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 TNFα for 24 h 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 TNFα was maximal (>55-fold) at 12 h. This response was markedly different to IL-1β (peak mRNA increase at 2 h; 5.4-fold) and 1L-18 (peak mRNA increase at 6 h; >1500-fold). Exposure of adipocytes to hypoxia (1% O₂, 24 h) 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 TNFα.     

URB is abundantly expressed in adipose tissue and dysregulated in obesity

Dysregulated production of adipocytokines in obesity is involved in the development of metabolic syndrome. URB/DRO1 contains N-terminal signal sequence and is thought to play a role in apoptosis of tumor cells. In the present study, we investigated the expression pattern of URB mRNA in adipose tissue and secretion from cultured adipocytes. In human and mouse, URB mRNA was predominantly expressed in adipose tissue and was downregulated in obese mouse models, such as ob/ob, KKAy, and diet-induced obese mice. In 3T3L1 adipocytes, insulin, TNF-α, H₂O₂ and hypoxia decreased URB mRNA level. This regulation was similar to that for adiponectin and opposite to MCP-1. URB protein was secreted in media of URB cDNA-stably transfected cells and endogenous URB was detected in media of cultured human adipocytes. In conclusion, the expression pattern of URB suggests its role in obesity and the results suggest that URB is secreted, at least in part, from adipocytes.     

Adipose-specific deletion of stearoyl-CoA desaturase 1 up-regulates the glucose transporter GLUT1 in adipose tissue

Stearoyl-CoA desaturase 1 (SCD1) deficiency protects mice from diet-induced obesity and insulin resistance. To understand the tissue-specific role of SCD1 in energy homeostasis, we have generated mice with an adipose-specific knockout of Scd1 (AKO), and report here that SCD1 deficiency increases GLUT1 expression in adipose tissue of AKO mice, but not global SCD1 knockout (GKO) mice. In 3T3-L1 adipocytes treated with an SCD inhibitor, basal glucose uptake and the cellular expression of GLUT1 were significantly increased while GLUT4 expression remained unchanged. Consistently, adipose-specific SCD1 knockout (AKO) mice had significantly elevated GLUT1 expression, but not GLUT4, in white adipose tissue compared to Lox counterparts. Concurrently, adiponectin expression was significantly diminished, whereas TNF-α expression was elevated. In contrast, in adipose tissue of GKO mice, GLUT4 and adiponectin expression were significantly elevated with lowered TNF-α expression and little change in GLUT1 expression, suggesting a differential responsiveness of adipose tissue to global- or adipose-specific SCD1 deletion. Taken together, these results indicate that adipose-specific deletion of SCD1 induces GLUT1 up-regulation in adipose tissue, associated with decreased adiponectin and increased TNF-α production, and suggest that GLUT1 may play a critical role in controlling glucose homeostasis of adipose tissue in adipose-specific SCD1-deficient conditions.     

Regulation of glucose transporter messenger RNA levels in rat adipose tissue by insulin

Analysis of glucose transporter mRNA levels in adipose tissue from streptozotocin (STZ)-induced diabetic rats demonstrated a specific decrease (10-fold) in adipose tissue GLUT-4 mRNA with no significant effect on GLUT-1 mRNA levels. Treatment of STZ-diabetic rats with twice daily injections of insulin for 1-3 days resulted in a 16-fold increase in the relative amount of GLUT-4 mRNA to levels approximately 2-fold greater than those in control animals. However, after 7 days of insulin therapy the amount of GLUT-4 mRNA decreased approximately 2-fold back to the levels in the control animals. Normalization of the STZ-induced serum hyperglycemia by phlorizin treatment, which inhibits renal tubular reabsorption of glucose, had no effect on GLUT-4 mRNA in the absence of insulin. Similar to STZ-diabetes, fasting for 48 h also reduced adipose GLUT-4 mRNA levels. Parenteral administration of insulin with glucose over 7.5 h, but not glucose alone, increased the levels of the GLUT-4 mRNA 3- to 4-fold. These studies demonstrate that the relative glycemic state does not influence GLUT-4 glucose transporter mRNA expression in vivo and strongly suggests that insulin is a major factor regulating the levels of GLUT-4 mRNA in adipose tissue.     

