Brown Adipose Tissue: A Short Historical Perspective.

Brown adipose tissue (BAT) was first identified by Conrad Gessner in 1551, but it was only in 1961 that it was firmly identified as a thermogenic organ. Key developments in the subsequent two decades demonstrated that: (1) BAT is quantitatively important to non-shivering thermogenesis in rodents, (2) uncoupling of oxidative phosphorylation through a mitochondrial proton conductance pathway is the central mechanism by which heat is generated, (3) uncoupling protein-1 is the critical factor regulating proton leakage in BAT mitochondria. Following pivotal studies on cafeteria-fed rats and obese ob/ob mice, BAT was then shown to have a central role in the regulation of energy balance and the etiology of obesity. The application of fluorodeoxyglucose positron emission tomography in the late 2000s confirmed that BAT is present and active in adults, resulting in renewed interest in the tissue in human energetics and obesity. Subsequent studies have demonstrated a broad metabolic role for BAT, the tissue being an important site of glucose disposal and triglyceride clearance, as well as of insulin action. BAT continues to be a potential target for the treatment of obesity and related metabolic disorders.

Adipokines: inflammation and the pleiotropic role of white adipose tissue.

I had been working on the endocrine and signalling role of white adipose tissue (WAT) since 1994 following the identification of the ob (Lep) gene(1), this after some 15 years investigating the physiological role of brown adipose tissue. The ob gene, a mutation in which it is responsible for the profound obesity of ob/ob (Lepob/Lepob) mice, is expressed primarily in white adipocytes and encodes the pleiotropic hormone leptin. The discovery of this adipocyte hormone had wide-ranging implications, including that white fat has multiple functions that far transcend the traditional picture of a simple lipid storage organ.

Oxygen in Metabolic Dysfunction and Its Therapeutic Relevance.

Significance: In recent years, a number of studies have shown altered oxygen partial pressure at a tissue level in metabolic disorders, and some researchers have considered oxygen to be a (macro) nutrient. Oxygen availability may be compromised in obesity and several other metabolism-related pathological conditions, including sleep apnea-hypopnea syndrome, the metabolic syndrome (which is a set of conditions), type 2 diabetes, cardiovascular disease, and cancer. Recent Advances: Strategies designed to reduce adiposity and its accompanying disorders have been mainly centered on nutritional interventions and physical activity programs. However, novel therapies are needed since these approaches have not been sufficient to counteract the worldwide increasing rates of metabolic disorders. In this regard, intermittent hypoxia training and hyperoxia could be potential treatments through oxygen-related adaptations. Moreover, living at a high altitude may have a protective effect against the development of abnormal metabolic conditions. In addition, oxygen delivery systems may be of therapeutic value for supplying the tissue-specific oxygen requirements. Critical Issues: Precise in vivo methods to measure oxygenation are vital to disentangle some of the controversies related to this research area. Further, it is evident that there is a growing need for novel in vitro models to study the potential pathways involved in metabolic dysfunction to find appropriate therapeutic targets. Future Directions: Based on the existing evidence, it is suggested that oxygen availability has a key role in obesity and its related comorbidities. Oxygen should be considered in relation to potential therapeutic strategies in the treatment and prevention of metabolic disorders. Antioxid. Redox Signal. 35, 642-687.

Through fat and thin - a journey with the adipose tissues.

The paper is based on the lecture that I gave on receiving the Nutrition Society's inaugural Gowland Hopkins Award for contributions to Cellular and Molecular Nutrition. It reviews studies on the adipose tissues, brown and white, conducted by the groups that I have led since entering nutrition research in 1975. The initial focus was on exploring metabolic factors that underpin the development of obesity using animal models. This resulted in an interest in non-shivering thermogenesis with brown adipose tissue being identified as the key effector of facultative heat production. Brown fat is less thermogenically active in various obese rodents, and major changes in activity are exhibited under physiological conditions such as lactation and fasting consistent with a general role for the tissue in nutritional energetics. My interests moved to white adipose tissue following the cloning of the Ob gene. Our initial contributions in this area included demonstrating nutritional regulation of Ob gene expression and circulating leptin levels, as well as a regulatory role for the sympathetic nervous system operating through ß3-adrenoceptors. My interests subsequently evolved to a wider concern with the endocrine/signalling role of adipose tissue. Inflammation is a characteristic of white fat in obesity with the release of inflammation-related adipokines, and we proposed that hypoxia underlies this inflammatory state. O2-deprivation was shown to have substantial effects on gene expression and cellular function in white adipocytes. The hypoxia studies led to the proposition that O2 should be considered as a critical macronutrient.

Is energy expenditure reduced in obese mice with mutations in the leptin/leptin receptor genes?

