) Heat generation is not significantly different and F) activity is not different between the Fgl1 containing and deficient mice. n = 6 for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058084#pone-0058084-g006" target="_blank">Figures 6B to 6F</a>.</p

The liver in the Fgl1 knockout mouse.

<p>A) Fgl1 transcript is absent in the livers of the knockout mouse pre and post PH. Note the expected induction of Fgl1 in the wild type mouse after PH (P<0.01 for Fgl1 at baseline and 48 h after PH, n = 3 per group) B) Fgl1 protein is absent in the livers of the knockout mouse before and after PH while it is detectable at baseline and induced after PH in the wild type mouse. C) <i>Fgl1^−/−</i> mice are larger than wild type mates as early as three weeks after birth (P<0.0001, n = 5 for each group). D) Representative graph of change in weight over time for <i>Fgl1^+/+</i> (n = 8 for first 4 weeks, n = 4 for last three weeks) and <i>Fgl1^−/−</i> (n = 9 for first 4 weeks and n = 8 for last three weeks). E) Liver mass is not different between <i>Fgl1^+/+</i> and <i>Fgl1^−/−</i> mice (n = 9 for each group). F) Liver weight to body weight ratio is smaller for <i>Fgl1^−/−</i> mice (P = 0.008, n = 9). G) Marked lipid accumulation in the livers of <i>Fgl1^−/−</i> mice after PH. Top: gross images of representative livers from <i>Fgl1^+/+</i> (left) and <i>Fgl1^−/−</i> (right). Arrows indicate remnant liver lobes. Middle: H&E images at 40× magnification of liver sections from <i>Fgl1^+/+</i> and <i>Fgl1^−/−</i> mice at 48 h post PH and bottom: similar H&E images at 96 h post PH. Note the resolution of steatosis in the <i>Fgl1^−/−</i> mouse by 96 h after PH. H) Triglyceride (TG) content of liver extracts from <i>Fgl1^+/+</i> and <i>Fgl1^−/−</i> mice before and after PH. The difference between <i>Fgl1^−/−</i> and <i>Fgl1^+/+</i> at 48 h after PH is significant (P = 0.014, n = 3–5 per cohort for experiments). I) mRNA levels of lipid regulatory genes at 48 h after PH (P = 0.011, 0.014 and 0.037 respectively for PPARα, PPARδ and FATP5 but is otherwise non significant). n = 4 for <i>Fgl1^+/+</i> except FATP5 where n = 3 per group. n = 5 for <i>Fgl1^−/−</i>.</p

Fgl1 in hepatic and brown adipose tissue.

<p>A) mRNA levels of Fgl1 in the liver at baseline (0 h) and at 24 h and 48 h after PH. Note the increased expression after PH. P = 0.002 between 0 h and 24 h and less than 0.0001 between 0 h and 48 h after PH. The difference between levels at 24 h and 48 h is not significant. B) mRNA levels of Fgl1 in brown adipose tissue (BAT) at baseline and at 24 h and 48 h after partial hepatectomy. Fgl1 is detectable in BAT prior to injury but expression is enhanced after PH. P = 0.026 between 0 and 24 h and P = 0.013 between 0 and 48 h after PH. The difference between levels at 24 h and 48 h is not significant. n = 3 for samples in 1A and B. C) Gel electrophoresis of amplified cDNA from BAT at baseline and at 24 h and 48 h after PH. Cyclophilin A is the loading control. D) Comparison of BAT Fgl1 levels pre and post PH with hepatic Fgl1 levels at baseline. Samples are normalized to hepatic Fgl1 at 100%. Fgl1 in BAT is 0.4%, 2.6% and 3.4% of hepatic levels at baseline and at 24 h and 48 h after PH (P<0.0001).</p

Plasma lipid, cholesterol and free fatty acid levels in the Fgl1 null mouse.

<p>A) There is no significant difference in steady state plasma TG levels of <i>Fgl1^+/+</i> and <i>Fgl1^−/−</i> mice. B) Free fatty acid levels are decreased in the Fgl1 null mouse (P = 0.001). C and D) Plasma cholesterol levels are levels are lower as determined by total cholesterol (P = 0.003) and FPLC analysis. n = 5 per group.</p