Short-term exercise enhances insulin-stimulated GLUT-4 translocation and glucose transport in adipose cells

This investigation examined the effectsof short-term exercise training on insulin-stimulated GLUT-4 glucosetransporter translocation and glucose transport activity in rat adiposecells. Male Wistar rats were randomly assigned to a sedentary (Sed) orswim training group (Sw, 4 days; final 3 days: 2 × 3 h/day). Adipose cell size decreased significantly but minimally(~20%), whereas total GLUT-4 increased by 30% in Sw vs. Sed rats.Basal3-O-methyl-D-[¹⁴C]glucosetransport was reduced by 62%, whereas maximally insulin-stimulated (MIS) glucose transport was increased by 36% in Sw vs. Sed rats. MIScell surface GLUT-4 photolabeling was 44% higher in the Sw vs. Sedanimals, similar to the increases observed in MIS glucose transportactivity and total GLUT-4. These results suggest that increases intotal GLUT-4 and GLUT-4 translocation to the cell surface contribute tothe increase in MIS glucose transport with short-term exercisetraining. In addition, the results suggest that the exercisetraining-induced adaptations in glucose transport occur more rapidlythan previously thought and with minimal changes in adipose cell size.

     

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.     

Visfatin in adipocytes is upregulated by hypoxia through HIF1alpha-dependent mechanism

Obesity is associated with metabolic disorders, such as insulin resistance. Visfatin is an adipose-derived secretory factor to exert insulin-mimetic effects. Plasma visfatin levels and mRNA levels of visfatin in adipose tissues are increased in obesity. However, the mechanism that mediates induction of visfatin mRNA in adipose tissue of obesity remains unknown. Recent studies have reported that fat tissue is hypoxia in obesity. In this study, we investigated the effects of hypoxia on mRNA expression of visfatin in adipocytes. Hypoxia increased visfatin mRNA expression. Desferoxamine and Cobaltous chloride, two hypoxia mimetic compounds, also increased visfatin mRNA levels. Dimethyloxallyl glycine, a stabilizer of hypoxia-inducible factor 1alpha (HIF1alpha), mimicked the hypoxia-mediated upregulation of visfatin, and YC1, an inhibitor of HIF1 cancelled the hypoxia-induced upregulation of visfatin mRNA. We identified two functional hypoxia responsive elements (HRE) in mouse visfatin promoter. Hypoxic treatment and overexpression of HIF1alpha increased the promoter activity, and mutation of the HRE blunted hypoxia-induced activation of visfatin promoter. Our results suggest that visfatin mRNA expression is upregulated in the fat tissue of obesity through the activation of HIF1alpha pathway due to hypoxia.     

Distinct hypoxic regulation of preadipocyte factor-1 (Pref-1) in preadipocytes and mature adipocytes

Preadipocyte factor-1 (Pref-1) is a secretory soluble protein, which exerts pleiotropic effects on maintenance of cancer stem cell characteristics and commitment of mesenchymal stem cell lineages by inhibiting adipogenesis. Observations that obesity renders the microenvironment of adipose tissues hypoxic and that hypoxia inhibits adipogenesis lead us to investigate whether hypoxia increases the expression of anti-adipogenic Pref-1 in preadipocytes, mature adipocytes, and adipose tissues from obese mouse. In 3T3-L1 preadipocytes, hypoxia induces Pref-1 by a hypoxia-inducible factor 1 (HIF-1)-dependent mechanism accompanied by increase in the levels of the active histone mark, acetylated H3K9/14 (H3K9/14Ac). Adipogenesis increased the levels of the heterochromatin histone mark, trimethylated H3K27 (H3K27me3), whereas it decreased the levels of H3K4me3 and H3K9/14Ac euchromatin marks of the mouse Pref-1 promoter. However, differently from preadipocytes, in mature adipocytes hypoxia failed to reverse the repressive epigenetic changes of Pref-1 promoter and to increase its expression. Short term (8 weeks) high fat diet (HFD) increased HIF-1α protein in subcutaneous and epididymal adipose tissues, but did not increase Pref-1 expression. Unlike in 3T3-L1 preadipocytes, HIF-1α did not increase Pref-1 expression in adipose tissues in which mature adipocytes constitute the main population. Interestingly, long term (35 weeks) HFD increased Pref-1 in serum but not in obese adipose tissues. This study suggests that Pref-1 is an endocrine factor which is synergistically increased by obesity and age.     