Rodents with mutations in the leptin, or leptin receptor, genes have been extensively used to investigate the regulation of energy balance and the factors that underlie the development of obesity. The excess energy gain of these mutants has long been considered as being due in part to increased metabolic efficiency, consequent to reduced energy expenditure, but this view has recently been challenged. We argue, particularly though not exclusively, from data on ob/ob mice, that three lines of evidence support the proposition that reduced expenditure is important in the aetiology of obesity in leptin pathway mutants (irrespective of the genetic background): (i) milk intake is similar in suckling ob/ob and +/? mice; (ii) ob/ob mice deposit excess energy when pair-fed to the ad libitum food intake of lean siblings; (iii) in several studies mutant mice have been shown to exhibit a lower RMR 'per animal' at temperatures below thermoneutrality. When metabolic rate is expressed 'per unit body weight' (inappropriately, because of body composition differences), then it is invariably lower in the obese than the lean. It is important to differentiate the causes from the consequences of obesity. Hyperphagic, mature obese animals weighing 2-3 times their lean siblings may well have higher expenditure 'per animal', reflecting the costs of being larger and of enhanced obligatory diet-induced thermogenesis resulting from the increased food intake. This cannot, however, be used to inform the aetiology of their obesity.

Oxygen-A Critical, but Overlooked, Nutrient.

Gaseous oxygen is essential for all aerobic animals, without which mitochondrial respiration and oxidative phosphorylation cannot take place. It is not, however, regarded as a "nutrient" by nutritionists and does not feature as such within the discipline of nutritional science. This is primarily a consequence of the route by which O2 enters the body, which is via the nose and lungs in terrestrial animals as opposed to the mouth and gastrointestinal tract for what are customarily considered as nutrients. It is argued that the route of entry should not be the critical factor in defining whether a substance is, or is not, a nutrient. Indeed, O2 unambiguously meets the standard dictionary definitions of a nutrient, such as "a substance that provides nourishment for the maintenance of life and for growth" (Oxford English Dictionary). O2 is generally available in abundance, but deficiency occurs at high altitude and during deep sea dives, as well as in lung diseases. These impact on the provision at a whole-body level, but a low pO2 is characteristic of specific tissues includings the retina and brain, while deficiency, or overt hypoxia, is evident in certain conditions such as ischaemic disease and in tumours - and in white adipose tissue in obesity. Hypoxia results in a switch from oxidative metabolism to increased glucose utilisation through anaerobic glycolysis, and there are extensive changes in the expression of multiple genes in O2-deficient cells. These changes are driven by hypoxia-sensitive transcription factors, particularly hypoxia-inducible factor-1 (HIF-1). O2 deficiency at a whole-body level can be treated by therapy or supplementation, but O2 is also toxic through the generation of reactive oxygen species. It is concluded that O2 is a critical, but overlooked, nutrient which should be considered as part of the landscape of nutritional science.

IL-33 stimulates expression of the GPR84 (EX33) fatty acid receptor gene and of cytokine and chemokine genes in human adipocytes.

Expression of GPCR fatty acid sensor/receptor genes in adipocytes is modulated by inflammatory mediators, particularly IL-1ß. In this study we examined whether the IL-1 gene superfamily member, IL-33, also regulates expression of the fatty acid receptor genes in adipocytes. Human fat cells, differentiated from preadipocytes, were incubated with IL-33 at three different dose levels for 3 or 24 h and mRNA measured by qPCR. Treatment with IL-33 induced a dose-dependent increase in GPR84 mRNA at 3 h, the level with the highest dose being 13.7-fold greater than in controls. Stimulation of GPR84 expression was transitory; the mRNA level was not elevated at 24 h. In contrast to GPR84, IL-33 had no effect on GPR120 expression. IL-33 markedly stimulated expression of the IL1B, CCL2, IL6, CXCL2 and CSF3 genes, but there was no effect on ADIPOQ expression. The largest effect was on CSF3, the mRNA level of which increased 183-fold over controls at 3 h with the highest dose of IL-33; there was a parallel increase in the secretion of G-CSF protein into the medium. It is concluded that in human adipocytes IL-33, which is synthesised in adipose tissue, has a strong stimulatory effect on the expression of cytokine and chemokine genes, particularly CSF3, and on the expression of GPR84, a pro-inflammatory fatty acid receptor.

IL-1ß and TNFα inhibit GPR120 (FFAR4) and stimulate GPR84 (EX33) and GPR41 (FFAR3) fatty acid receptor expression in human adipocytes: implications for the anti-inflammatory action of n-3 fatty acids.

Regulation of the expression of GPCR fatty acid receptor genes has been examined in human adipocytes differentiated in culture. TNFα and IL-1ß induced a marked reduction in GPR120 expression, mRNA level falling 17-fold at 24 h in adipocytes incubated with TNFα. In contrast, GPR84 mRNA was dramatically increased by these cytokines (>500-fold for IL-1ß at 4 h); GPR41 expression was also stimulated. Rosiglitazone did not affect GPR84 expression, but GPR120 and GPR41 expression increased. Dexamethasone, insulin, linoleic and docosahexaenoic acids (DHA), and TUG891 (GPR120 agonist) had little effect on GPR120 and GPR84 expression. TUG891 did not attenuate the pro-inflammatory actions of TNFα and IL-1ß. DHA slightly countered the actions of IL-1ß on CCL2, IL6 and ADIPOQ expression, though not on secretion of these adipokines. GPR120 and GP84 gene expression in human adipocytes is highly sensitive to pro-inflammatory mediators; the inflammation-induced inhibition of GPR120 expression may compromise the anti-inflammatory action of GPR120 agonists.

