Modulation of adipokine production, glucose uptake and lactate release in human adipocytes by small changes in oxygen tension.

Adipose tissue becomes hypoxic in obesity, and cell culture studies have demonstrated that hypoxia leads to major changes in adipocyte function. Studies on the response of adipocytes to low O2 tension have employed marked hypoxia (1% O2). Here, we have examined the effects of modest hypoxia, utilising differing concentrations of O2 (1-21%), on adipokine production and glucose uptake by human adipocytes. Incubation with 10% O2 (24 h) increased expression of the leptin, vascular endothelial growth factor (VEGF) and Angptl4 genes, while leptin expression was elevated even at 15% O2 (compared to 'normoxia'-21% O2). Overall, there was a concentration-dependent increase in the expression of these genes as O2 fell, with the highest mRNA level evident at 1% O2. Parallel changes were observed in the secretion of leptin, VEGF and IL-6 into the medium, an increased release being evident at 10% O2 (15% O(2) for leptin). Adiponectin gene expression was reduced at 15% O2 and below, while adiponectin release was significantly reduced at 5% O2. Both 2-deoxy-D: -glucose uptake and lactate release showed progressive increases as O2 concentration fell, being significantly raised at 10% and 5% O2, respectively. The alterations in substrate transport were accompanied by parallel changes in transporter gene expression, GLUT1 and MCT1 mRNA level increasing from 15% and 10% O2, respectively. These results indicate that marked responses to reduced O2 concentration are exhibited by human adipocytes at O2 levels well above those associated with hypoxia and employed in cell culture studies. Adipocytes are sensitive to small changes in O2 tension.

Hypoxia stimulates lactate release and modulates monocarboxylate transporter (MCT1, MCT2, and MCT4) expression in human adipocytes.

Hypoxia modulates white adipose tissue function, and this includes stimulating glucose uptake and the expression of facilitative glucose transporters (particularly GLUT1) in adipocytes. This study has examined the effect of hypoxia on lactate release from adipocytes and whether the monocarboxylate transporters that mediate lactate transport (MCTs1-4) are expressed in human adipocytes and are induced by low O(2) tension. Exposure of human Simpson-Golabi-Behmel syndrome adipocytes to 1% O(2) for 24 h resulted in increased lactate release (2.3-fold) compared with cells in normoxia (21% O(2)). Screening by reverse transcription polymerase chain reaction indicated that the genes encoding MCT1, MCT2, and MCT4 are expressed in human adipose tissue, and in adipocytes and preadipocytes in culture. Hypoxia (48 h) increased MCT1 (8.5-fold) and MCT4 (14.3-fold) messenger RNA (mRNA) levels in human adipocytes, but decreased MCT2 mRNA (fourfold). MCT1 protein level was also increased (2.7-fold at 48 h) by hypoxia, but there was no change in MCT4 protein. The changes in MCT gene expression induced by hypoxia were reversed on return to normoxia. Treatment with the hypoxia mimetic CoCl(2) resulted in up-regulation of MCT1 (up to twofold) and MCT4 (fivefold) mRNA level, but there was no significant effect on MCT2 expression. It is concluded that hypoxia increases lactate release from adipocytes and modulates MCT expression in a type-specific manner, with MCT1 and MCT4 expression being hypoxia-inducible transcription factor-1 (HIF-1) dependent. Increased lactate production and monocarboxylate transporter expression are likely to be key components of the adaptive response of adipocytes to low O(2) tension as adipose tissue mass expands in obesity.

Adipokine expression and secretion by canine adipocytes: stimulation of inflammatory adipokine production by LPS and TNFalpha.

