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In-vivo metabolic studies of regional adipose tissue

Melvin, Audreya; McQuaid, Siobhán E.b,c

Cardiovascular Endocrinology & Metabolism: December 2018 - Volume 7 - Issue 4 - p 75–79
doi: 10.1097/XCE.0000000000000154
Review articles

The accumulation of abdominal adipose tissue has long been associated with adverse cardiovascular outcomes. Paradoxically, increased gluteofemoral adipose tissue, which is predominantly subcutaneous fat, seems to play a protective role. There has been significant scientific interest in understanding how abdominal and gluteofemoral depots confer opposing metabolic risks. However, the study of regional adipose physiology in vivo remains challenging. We discuss some of the methodologies used. We focus specifically on the arteriovenous difference technique and present some insights into gluteofemoral adipose physiology.

aDepartment of Diabetes and Endocrinology and Metabolic Research Laboratory, Wellcome Trust-MRC Institute of Metabolic Science, University of Cambridge, Cambridge, UK

bDepartment of Diabetes and Endocrinology, Mater Misericordiae University Hospital

cUniversity College Dublin, Dublin, Ireland

Correspondence to Siobhán E. McQuaid, DPhil, FRCPI, Department of Diabetes and Endocrinology, Mater Misericordiae University Hospital, Dublin 7, D07 XA09, Ireland Tel: +353 18 034 610; fax: +353 18 034 637; e-mail: smcquaid@mater.ie

Received December 18, 2017

Accepted February 20, 2018

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Introduction

Adipose tissue (AT) is a metabolically active tissue whose primary function is the storage and mobilization of lipid energy. It also plays a role in insulation, mechanical support, in immunity and as an endocrine organ.

White AT is distributed throughout the body, mainly in subcutaneous and visceral depots. In lean patients, subcutaneous fat makes up about 80% and visceral fat about 5–10% of the total body fat 1. Lower body or gluteofemoral fat accumulation (also called ‘pear’ shape, gynoid obesity) refers to the distribution of fat on the lower body and is predominantly subcutaneous fat of the gluteal and femoral regions (GFSAT). It is commonly delineated as all AT caudad to the inguinal ligament anteriorly and the iliac crest posteriorly 2. Clinically, it can be measured using hip, thigh or leg circumferences. Central or abdominal fat accumulation consists of subcutaneous and visceral fat and can be measured with a waist circumference. This is also known as the ‘apple’ shape or android obesity.

Although the prevalence of obesity has increased markedly, it is now recognized that it is not just the total amount of AT that is important, but also where that fat is distributed. The relationship between increased abdominal fat and altered lipid and glucose metabolism has long been reported in the literature, with large-scale epidemiological studies confirming the correlation between increased waist-to-hip ratio and adverse cardiovascular outcomes 3,4. Indeed, both waist circumference and BMI are embedded in most definitions of the metabolic syndrome.

Paradoxically, increased gluteofemoral fat has been shown to correlate with protection from adverse metabolic consequences, a relationship that is independent of BMI 5,6. There is significant variation in fat distribution between men and women, with differences in the hormonal milieu between sexes exerting an influence over AT depots 7. Recently, genomewide analysis of anthropometric traits has identified a number of genetic loci associated with waist-related phenotypes. Interestingly, there is a female-specific single-nucleotide polymorphism (SNP) effect that is evident for these loci 8,9. This observed sexual dimorphism in the heritability of AT distribution, in addition to observations of hormone dependant depot regulation, supports a relationship between genetic and hormonal factors in the determination of AT distribution. The study of individuals with monogenic partial lipodystrophies, such as those associated with mutations in PPARG, who have a phenotype of reduced peripheral subcutaneous AT complicated by ectopic fat storage, nonalcoholic fatty liver disease, insulin resistance (IR), diabetes mellitus and dyslipidaemia, further highlights the protective benefits of gluteofemoral fat depots 10. More recently, individuals enriched for SNPs in 53 genomic regions significantly associated with an IR phenotype (adjusted for body mass) were reported to have reduced gynoid and leg fat mass compared with those with a lower SNP score. Interestingly, among more than 9000 individuals followed for a median of 3.7 years, those with a higher IR SNP score were less likely to have fat deposit in the gynoid/leg region when corrected for the degree of weight gain 11. Epidemiological and genetic evidence supports the protective role of GFSAT expansion in protection against adverse metabolic sequelae, type 2 diabetes and cardiovascular disease. This has led us to reconsider abdominal obesity as a marker of impaired gluteofemoral function, representing ectopic fat deposition in cases where gluteofemoral storage capacity has been exhausted 12. These observations may reflect a process of adipose expandability, where humans have a predetermined threshold for AT storage and once met, additional lipid storage requirements are accommodated by ectopic nonadipocyte cells 13.