Obesity and weight loss result in increased adipose tissue ABCG1 expression in db/db mice

The prevalence of obesity has reached epidemic proportions and is associated with several co-morbid conditions including diabetes, dyslipidemia, cancer, atherosclerosis and gallstones. Obesity is associated with low systemic inflammation and an accumulation of adipose tissue macrophages (ATMs) that are thought to modulate insulin resistance. ATMs may also modulate adipocyte metabolism and take up lipids released during adipocyte lipolysis and cell death. We suggest that high levels of free cholesterol residing in adipocytes are released during these processes and contribute to ATM activation and accumulation during obesity and caloric restriction. Db/db mice were studied for extent of adipose tissue inflammation under feeding conditions of ad libitum (AL) and caloric restriction (CR). The major finding was a marked elevation in epididymal adipose ABCG1 mRNA levels with obesity and CR (6-fold and 16-fold, respectively) over that seen for lean wild-type mice. ABCG1 protein was also elevated for CR as compared to AL adipose tissue. ABCG1 is likely produced by cholesterol loaded ATMs since this gene is not highly expressed in adipocytes and ABCG1 expression is sterol mediated. Our data supports the concept that metabolic changes in adipocytes due to demand lipolysis and cell death lead to cholesterol loading of ATMs. Based on finding cholesterol-loaded peritoneal leukocytes with elevated levels of ABCG1 in CR as compared to AL mice, we suggest that pathways for cholesterol trafficking out of adipose tissue involve ATM egress as well as ABCG1 mediated cholesterol efflux. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

Regulation of glucose transport in Clone 9 cells by thyroid hormone.

Triiodothyronine (T3) is found to stimulate cytochalasin B-inhibitable glucose transport in Clone 9 cells, a 'non-transformed' rat liver cell line. After an initial lag period of more than 3 h, glucose transport rate is significantly increased at 6 h and reaches more than 3-times the control rate at 24 h. The enhancement of glucose transport by T3 is due to an increase in transport Vmax and occurs in the absence of a change in either the Km for glucose transport (approximately 3 mM) or the Ki for inhibition of transport by cytochalasin B ((1-2).10(-7) M). Consistent with the observed Ki for cytochalasin B, Northern blot analysis of RNA from control and T3-treated cells employing cDNA probes encoding GTs of the human erythrocyte/rat brain/HepG2 cell transporter (GLUT-1), rat muscle/fat cell transporter (GLUT-4), and rat liver transporter (GLUT-2) types indicates expression of only the GLUT-1 mRNA isoform in these cells. The abundance of GLUT-1 mRNA increases approx. 1.9-fold after 24 h of T3 treatment and is accompanied by an approx. 1.3-fold increase in the abundance of GLUT-1 in whole-cell extracts as demonstrated by Western blot analysis employing a polyclonal antibody directed against the 13 amino acid C-terminal peptide of GLUT-1. The more than 3-fold stimulation of glucose transport at 24 h substantially exceeds the fractional increment in transporter abundance suggesting that, in addition to increasing total GLUT-1 abundance, exposure to T3 may result in a translocation of transporters to the plasma membrane or an activation of pre-existing membrane transporter sites.     

The PPARγ agonist rosiglitazone promotes the induction of brite adipocytes,increasing β-adrenoceptor-mediated mitochondrial function and glucose uptake

Recruitment and activation of brite (or beige) adipocytes has been advocated as a potential avenue for manipulating whole-body energy expenditure. Despite numerous studies illustrating the differences in gene and protein markers between brown, brite and white adipocytes, there is very little information on the adrenergic regulation and function of these brite adipocytes. We have compared the functional (cyclic AMP accumulation, oxygen consumption rates, mitochondrial function, glucose uptake, extracellular acidification rates, calcium influx) profiles of mouse adipocytes cultured from three contrasting depots, namely interscapular brown adipose tissue, and inguinal or epididymal white adipose tissues, following chronic treatment with the peroxisome proliferator-activated receptor γ (PPARγ) agonist rosiglitazone. Prototypical brown adipocytes readily express β₃-adrenoceptors, and β₃-adrenoceptor stimulation increases cyclic AMP accumulation, oxygen consumption rates, mitochondrial function, glucose uptake, and extracellular acidification rates. Treatment of brown adipocytes with rosiglitazone increases uncoupling protein 1 (UCP1) levels, and increases β₃-adrenoceptor mitochondrial function but does not affect glucose uptake responses. In contrast, inguinal white adipocytes only express UCP1 and β₃-adrenoceptors following rosiglitazone treatment, which results in an increase in all β₃-adrenoceptor-mediated functions. The effect of rosiglitazone in epididymal white adipocytes, was much lower compared to inguinal white adipocytes. Rosiglitazone also increased α₁-adrenoceptor mediated increases in calcium influx and glucose uptake (but not mitochondrial function) in inguinal and epididymal white adipocytes. In conclusion, the PPARγ agonist rosiglitazone promotes the induction and function of brite adipocytes cultured from inguinal and epididymal white adipose depots.     