Adiposity and obesity are increasing in dogs. We have examined here the endocrine function of canine adipose tissue and the regulation of production of inflammation-related adipokines by dog adipocytes. Adiponectin, leptin, IL-6, MCP-1 and TNFalpha genes were expressed in the main adipose depots of dogs, but there were no major depot differences in mRNA levels. Each adipokine was expressed in canine adipocytes differentiated in culture and secreted into the medium (leptin undetected). IL-6, MCP-1 and TNFalpha were also expressed and secreted by preadipocytes; adiponectin and leptin were only expressed after adipocyte differentiation. The inflammatory mediators LPS and TNFalpha had major stimulatory effects on the expression and secretion of IL-6, MCP-1 and TNFalpha; there was a >5,000-fold increase in IL-6 mRNA level with LPS. IL-6 release into the medium was increased >50-fold over 24 h with LPS and TNFalpha, while MCP-1 release was increased 23- and 40-fold by TNFalpha and LPS, respectively. However, there was no effect, or small reductions, in adiponectin and leptin mRNA levels with the inflammatory mediators. Dexamethasone-stimulated leptin gene expression, had no effect on adiponectin expression, but decreased the expression and secretion of IL-6 and MCP-1. The PPARgamma agonist rosiglitazone stimulated both adiponectin and leptin expression and inhibited the expression of IL-6, MCP-1 and TNFalpha; MCP-1 secretion was reduced. These results demonstrate that canine adipocytes express and secrete key adipokines and show that adipocytes of this species are highly responsive to inflammatory mediators with the induction of major increases in the production of inflammation-related adipokines.

IL-33, a recently identified interleukin-1 gene family member, is expressed in human adipocytes.

Inflammation occurs in adipose tissue in obesity. We have examined whether IL-33, a recently identified IL-1 gene family member, and its associated receptors are expressed in human adipocytes. IL-33, IL-1RL1 and IL-1RAP gene expression was observed in human visceral white fat, in preadipocytes and in adipocytes (SGBS cells). Treatment with TNFalpha for 24h induced a 6-fold increase in IL-33 mRNA level in preadipocytes and adipocytes. Time-course studies with adipocytes showed that the increase in IL-33 mRNA with TNFalpha was maximal (>55-fold) at 12h. This response was markedly different to IL-1beta (peak mRNA increase at 2h; 5.4-fold) and 1L-18 (peak mRNA increase at 6h; >1500-fold). Exposure of adipocytes to hypoxia (1% O(2), 24h) did not alter IL-33 mRNA level; in preadipocytes, however, there was a 3-fold increase. Human adipocytes and preadipocytes express IL-33, but the various IL-1 family members exhibit major differences in responsiveness to TNFalpha.

PCR arrays identify metallothionein-3 as a highly hypoxia-inducible gene in human adipocytes.

Hypoxia-signalling pathway PCR arrays were used to examine the integrated response of human adipocytes to low O(2) tension. Incubation of adipocytes in 1% O(2) for 24h resulted in no change in the expression of 63 of the 84 genes on the arrays, a reduction in expression of 9 genes (including uncoupling protein 2) and increased expression of 12 genes. Substantial increases (>10-fold) in leptin, angiopoietin-like protein 4, VEGF and GLUT-1 mRNA levels were observed. The expression of one gene, metallothionein-3 (MT-3), was dramatically (>600-fold) and rapidly (by 60 min) increased by hypoxia. MT-3 gene expression was also substantially induced by hypoxia mimetics (CoCl(2), desferrioxamine, dimethyloxalylglycine), indicating transcriptional regulation through HIF-1. Hypoxia additionally induced MT-3 expression in preadipocytes, and MT-3 mRNA was detected in human (obese) subcutaneous and omental adipose tissue. MT-3 is a highly hypoxia-inducible gene in human adipocytes; the protein may protect adipocytes from hypoxic damage.

Hypoxia in adipose tissue: a basis for the dysregulation of tissue function in obesity?

