Advantages of dynamic “closed loop” stable isotope flux phenotyping over static “open loop” clamps in detecting silent genetic and dietary phenotypes

In vivo insulin sensitivity can be assessed using “open loop” clamp or “closed loop” methods. Open loop clamp methods are static, and fix plasma glucose independently from plasma insulin. Closed loop methods are dynamic, and assess glucose disposal in response to a stable isotope labeled glucose tolerance test. Using PPARα−/− mice, open and closed loop assessments of insulin sensitivity/glucose disposal were compared. Indirect calorimetry done for the assessment of diurnal substrate utilization/metabolic flexibility showed that chow fed PPARα−/− mice had increased glucose utilization during the light (starved) cycle. Euglycemic clamps showed no differences in insulin stimulated glucose disposal, whether for chow or high fat diets, but did show differences in basal glucose clearance for chow fed PPARα−/− versus SV129J-wt mice. In contrast, the dynamic stable isotope labeled glucose tolerance tests reveal enhanced glucose disposal for PPARα−/− versus SV129J-wt, for chow and high fat diets. Area under the curve for plasma labeled and unlabeled glucose for PPARα−/− was ≈1.7-fold lower, P < 0.01 during the stable isotope labeled glucose tolerance test for both diets. Area under the curve for plasma insulin was 5-fold less for the chow fed SV129J-wt (P < 0.01) but showed no difference on a high fat diet (0.30 ± 0.1 for SV129J-wt vs. 0.13 ± 0.10 for PPARα−/−, P = 0.28). This study demonstrates that dynamic stable isotope labeled glucose tolerance test can assess “silent” metabolic phenotypes, not detectable by the static, “open loop”, euglycemic or hyperglycemic clamps. Both open loop and closed loop methods may describe different aspects of metabolic inflexibility and insulin sensitivity

Peripheral Effects of FAAH Deficiency on Fuel and Energy Homeostasis: Role of Dysregulated Lysine Acetylation

FAAH (fatty acid amide hydrolase), primarily expressed in the liver, hydrolyzes the endocannabinoids fatty acid ethanolamides (FAA). Human FAAH gene mutations are associated with increased body weight and obesity. In our present study, using targeted metabolite and lipid profiling, and new global acetylome profiling methodologies, we examined the role of the liver on fuel and energy homeostasis in whole body FAAH(-/-) mice.FAAH(-/-) mice exhibit altered energy homeostasis demonstrated by decreased oxygen consumption (Indirect calorimetry). FAAH(-/-) mice are hyperinsulinemic and have adipose, skeletal and hepatic insulin resistance as indicated by stable isotope phenotyping (SIPHEN). Fed state skeletal muscle and liver triglyceride levels was increased 2-3 fold, while glycogen was decreased 42% and 57% respectively. Hepatic cholesterol synthesis was decreased 22% in FAAH(-/-) mice. Dysregulated hepatic FAAH(-/-) lysine acetylation was consistent with their metabolite profiling. Fasted to fed increases in hepatic FAAH(-/-) acetyl-CoA (85%, p<0.01) corresponded to similar increases in citrate levels (45%). Altered FAAH(-/-) mitochondrial malate dehydrogenase (MDH2) acetylation, which can affect the malate aspartate shuttle, was consistent with our observation of a 25% decrease in fed malate and aspartate levels. Decreased fasted but not fed dihydroxyacetone-P and glycerol-3-P levels in FAAH(-/-) mice was consistent with a compensating contribution from decreased acetylation of fed FAAH(-/-) aldolase B. Fed FAAH(-/-) alcohol dehydrogenase (ADH) acetylation was also decreased.Whole body FAAH deletion contributes to a pre-diabetic phenotype by mechanisms resulting in impairment of hepatic glucose and lipid metabolism. FAAH(-/-) mice had altered hepatic lysine acetylation, the pattern sharing similarities with acetylation changes reported with chronic alcohol treatment. Dysregulated hepatic lysine acetylation seen with impaired FAA hydrolysis could support the liver's role in fostering the pre-diabetic state, and may reflect part of the mechanism underlying the hepatic effects of endocannabinoids in alcoholic liver disease mouse models

Indirect calorimetry of FAAH^−/− and wild-type mice.

<p>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.</p

General body composition, basal glucose and insulin.

<p>Data are mean±SEM.</p>*<p>p<0.05,</p>**<p>p<0.01 by Student's t-test for wild-type vs. FAAH^−/− mice.</p

Fasted/Re-fed hepatic triose-p metabolites profile.

<p>Values are data±SEM,</p>*<p>p<0.05, FAAH^−/− vs. wild-type mice by Student's t-test.</p

FAAH deficiency affects fuel storage.

<p>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. <i>c.</i> 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.</p

FAAH deficiency causes skeletal muscle insulin resistance.

<p>Glucose disposal measured during the Stable isotope Glucose Tolerance Test (SipGTT) for chow fed, overnight fasted, FAA^H−/− 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 (FAA^H−/− vs. wild-type). Muscle (quadriceps) protein synthesis in FAA^H−/− 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 FAA^H−/− mice by Student's t-test.</p