There has been significant scientific interest in understanding how abdominal subcutaneous AT (ASAT) and GFSAT confer opposing metabolic risks. Certainly, both depots respond differently to increased energy intake, with lower body subcutaneous depots expanding through increased adipogenesis, whereas adipocyte hypertrophy is observed in ASAT 14. The distinctive responses are likely related to the differing subtypes of the adipocyte progenitor preadipocytes within these regions 15. It may be that ASAT and GFSAT are developmentally distinct on the basis of the differential developmental gene expression observed in preadipocytes from both depots 15,16. Determining how these depots functionally behave in humans is critical not only to understanding how GFSAT confers its protective properties but also to ascertain the potential for manipulating GFSAT physiology for therapeutic benefits.

The study of regional adipose physiology in humans, however, remains challenging. In this review, we will briefly discuss a number of techniques that have been utilized to study AT physiology in vivo and specifically focus on the role of direct measurement of GFSAT metabolism using the arteriovenous (AV) concentration difference method.

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Techniques used in the study of adipose metabolism in humans in vivo

Although the study of AT physiology in rodent models has been informative, the striking anatomical differences in fat distribution between animals and humans makes it difficult to draw comparisons between the species. As such, a number of dynamic techniques, outlined below, have been used in the interrogation of AT physiology in vivo that have advanced our understanding of AT function.

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Isotopes/tracer studies

Tracer studies have been used extensively in the study of AT metabolism. They use isotopes, which are elements that are identical in chemical structure, but with a different mass to the commonly occurring element in a molecule. Isotopes of carbon, hydrogen and nitrogen are most frequently incorporated into molecules to create a tracer for metabolic studies 16. The principle of this technique is based on the tracer-dilution method. During the continuous infusion of a specific tracer, the rate of appearance of new molecules in the blood stream can be calculated using the dilution of the tracer by Steele’s equation, where the infusion rate of the tracer is known and the plasma level can be measured commonly using liquid or gas chromatography/mass spectroscopy 17,18. Tracer techniques using glycerol or fatty acids can be labelled with stable or radioactive isotopes, enabling determination of whole-body lipolysis rates 19,20. The administration of tracers followed by biopsy of different adipose depots has been used to provide information about substrate turnover in regional depots 21,22. However, a disadvantage of isotope methods is that an indirect measure of triglyceride kinetics is obtained and is founded on a number of assumptions.

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Microdialysis

Microdialysis is based on the measurement of substrate transport across a semipermeable membrane of a microdialysis probe 23. Microdialysis was adapted for use in AT where the tube is sited in subcutaneous AT and perfused with a solvent, which is collected, and the glycerol content is assayed as a measure of intracellular lipolysis 24. One particular advantage of this technique is that the system allows for the delivery of pharmacological agents whose influence over AT metabolism can be determined. However, microdialysis can only measure hydrophilic molecules and not hydrophobic ones such as nonesterified fatty acids (NEFA) and triglyceride (TG), which are substrates of major importance within AT metabolism. This method may overestimate lipolysis. Without the simultaneous quantification of the tissue blood flow, it is difficult to estimate fatty acid release from AT as the microdialysis method does not differentiate between hormone sensitive-derived and lipoprotein lipase-derived sources of glycerol 25.

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Arteriovenous concentration difference

The AV difference technique was initially described in the 1950s 26 before being adapted to directly study the function of AT depots in humans 27. By measuring differences in the composition between the afferent and efferent blood flow within subcutaneous AT, determinations of the metabolic activity of that tissue can be made. If one subsequently factors in blood flow, then the rate of substrate exchange in a given volume of tissue (flux) can be calculated. The AV difference technique, although technically more challenging than microdialysis, when applied to AT, enables (i) incorporation of AT blood flow (ATBF) measurement, (ii) the assessment of a relatively homogenous isolated AT depot, (iii) the measurement of hydrophobic molecules (NEFA, TG) and (iv) direct comparison of adipokine release by different AT depots. It is essential to the success of this technique that the efferent venous sampling site specifically drains the depot of interest. In the case of ASAT, a branch of the superficial inferior epigastric vein is cannulated in an anterograde direction, with the tip of the cannula lying above the inguinal ligament 27. Its patency is maintained with a saline infusion, allowing for serial sampling. The retrograde cannulation of a hand vein heated in air at 60°C 28 using a hot box enables the collection of arterialized blood, reflecting afferent flow to the tissue of interest. Arterial cannulation is also suitable. To adapt the technique to GFSAT, a modified AV difference technique capturing the venous drainage of GFSAT was developed, whereby a branch of the great saphenous vein is cannulated. This technique was validated against abdominal subcutaneous tissue drainage under postabsorptive, postprandial and β-adrenergic stimulation conditions 29. This particular technique and the direction of catheter placement avoided the potential risk of effluent from the inferior epigastric artery (abdominal subcutaneous tissue drainage) being incorporated into samples.

ATBF is integral to the dynamic function of AT and is highly regulated with a number of nutritive factors known to elicit a response 30. The gold-standard measurement of ATBF is considered the Xenon-133 washout method, which exploits the inert and lipophilic properties of Xenon-133, a γ-emitting radioisotope 31. The technique, initially described by Larsen and colleagues, requires the injection of radiolabelled Xenon into subcutaneous AT, with the rate of radioactive decay measured using a detector close to the skin 32,33. ATBF is determined by the product of the slope of radioactive decay and the partition coefficient of AT 34.