White adipose tissue is a key endocrine and secretory organ, releasing multiple adipokines, many of which are linked to inflammation and immunity. During the expansion of adipose tissue mass in obesity there is a major inflammatory response in the tissue with increased expression and release of inflammation-related adipokines, including IL-6, leptin, monocyte chemoattractant protein-1 and TNF-alpha, together with decreased adiponectin production. We proposed in 2004 (Trayhurn & Wood, Br J Nutr 92, 347-355) that inflammation in adipose tissue in obesity is a response to hypoxia in enlarged adipocytes distant from the vasculature. Hypoxia has now been directly demonstrated in adipose tissue of several obese mouse models (ob/ob, KKAy, diet-induced) and molecular studies indicate that the level of the hypoxia-inducible transcription factor, hypoxia-inducible factor-1 alpha, is increased, as is expression of the hypoxia-sensitive marker gene, GLUT1. Cell- culture studies on murine and human adipocytes show that hypoxia (induced by low O2 or chemically) leads to stimulation of the expression and secretion of a number of inflammation-related adipokines, including angiopoietin-like protein 4, IL-6, leptin, macrophage migration inhibitory factor and vascular endothelial growth factor. Hypoxia also stimulates the inflammatory response of macrophages and inhibits adipocyte differentiation from preadipocytes. GLUT1 gene expression, protein level and glucose transport by human adipocytes are markedly increased by hypoxia, indicating that low O2 tension stimulates glucose utilisation. It is suggested that hypoxia has a pervasive effect on adipocyte metabolism and on overall adipose tissue function, underpinning the inflammatory response in the tissue in obesity and the subsequent development of obesity-associated diseases, particularly type 2 diabetes and the metabolic syndrome.

A microarray analysis of the hypoxia-induced modulation of gene expression in human adipocytes.

The effect of hypoxia on global gene expression in human adipocytes has been examined using DNA microarrays. Adipocytes (Zen-Bio, day 12 post-differentiation) were exposed to hypoxia (1% O(2)) or 'normoxia' (21% O(2)) for 24 h and extracted RNA probed with Agilent arrays containing 41,152 probes. A total of 1346 probes were differentially expressed (>2.0-fold change, P < 0.01) in response to hypoxia; 650 genes were up-regulated (including LEP, IL6, VEGF, ANGPTL4) and 650 down-regulated (including ADIPOQ, UCP2). Major genes not previously identified as hypoxia-sensitive in adipocytes include AQP3, FABP3, FABP5 and PPARGC1A. Ingenuity analysis indicated that several pathways and functions were modulated by hypoxia, including glucose utilization, lipid oxidation and cell death. Network analysis indicated a down-regulation of p38/MAPK and PGC-1α signalling in the adipocytes. It is concluded that hypoxia has extensive effects on human adipocyte gene expression, consistent with low O(2) tension underlying adipose tissue dysfunction in obesity.

Obesity, its associated disorders and the role of inflammatory adipokines in companion animals.

Obesity is characterised by an expansion of white adipose tissue mass that can lead to adverse health effects, such as decreased longevity, diabetes mellitus, orthopaedic and respiratory disease and neoplasia. Once thought a passive fuel depot, adipose tissue is now recognised as an active endocrine organ that communicates with the brain and peripheral tissues by secreting a wide range of hormones and protein factors, collectively termed adipokines. Examples include leptin, adiponectin, cytokines (tumour necrosis factor-alpha, interleukin-6), chemokines, acute phase proteins, haemostatic and haemodynamic factors and neurotrophins. Adipokines can influence various body systems, and perturbation of normal endocrine function is thought central to the development of many associated conditions. This review focuses on the medical consequences of obesity in companion animals, assesses the endocrine function of adipose tissue in disease pathogenesis, and highlights the potential role of adipokines as biomarkers of obesity-associated disease.

Cellular hypoxia and adipose tissue dysfunction in obesity.