Oxygen - the forgotten nutrient.

O2 is essential for the maintenance and growth of aerobic animals, similar to the essentiality of what are classically considered nutrients. Nevertheless, O2 is not customarily regarded as a nutrient, this reflecting the route by which it enters the body - through the lungs or gills in vertebrates, rather than via the mouth and gastrointestinal tract. A relative deficiency of O2 occurs at high altitudes and during deep-sea diving, to which distinct adaptations occur. Deficiency is also evident in lung diseases such as emphysema. Without O2, mitochondrial respiration and oxidative phosphorylation cannot take place. At a molecular level, cells adapt to O2 deficiency by switching from oxidative metabolism to anaerobic glycolysis and there are changes in the expression of a multiplicity of genes, driven by hypoxia-sensitive transcription factors, particularly hypoxia-inducible factor-1. It is argued that O2 should be fully included within the remit of nutritional science alongside the other essential macronutrients.

Origins and early development of the concept that brown adipose tissue thermogenesis is linked to energy balance and obesity.

Brown adipose tissue (BAT) was identified as a thermogenic organ in 1961, and in 1978 shown to be the major site of thermoregulatory non-shivering thermogenesis in rats acclimated to the cold. Investigations in the mid-late 1970s established the uncoupling of oxidative phosphorylation through a proton conductance pathway across the mitochondrial inner membrane as the mechanism for heat production in BAT, this being regulated by UCP1 which was first discovered as a 32,000 Mr cold-inducible protein. These developments came when those concerned with nutritional energetics were proposing that thermogenesis is a significant factor in energy balance and the aetiology of obesity. A link with BAT was first demonstrated in obese ob/ob mice, which were shown to have decreased thermogenic activity in the tissue, and in rats exhibiting diet-induced thermogenesis (DIT) during overfeeding on a cafeteria diet where an activation of brown fat was evident. These pioneering observations led to extensive studies on BAT in different animal models of obesity, both genetic (particularly ob/ob and db/db mice, fa/fa rats) and experimentally-induced. In each case, indices of BAT activity and capacity (mitochondrial content, GDP binding, amount of UCP1) indicated that the tissue plays a role in DIT and that obesity is characterised by reduced thermogenesis. Links between BAT and whole-body energetics were also made in physiological situations such as lactation and fasting. Studies in the 1980s also provided clear evidence for the presence of BAT in adult humans, particularly through the detection of UCP1, and its activation in patients with phaeochromocytoma. Interest in BAT in energetics and obesity waned by the 1990s; the current major renewal of interest has undoubtedly been contingent on the pioneering developments that emerged some 40 years ago.

PCR arrays indicate that the expression of extracellular matrix and cell adhesion genes in human adipocytes is regulated by IL-1ß (interleukin-1ß).

The role of IL-1ß in regulating the expression of extracellular matrix (ECM) and cell adhesion genes in human adipocytes has been examined. Adipocytes differentiated in culture were incubated with IL-1ß for 4 or 24 h and RNA probed with PCR arrays for 84 ECM and cell adhesion genes. Treatment with IL-1ß resulted in changes in the expression at one or both time points of ∼50% of the genes probed by the arrays, the majority being down-regulated. Genes whose expression was down-regulated by IL-1ß included those encoding several collagen chains and integrin subunits. In contrast, IL-1ß induced substantial increases (>10-fold) in the expression of ICAM1, VCAM1, MMP1 and MMP3; the secretion of the encoded proteins was also markedly stimulated. IL-1ß has a pervasive effect on the expression of ECM and cell adhesion genes in human adipocytes, consistent with the derangement of tissue structure during inflammation in white fat.

IL-1ß (interleukin-1ß) stimulates the production and release of multiple cytokines and chemokines by human preadipocytes.

The effect of IL-1ß on cytokine and chemokine production by human preadipocytes has been examined. Preadipocytes were incubated with IL-1ß, and cytokine and chemokine release was measured at 24 h by protein arrays, while the expression of cytokine/chemokine genes was assessed by qPCR at 4 and 24 h. IL-1ß stimulated the secretion of multiple cytokines/chemokines, including IL-6, IL-8, IL-10, IL-13, MCP-4, TNFα and IP-10. IL-10 was not released by un-stimulated preadipocytes, while IL-6 exhibited the greatest response to IL-1ß (453-fold increase). IL-16 and IL-12p40 did not respond to IL-1ß. qPCR demonstrated that IL-1ß markedly stimulated CCL3, CSF3 and CXCL10 expression at 4 h (>900-fold mRNA increase). A time-course indicated that while CCL13 (encoding MCP-4) exhibited minimal basal expression in preadipocytes, expression increased progressively following differentiation. Human preadipocytes are highly sensitive to IL-1ß, the cytokine stimulating a major inflammatory response in these cells similar to that in mature adipocytes.