It is often a challenge to undertake human studies of metabolic physiology specific enough to inform us about organ or tissue function. For example, as the transition from fasted to fed state reflects key adaptations to sustain life, there can be substantial change in the flux of metabolic substrates. Indeed, in some organs, a complete shift in the preference for metabolic substrate may occur. Thus, studying human organ/tissue metabolism in the fasted state may demonstrate only one facet of metabolic physiology.
Here, we describe methods we have used to enhance understanding of human tissue-specific metabolism, focusing in particular on adipose depots.
ARTERIO-VENOUS (A-V) BLOOD SAMPLING
This is a classic method of isolating an organ as a metabolic system. It can be highly informative for certain organs, but hard to apply to systems where sampling is difficult or when the organ has cellular or metabolic heterogeneity. The A-V technique is based on the Fick principle, and has been used to study leg and forearm (approximating to skeletal muscle)1–3 and heart,4 with hepatic vein sampling used to study the liver.5,6 Sampling of the inferior epigastric vein and the saphenous vein has been used to study subcutaneous abdominal and femoral adipose tissue respectively.2,3,7–10
'Rapid progress in imaging is providing less invasive techniques for tissue-specific metabolic physiology studies'
Where arterial blood cannot be obtained, the arterialised-venous (heated hand) technique has been used. The potential effects of body temperature and skin blood flow must be taken into account in this method, and it may be unsuitable when studying a tissue’s oxygen consumption.11,12 The position of venous catheters also needs careful consideration as some veins, such as the femoral vein, receive blood from leg muscles as well as adipose tissue and skin.12
The A-V difference technique, combined with measuring the blood flow of the organ/tissue of interest, allows for measurement of net flux of a substance, along with the clearance of a substance by the tissue. We have used A-V difference methodology, with selective venous catheterisation of subcutaneous abdominal adipose tissue and forearm skeletal muscle, in studies with stable isotopes to describe the metabolic characteristics of these tissues after an overnight fast and after a mixed test meal.3 Chylomicron (dietary/exogenous) and very low-density lipoprotein (VLDL; endogenous) triacylglycerol (TG) were both cleared across adipose tissue and muscle, with the fractional extraction of chylomicron-TG being notably greater than that of VLDL-TG.3
Using A-V difference across subcutaneous abdominal adipose tissue and feeding mixed meals 5 hours apart, we found the adipose tissue of lean males was more metabolically active over a 24-hour period than that of abdominally obese subjects.13 In particular, abdominally obese males stored a significantly lower proportion of dietary fat in subcutaneous abdominal adipose tissue than did lean men, which may increase the amount of fat stored in liver and skeletal muscle.13
We have also used A-V methodology to describe the metabolic characteristics of subcutaneous abdominal and gluteofemoral adipose tissue in the fasting and postprandial states14 and after adrenergic stimulation.15 We found distinct metabolic differences in fatty acid flux and blood flow between the fat depots, with the gluteofemoral depot being more metabolically inactive compared with the abdominal depot.14,15
USING METABOLIC TRACERS
Metabolic tracers (stable and radioisotopes) provide the opportunity to specifically study tissue metabolism or probe specific metabolic pathways. Factors to be considered include the method of delivery, the natural abundance of the tracer, the metabolic handling of the tracer, the molecules being labelled, background meal effects and the time between repeat study visits.
Rapid progress in imaging is providing less invasive techniques for tissue-specific metabolic physiology studies. Metabolic imaging modalities include positron emission tomography (PET), magnetic resonance imaging/ spectroscopy (MRI/S), computed tomography (CT) and ultrasound (US). Along with assessing substrate metabolism, imaging modalities can measure organ-specific perfusion (PET), tissue density and type of adipose tissue (CT), ectopic fat deposition, metabolite content and blood oxygenation levels in the brain (MRI/S) and liver stiffness/fat (US).16
Tracers have been used in combination PET and MRI/S to investigate fatty acid and glucose metabolism in vivo in humans. For example, by feeding individuals a meal containing 13C-fatty acids and then measuring the 13C signal in the liver with MRI/S at intervals postprandially, the flux of dietary fatty acids across the liver was found to be notably slower in individuals with high compared with low liver fat content.17
Hyperpolarised MRI (HP MRI) is an emerging technique allowing detection of 13C-enriched molecules with a significantly increased signal compared with conventional techniques. HP MRI will make it possible to follow single and multiple metabolic pathways using single or multiple hyperpolarised probes, along with tracing the real-time conversion of substrate to its metabolic products,18 but the technical platforms are expensive and experience in humans is limited.
PET is an alternative non-invasive imaging approach, which has been used to investigate organ- or tissue-specific metabolism. This has typically been undertaken in the fasting state. Using PET and 11C-palmitate, obese subjects were shown to have increased hepatic fatty acid oxidation, with no difference in hepatic fatty acid uptake and esterification rates, compared with non-obese controls in the fasting state.19
More recently, the metabolic probe 18F-FTHA and PET were used to study metabolic changes in adipose tissue in obese individuals before and after weight loss.20 After weight loss, there was no change in the uptake of non-esterified fatty acids into visceral and subcutaneous abdominal adipose tissues, but uptake by femoral adipose tissue was significantly decreased.
The short half-life of the radioisotopes suitable for PET restricts this technique to studying metabolic pathways with a short time frame, such as glucose or fatty acid uptake. Metabolic conversions, such as glycolysis or fatty acid oxidation, cannot be studied. A final consideration is that PET can detect incorporation of radiolabelled tracers into a tissue but cannot distinguish the parent compound from the formed radiolabelled product.18
In summary, studies of tissue-specific metabolic function have classically relied on complex A-V balance techniques, preferably using labelled metabolic tracers. However, novel metabolic imaging techniques are creating new opportunities for such studies in humans.
Leanne Hodson and Fredrik Karpe
Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford
- Olsen DB et al. 2005 Journal of Physiology 565 555–562.
- Coppack SW et al. 1990 Metabolism 39 384–390.
- Bickerton AS et al. 2007 Diabetes 56 168–176.
- Funada J et al. 2009 PLoS One 4 e7533.
- Havel RJ et al. 1970 Journal of Clinical Investigation 49 2017–2035.
- Westerbacka J et al. 2010 Gastroenterology 139 1961–1971 e1.
- Frayn KN & Coppack SW 2001 Methods in Molecular Biology 155 269–279.
- Frayn KN et al. 1993 International Journal of Obesity & Related Metabolic Disorders 17 Suppl 3 S18–S21, S22.
- Frayn KN et al. 1994 American Journal of Physiology 226 E308–E317.
- McQuaid SE et al. 2010 Obesity (Silver Spring) 18 1055–1058.
- Frayn KN 1999 Proceedings of the Nutrition Society 58 877–886.
- Macdonald IA 1999 Proceedings of the Nutrition Society 58 871–875.
- McQuaid SE et al. 2011 Diabetes 60 47–55.
- McQuaid SE et al. 2010 Diabetes 59 2465–2473.
- Manolopoulos KN et al. 2012 Diabetologia 55 3029–3037.
- Iozzo P 2015 Annals of the New York Academy of Sciences 1353 21–40.
- Ravikumar B et al. 2005 American Journal of Physiology – Endocrinology & Metabolism 288 E789–E797.
- Di Gialleonardo V et al. 2016 Seminars in Nuclear Medicine 46 28–39.
- Iozzo P et al. 2010 Gastroenterology 139 846–856, 856.e1–856.e6.
- Bucci M et al. 2015 Diabetologia 58 158–164.