Expansion of adipose tissue mass, the distinctive feature of obesity, is associated with low-grade inflammation. White adipose tissue secretes a diverse range of adipokines, a number of which are inflammatory mediators (such as TNFalpha, IL-1beta, IL-6, monocyte chemoattractant protein 1). The production of inflammatory adipokines is increased with obesity and these adipokines have been implicated in the development of insulin resistance and the metabolic syndrome. However, the basis for the link between increased adiposity and inflammation is unclear. It has been proposed previously that hypoxia may occur in areas within adipose tissue in obesity as a result of adipocyte hypertrophy compromising effective O2 supply from the vasculature, thereby instigating an inflammatory response through recruitment of the transcription factor, hypoxic inducible factor-1. Studies in animal models (mutant mice, diet-induced obesity) and cell-culture systems (mouse and human adipocytes) have provided strong support for a role for hypoxia in modulating the production of several inflammation-related adipokines, including increased IL-6, leptin and macrophage migratory inhibition factor production together with reduced adiponectin synthesis. Increased glucose transport into adipocytes is also observed with low O2 tension, largely as a result of the up-regulation of GLUT-1 expression, indicating changes in cellular glucose metabolism. Hypoxia also induces inflammatory responses in macrophages and inhibits the differentiation of preadipocytes (while inducing the expression of leptin). Collectively, there is strong evidence to suggest that cellular hypoxia may be a key factor in adipocyte physiology and the underlying cause of adipose tissue dysfunction contributing to the adverse metabolic milieu associated with obesity.

Hypoxia induces leptin gene expression and secretion in human preadipocytes: differential effects of hypoxia on adipokine expression by preadipocytes.

The effect of hypoxia on the expression and secretion of major adipokines by human preadipocytes has been examined. Hypoxia (1% O(2)) led to an increase in the HIF-1 alpha transcription factor subunit in cultured preadipocytes, as did incubation with the hypoxia mimetic CoCl(2). Leptin mRNA was essentially undetectable in preadipocytes incubated under normoxia (21% O(2)), but exposure to 1% O(2), or CoCl(2), for 4 or 24 h resulted in an induction of leptin gene expression (measured by real-time PCR). Immunoreactive leptin was not detected in the medium from normoxic preadipocytes, but was present in the medium from the hypoxic cells. Hypoxia stimulated expression of the GLUT-1 facilitative glucose transporter gene and the vascular endothelial growth factor (VEGF) gene in preadipocytes, as in adipocytes. PPAR gamma and aP2 mRNA levels, markers of adipocyte differentiation, were reduced by hypoxia in both cell types. In marked contrast to adipocytes, interleukin-6 (IL-6), angiopoietin-like protein 4, and plasminogen activator inhibitor-1 expression by preadipocytes was not stimulated by low O(2) tension. Consistent with the gene expression results, VEGF release into the medium from preadipocytes was increased by hypoxia, but there was no change in IL-6 secretion. It is concluded that hypoxia induces human preadipocytes to synthesize and secrete leptin. Preadipocytes and adipocytes differ in their responsiveness to low O(2) tension, maturation of the response to hypoxia developing on differentiation.

Hypoxia and the endocrine and signalling role of white adipose tissue.

White adipose tissue is a major endocrine and signalling organ. It secretes multiple protein hormones and factors, termed adipokines (such as adiponectin, leptin, IL-6, MCP-1, TNFalpha) which engage in extensive cross-talk within adipose tissue and with other tissues. Many adipokines are linked to inflammation and immunity and these include cytokines, chemokines and acute phase proteins. In obesity, adipose tissue exhibits a major inflammatory response with increased production of inflammation-related adipokines. It has been proposed that hypoxia may underlie the inflammatory response in adipose tissue and evidence that the tissue is hypoxic in obesity has been obtained in animal models. Cell culture studies have demonstrated that the expression and secretion of key adipokines, including leptin, IL-6 and VEGF, are stimulated by hypoxia, while adiponectin (with an anti-inflammatory action) production falls. Hypoxia also stimulates glucose transport by adipocytes and may have a pervasive effect on cell function within adipose tissue.

Dysregulation of the expression and secretion of inflammation-related adipokines by hypoxia in human adipocytes.

