I. Stuart Wood

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.

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.

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.

Identification and characterization of a monocarboxylate transporter (MCT1) in pig and human colon: its potential to transport l‐lactate as well as butyrate

1 Oligonucleotide primers based on the human heart monocarboxylate transporter (MCT1) cDNA sequence were used to isolate a 544 bp cDNA product from human colonic RNA by reverse transcription‐polymerase chain reaction (RT‐PCR). The sequence of the RT‐PCR product was identical to that of human heart MCT1. Northern blot analysis using the RT‐PCR product indicated the presence of a single transcript of 3.3 kb in mRNA isolated from both human and pig colonic tissues. Western blot analysis using an antibody to human MCT1 identified a specific protein with an apparent molecular mass of 40 kDa in purified and well‐characterized human and pig colonic lumenal membrane vesicles (LMV). 2 Properties of the colonic lumenal membrane l‐lactate transporter were studied by the uptake of L‐[U‐14C]lactate into human and pig colonic LMV. l‐lactate uptake was stimulated in the presence of an outward‐directed anion gradient at an extravesicular pH of 5.5. Transport of l‐lactate into anion‐loaded colonic LMV appeared to be via a proton‐activated, anion exchange mechanism. 3 l‐lactate uptake was inhibited by pyruvate, butyrate, propionate and acetate, but not by Cl− and SO42−. The uptake of l‐lactate was inhibited by phloretin, mercurials and α‐cyano‐4‐hydroxycinnamic acid (4‐CHC), but not by the stilbene anion exchange inhibitors, 4,4′‐diisothiocyanostilbene‐2,2′‐disulphonic acid (DIDS) and 4‐acetamido‐4′‐isothiocyanostilbene‐2,2′‐disulphonic acid (SITS). 4 The results indicate the presence of a MCT1 protein on the lumenal membrane of the colon that is involved in the transport of l‐lactate as well as butyrate across the colonic lumenal membrane. Western blot analysis showed that the abundance of this protein decreases in lumenal membrane fractions isolated from colonic carcinomas compared with that detected in the normal healthy colonic tissue.

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.

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.

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 O2 tension. Incubation of adipocytes in 1% O2 for 24 h 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 (CoCl2, 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.

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.

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% O2) or ‘normoxia’ (21% O2) 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 O2 tension underlying adipose tissue dysfunction in obesity.

Expression of the Na+/glucose co-transporter (SGLT1) in the intestine of domestic and wild ruminants

Abstract. The activity and abundance of the Na+/glucose co-transporter (SGLT1) was assessed in brush-border-membrane vesicles (BBMV) isolated from the intestine of grass- and roughage- (GR) consuming ruminants (sheep and dairy cattle), during the transition from the pre-ruminant to the mature ruminant state. The abundance of SGLT1 messenger ribonucleic acid (mRNA) was also compared in the intestinal tissue of the same animals. The dramatic developmental decline in the activity and expression of SGLT1 appears to be typical of GR-consuming ruminants and is coincident with the significant decline in the levels of lumenal monosaccharides. Expression of the ovine SGLT1 complementary deoxyribonucleic acid (cDNA) in Xenopus laevis oocytes confirmed that the isolated cDNA encodes for a functional Na+/glucose co-transporter. Determination of a bovine intestinal SGLT1 protein sequence (amino acids 347–658) indicated 99% similarity to the ovine SGLT1 protein with differences in the carboxyl terminus. In contrast to GR-consuming ruminants, the abundance of SGLT1 protein and SGLT1 mRNA remained significantly high in the intestine of ruminants in both the intermediate-mixed (IM) feeding goat and fallow deer and the concentrate-selecting (CS) moose and roe deer, dietary groups correlating with the availability of monosaccharides in the intestinal lumen.