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Insights into GFSAT physiology using an AV concentration difference approach

In the assessment of metabolic function in adipose depots, AV concentration difference allows for the direct measurement of metabolites. The incorporation of ATBF and stable isotope measurements into AV protocols enables a more detailed interrogation of the physiology.

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Lipid handling

One of the essential functions of AT is the storage of fatty acids and given the differential effects of ASAT and GFSAT on metabolic risk, it was paramount to determine the relative contribution of both depots to the NEFA pool and their ability to sequester lipids. Tan and colleagues explored this in healthy male volunteers, using the AV concentration difference approach to enable direct measurement of TG and NEFA across each depot. Simultaneous measurement did not show a difference in the rates of TG uptake between depots when the lower rate of gluteofemoral blood flow was accounted for. However, NEFA release was significantly lower in the gluteofemoral depot compared with abdominal tissue 35. A direct comparison between ASAT and GFSAT following meal ingestion was performed using a similar approach. GFSAT behaved similarly to ASAT following a mixed meal with NEFA suppression, increased glucose uptake and TG storage. However, the addition of isotope-labelled palmitate allowed for the determination of very low-density lipoprotein-derived TG uptake across each depot. GFSAT, unlike the abdominal subcutaneous depot, did not differentiate between dietary chylomicron-TG and very low-density lipoprotein-TG to the same extent as ASAT, which preferentially stored chylomicron-derived TG 36.

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Adrenergic stimulation

Adrenergic activity is a recognized stimulus for AT lipolysis and it was hypothesized that the GFSAT lipolytic response to endogenous catecholamines may differ from ASAT. Regional lipolytic differences in AT responsiveness to sympathetic stimuli have been measured indirectly using microdialysis and tracer techniques, but the results were inconsistent 37–39. To resolve this, during a systemic infusion of adrenaline, NEFA, as a marker of lipolysis, was measured from a branch of the great saphenous vein using an AV concentration difference method. Gluteofemoral efferent NEFA were significantly lower than that observed from abdominal venous drainage. The lipolysis-suppressing effect of adrenaline as an α-adrenergic and β-adrenergic receptor agonist was ameliorated by the local micro perfusion of an α-blocking agent into femoral AT. This suggests that GFSAT, in contrast to ASAT, was predominantly regulated by α-adrenergic activity that exerted a suppressive effect over lipolysis, resolving the controversies of earlier in-vivo studies 40.

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Adipokine release

Differences in adipokine secretion could contribute toward regional variations in AT function. Depot-specific adipokine release (interleukin-6, monocyte chemoattractant protein-1 and leptin) was measured in age-matched and BMI-matched patients using the AV difference technique. Release of leptin and monocyte chemoattractant protein-1 was similar across both ASAT and GFSAT. However, AV release of the proinflammatory cytokine interleukin-6 was lower from GFSAT than ASAT 41, suggesting that GFSAT is more resistant to the proinflammatory state.

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Hypercortisolaemia

A partial lipodystrophic phenotype has been observed in states of endogenous and exogenous cortisol excess in addition to high circulating NEFA. It had been hypothesized that hypercortisolaemia induces peripheral adipose depot lipolysis mediating the lipodystrophic appearance and contributing towards the circulating NEFA pool. Microdialysis techniques have been used to determine the depot-specific effects of hypercortisolaemia on AT in vivo, with conflicting results. These studies, which calculate lipolysis rates on the basis of glycerol measurement, have reported either no difference in lipolysis rates between depots or increased lipolysis within abdominal subcutaneous adipose depots in response to pharmacological hypercortisolaemia 42,43. Recently, an AV concentration difference approach was used to address this controversy. Investigators directly measured lipolysis within the subcutaneous adipose depots following a hydrocortisone infusion. Acute hypercortisolaemia was associated with a significant increase in lipolysis within abdominal adipose depots contributing towards the systemic increase in NEFA observed in hypercortisolaemia; however, there was no evidence of increased lipolysis in femoral AT 44. These findings are in conflict with the authors’ hypothesis, but provide direct evidence of the influence of cortisol on specific adipose depot function as well as reaffirming the role of gluteofemoral AT in sequestering fatty acid stores.

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Conclusion

The terms ‘android’ and ‘gynoid’ fat were initially coined by Jean Vague in the 1940s. In the 21st century, the AV difference technique in conjunction with stable isotopically-labelled fatty acid methodology, as applied to the abdominal and gluteofemoral AT depots, can help to further dissect and understand the relationship between these two AT depots. Determining how GFSAT exerts its protective effects could have potential health implications and reveal new therapeutic targets for the management of diabetes, insulin-resistant states and lipodystrophy.

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Acknowledgements

Conflicts of interest

There are no conflicts of interest.

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Keywords:

abdominal; adipose; arteriovenous; gluteofemoral; subcutaneous fat

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