The effect of hypoxia, induced by incubation under low (1%) oxygen tension or by exposure to CoCl(2), on the expression and secretion of inflammation-related adipokines was examined in human adipocytes. Hypoxia led to a rapid and substantial increase (greater than sevenfold by 4 h of exposure to 1% O(2)) in the hypoxia-sensitive transcription factor, HIF-1alpha, in human adipocytes. This was accompanied by a major increase (up to 14-fold) in GLUT1 transporter mRNA level. Hypoxia (1% O(2) or CoCl(2)) led to a reduction (up to threefold over 24 h) in adiponectin and haptoglobin mRNA levels; adiponectin secretion also decreased. No changes were observed in TNFalpha expression. In contrast, hypoxia resulted in substantial increases in FIAF/angiopoietin-like protein 4, IL-6, leptin, MIF, PAI-1 and vascular endothelial growth factor (VEGF) mRNA levels. The largest increases were with FIAF (maximum 210-fold), leptin (maximum 29-fold) and VEGF (maximum 23-fold); these were reversed on return to normoxia. The secretion of IL-6, leptin, MIF and VEGF from the adipocytes was also stimulated by exposure to 1% O(2). These results demonstrate that hypoxia induces extensive changes in human adipocytes in the expression and release of inflammation-related adipokines. Hypoxia may underlie the development of the inflammatory response in adipocytes, leading to obesity-associated diseases.

Hypoxia increases expression of selective facilitative glucose transporters (GLUT) and 2-deoxy-D-glucose uptake in human adipocytes.

Hypoxia modulates the production of key inflammation-related adipokines and may underlie adipose tissue dysfunction in obesity. Here we have examined the effects of hypoxia on glucose transport by human adipocytes. Exposure of adipocytes to hypoxia (1% O(2)) for up to 24 h resulted in increases in GLUT-1 (9.2-fold), GLUT-3 (9.6-fold peak at 8 h), and GLUT-5 (8.9-fold) mRNA level compared to adipocytes in normoxia (21% O(2)). In contrast, there was no change in GLUT-4, GLUT-10 or GLUT-12 expression. The rise in GLUT-1 mRNA was accompanied by a substantial increase in GLUT-1 protein (10-fold), but there was no change in GLUT-5; GLUT-3 protein was not detected. Functional studies with [(3)H]2-deoxy-D-glucose showed that hypoxia led to a stimulation of glucose transport (4.4-fold) which was blocked by cytochalasin B. These results indicate that hypoxia increases monosaccharide uptake capacity in human adipocytes; this may contribute to adipose tissue dysregulation in obesity.

Adipose tissue and adipokines--energy regulation from the human perspective.

There has been a rapid rise in the incidence of obesity, primarily as a result of changes in lifestyle (diet and activity levels). Obesity has provided considerable impetus for the investigation of the fundamental mechanisms involved in the regulation of energy balance. Important developments include the identification of novel factors involved in the control of appetite, such as ghrelin, orexin A, and the endogenous cannabinoids, and the emergence of the concept of "nonexercise activity thermogenesis" (NEAT) provided new perspectives on energy expenditure. Studies on white adipose tissue have led to the recognition that it is an important endocrine organ, communicating with the brain and peripheral tissues through the secretion of leptin and other adipokines. There is a rapidly expanding list of protein factors released by white adipose tissue, including the key hormone, adiponectin. Of particular note is the range of cytokines, chemokines, and other inflammation-related proteins secreted by white fat as tissue mass rises; indeed, obesity is characterized by chronic mild inflammation. The adipokines provide an extensive network of communication both within adipose tissue and with other organs, and some are implicated directly in the pathologies associated with obesity, particularly the metabolic syndrome. Although the focus remains very much on obesity in humans, the disorder and its sequelae are also a growing concern in companion animals.

The pro-inflammatory cytokine IL-18 is expressed in human adipose tissue and strongly upregulated by TNFalpha in human adipocytes.

White adipocytes have been examined as a potential source of interleukin-18 (IL-18), the circulating levels of which are increased in obesity. IL-18 gene expression was evident in human subcutaneous and visceral adipose tissue, and expression occurred in mature adipocytes and the stromal-vascular fraction. Expression of the IL-18 receptor complex (IL-18Ralpha and IL-18Rbeta) and the IL-18 binding protein (IL-18BP) genes was also observed, mirroring that of IL-18. IL-18 mRNA level increased rapidly (within 2h) and dramatically (>900-fold) in response to TNFalpha in human adipocytes differentiated in culture. IL-18 protein was detected in lysates of cultured adipocytes, though not in the medium. There was a small increase in IL-18 in lysates of adipocytes treated with TNFalpha, but the protein was again undetectable in the medium. IL-18 may be part of the inflammatory cascade within adipose tissue; however, human adipocytes do not appear to secrete significant amounts of IL-18.

