Table 1.
General body composition, basal glucose and insulin.
Figure 1.
Indirect calorimetry of FAAH−/− and wild-type mice.
A) hr-hr B) 12 hr average. a) Oxygen consumption (VO2), b) Respiratory exchange ratio (RER) and c) Activity during the diurnal cycle and fasted to fed transitions. Day (light cycle) and night (dark cycle) 12 hours, (over) night fast −15 h, day re-fed −5 h in duration. n = 8, data are mean ± SEM, *p<0.05, **p<0.01 by Student's t-test.
Figure 2.
FAAH deficiency affects fuel storage.
a. Fed triglycerides and glycogen of liver and skeletal muscle. Upper panel shows thin layer chromatography (TLC) for hepatic and intra-muscular triglycerides with corresponding densitometry. The lower panel shows the amount of glycogen in the same tissues. Data are mean ± SEM, n = 4, *p<0.05, **p<0.01, ***p<0.001 FAAH−/− vs. wild-type by Student's t-test. b. Immunoblot analysis for Lipin 1 and DGAT 1 in overnight fasted (18 h) and 5 h re-fed liver (Top). Quantification normalized by actin content and arbitary units expressed relative to wild-type (Bottom). n = 4, data are mean ± SEM. *p<0.05, **p<0.01 by Student's t-test. c. De novo lipogenesis and Cholesterol synthesis. Synthesis rates measured in fed FAAH−/− mice vs. wild-type mice over a 10 day period. Data are mean ± SEM, n = 6, *p<0.05 by Student's t-test.
Figure 3.
FAAH deficiency affects disposition of hepatic acyl carnitine and acetyl-CoA.
a. Hepatic acyl carnitine profile in 18 h fasted and 5 h re-fed mice. Data are mean ± SEM, n = 4 for FAAH−/− vs wild-type mice. Non-repeated measures 2-way ANOVA was done to calculate statistical significance. P<0.001 for both row and columns factors. Bonferroni post-tests showed P<0.001 for FAAH−/− acetylcarnitine. b. Hepatic acetyl-CoA levels in 18 h fasted and 5 h re-fed mice. Data are mean ± SEM, n = 4, **p<0.01 by Student's t-test for FAAH−/− vs wild-type mice.
Figure 4.
FAAH deficiency causes dyslipidemia.
Fasted/re-fed plasma glucose and lipid profile for the FAAH−/− and wild-type mice. Plasma samples used were from [U-13C6] glucose (18 h fast) and [2-13C] glycerol (5 h re-feed) tracer infusion experiments. n = 5, data are mean ± SEM. **p<0.01, ***p<0.001 FAAH−/− vs. wild-type (fasted) and a p<0.05, b p<0.01, FAAH−/− vs. wild-type (fed) by Student's t-test.
Figure 5.
FAAH deficiency causes hepatic and adipose insulin resistance.
Glycerol production, and hepatic glucose production from glycerol, assessed using a [2-13C] glycerol infusion administered by Alza miniosmotic pump. Glycerol production represents mainly in vivo lipolysis, and was measured after 18 h of overnight fast. Fasted plasma glucose and insulin levels are given in the table. Glycerol production rate is expressed in terms of mg produced/kg of body weight/minute. n = 5, data are mean ± SEM. *p<0.05, ***p<0.001 wild-type vs. FAAH−/− mice by Student's t-test.
Table 2.
Fasted/Re-fed hepatic triose-p metabolites profile.
Figure 6.
FAAH deficiency causes fasted/re-fed dysregulation of hepatic TCA intermediates.
Hepatic TCA intermediates in 18 h fasted and 5 h re-fed mice. Data are mean ± SEM, n = 4, *p<0.05, **p<0.01, ***p<0.001 by Student's t-test in FAAH−/− mice vs. wild-type mice respectively.
Table 3.
Plasma glucose and insulin during [U-13C6] glucose pump assessment following an 18 hr fast.
Figure 7.
FAAH deficiency causes skeletal muscle insulin resistance.
Glucose disposal measured during the Stable isotope Glucose Tolerance Test (SipGTT) for chow fed, overnight fasted, FAAH−/− vs. wild-type mice. Time courses of plasma glucose (a), insulin (b) and [6, 6-2H2]-glucose (c) normalized to wild-type basal levels are shown (Left). Each point shown represents the mean ± SEM, n = 5. Integrated responses for the areas under the curve (AUC) are presented in the table shown (Top right). *p<0.05, **p<0.01 for (FAAH−/− vs. wild-type). Muscle (quadriceps) protein synthesis in FAAH−/− and wild-type mice (Bottom right) (d). Protein synthesis is represented as % newly made alanine made over the entire 10 day study. Data are mean ± SEM. *p<0.05, comparing n = 6 for wild-type and FAAH−/− mice by Student's t-test.
Figure 8.
FAAH deficiency causes dyregulates the hepatic acetylome.
Log10 ratios of the fasted and re-fed, wild-type and FAAH−/− liver acetylated peptides. The top 5% dramatically changed acetylated peptides are marked with red *. Acetylated peptides from the same proteins are grouped and shaded with gray background color. The 12 proteins are indicated with single letters on top of each group of peptides in the figure and their names are as follows: A. Superoxide dismutase; B. Aspartate aminotransferase; C. T-lymphoma invasion and metastasis-inducing protein 1; D. ATP synthase coupling factor 6; E. Fructose-bisphosphate aldolase B; F. Acetyl-CoA acetyltransferase; G. GTP:AMP phosphotransferase mitochondrial; H. 3-ketoacyl-CoA thiolase; I. ATP synthase D chain; J. Phosphoglycerate mutase 2; K. Malate dehydrogenase; L. Alcohol dehydrogenase.