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

White adipose tissue is now recognised to be a multifunctional organ; in addition to the central role of lipid storage, it has a major endocrine function secreting several hormones, notably leptin and adiponectin, and a diverse range of other protein factors. These various protein signals have been given the collective name 'adipocytokines' or 'adipokines'. However, since most are neither 'cytokines' nor 'cytokine-like', it is recommended that the term 'adipokine' be universally adopted to describe a protein that is secreted from (and synthesised by) adipocytes. It is suggested that the term is restricted to proteins secreted from adipocytes, excluding signals released only by the other cell types (such as macrophages) in adipose tissue. The adipokinome (which together with lipid moieties released, such as fatty acids and prostaglandins, constitute the secretome of fat cells) includes proteins involved in lipid metabolism, insulin sensitivity, the alternative complement system, vascular haemostasis, blood pressure regulation and angiogenesis, as well as the regulation of energy balance. In addition, there is a growing list of adipokines involved in inflammation (TNFalpha, IL-1beta, IL-6, IL-8, IL-10, transforming growth factor-beta, nerve growth factor) and the acute-phase response (plasminogen activator inhibitor-1, haptoglobin, serum amyloid A). Production of these proteins by adipose tissue is increased in obesity, and raised circulating levels of several acute-phase proteins and inflammatory cytokines has led to the view that the obese are characterised by a state of chronic low-grade inflammation, and that this links causally to insulin resistance and the metabolic syndrome. It is, however, unclear as to the extent to which adipose tissue contributes quantitatively to the elevated circulating levels of these factors in obesity and whether there is a generalised or local state of inflammation. The parsimonious view is that the increased production of inflammatory cytokines and acute-phase proteins by adipose tissue in obesity relates primarily to localised events within the expanding fat depots. It is suggested that these events reflect hypoxia in parts of the growing adipose tissue mass in advance of angiogenesis, and involve the key controller of the cellular response to hypoxia, the transcription factor hypoxia inducible factor-1.

Glucose transporters (GLUT and SGLT): expanded families of sugar transport proteins.

The number of known glucose transporters has expanded considerably over the past 2 years. At least three, and up to six, Na+-dependent glucose transporters (SGLT1-SGLT6; gene name SLC5A) have been identified. Similarly, thirteen members of the family of facilitative sugar transporters (GLUT1-GLUT12 and HMIT; gene name SLC2A) are now recognised. These various transporters exhibit different substrate specificities, kinetic properties and tissue expression profiles. The number of distinct gene products, together with the presence of several different transporters in certain tissues and cells (for example, GLUT1, GLUT4, GLUT5, GLUT8, GLUT12 and HMIT in white adipose tissue), indicates that glucose delivery into cells is a process of considerable complexity.

Expression of Class III facilitative glucose transporter genes (GLUT-10 and GLUT-12) in mouse and human adipose tissues.

We have examined whether GLUT-10 and GLUT-12, members of the Class III group of the recently expanded family of facilitative glucose transporters, are expressed in adipose tissues. The mouse GLUT-12 gene, located on chromosome 10, comprises at least five exons and encodes a 622 amino acid protein exhibiting 83% sequence identity and 91% sequence similarity to human GLUT-12. Expression of the GLUT-12 gene was evident in all the major mouse adipose tissue depots (epididymal, perirenal, mesenteric, omental, and subcutaneous white; interscapular brown). The GLUT-10 gene is also expressed in mouse adipose tissues and as with GLUT-12 expression occurred in the mature adipocytes as well as the stromal vascular cells. 3T3-L1 adipocytes express GLUT-10, but not GLUT-12, and expression of GLUT-12 was not induced by insulin or glucose. Both GLUT-10 and GLUT-12 expression was also found in human adipose tissue (subcutaneous and omental) and SGBS adipocytes. It is concluded that white fat expresses a wide range of facilitative glucose transporters.