Effects of Barbiturates on Facilitative Glucose Transporters are Pharmacologically Specific and Isoform Selective

Barbiturates inhibit GLUT-1-mediated glucose transport across the blood-brain barrier, in cultured mammalian cells, and in human erythrocytes. Barbiturates also interact directly with GLUT-1. The hypotheses that this inhibition of glucose transport is (i) selective, preferring barbiturates over halogenated hydrocarbon inhalation anesthetics, and (ii) specific, favoring some GLUT-# isoforms over others were tested. Several oxy- and thio-barbiturates inhibited [³H]-2-deoxyglucose uptake by GLUT-1 expressing murine fibroblasts with IC₅₀s of 0.2–2.9 mm. Inhibition of GLUT-1 by barbiturates correlates with their overall lipid solubility and pharmacology, and requires hydrophobic side chains on the core barbiturate structure. In contrast, several halogenated hydrocarbons and ethanol (all ≤10 mm) do not significantly inhibit glucose transport. The interaction of these three classes of anesthetics with purified GLUT-1 was evaluated by quenching of intrinsic protein fluorescence and displayed similar specificities and characteristics. The ability of barbiturates to inhibit other facilitative glucose transporters was determined in cell types expressing predominantly one isoform. Pentobarbital inhibits [³H]-2-deoxyglucose and [¹⁴C]-3-O-methyl-glucose uptake in cells expressing GLUT-1, GLUT-2, and GLUT-3 with IC₅₀s of ∼1 mm. In contrast, GLUT-4 expressed in insulin-stimulated rat adipocytes was much less sensitive than the other isoforms to inhibition by pentobarbital (IC₅₀ of >10 mm). Thus, barbiturates selectively inhibit glucose transport by some, but not all, facilitative glucose transporter isoforms. Received: 10 November 1998/Revised: 3 February 1999     

Contribution of glucocorticoid-mineralocorticoid receptor pathway on the obesity-related adipocyte dysfunction

AimsMineralocorticoid receptor (MR) blockade ameliorated insulin resistance with improvements in adipocytokine dysregulation, inflammation, and excess of reactive oxygen species (ROS) in obese adipose tissue and adipocytes, but its mechanism has not been clarified. The 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), producing active glucocorticoids, is highly expressed in adipocytes and glucocorticoids bind to MR with higher affinity than to glucocorticoid receptor (GR). We investigated whether glucocorticoids effect on adipocytokines and ROS through MR in adipocytes. In addition, fat distributions of MR and GR were investigated in human subjects.Methods and ResultsCorticoid receptors and their target genes were examined in adipose tissue of obese db/db mice. 3T3-L1 adipocytes were treated with glucocorticoids, H₂O₂, MR antagonist eplerenone (EP), GR antagonist RU486 (RU), MR-siRNA, and/or N-acetylcysteine. Human adipose tissues were obtained from seven patients who underwent abdominal surgery. The mRNA levels of MR and its target gene were higher in db/db mice than in control db/m + mice. In 3T3-L1 adipocytes, glucocorticoids, similar to H₂O₂, caused the dysregulation of mRNA levels of various genes related to adipocytokines and the increase of intracellular ROS. Such changes were rectified by MR blockade, not by GR antagonist. In human fat, MR mRNA level was increased in parallel with the increase of body mass index (BMI) and its increase was more significant in visceral fat, while there were no apparent correlations of GR mRNA level to BMI or fat distribution.ConclusionGlucocorticoid-MR pathway may contribute to the obesity-related adipocytokine dysregulation and adipose ROS.     

Reduced expression of PGC-1 and insulin-signaling molecules in adipose tissue is associated with insulin resistance

Peroxisome proliferator-activated receptor gamma (PPAR gamma) co-activator 1 (PGC-1) regulates glucose metabolism and energy expenditure and, thus, potentially insulin sensitivity. We examined the expression of PGC-1, PPAR gamma, insulin receptor substrate-1 (IRS-1), glucose transporter isoform-4 (GLUT-4), and mitochondrial uncoupling protein-1 (UCP-1) in adipose tissue and skeletal muscle from non-obese, non-diabetic insulin-resistant, and insulin-sensitive individuals. PGC-1, both mRNA and protein, was expressed in human adipose tissue and the expression was significantly reduced in insulin-resistant subjects. The expression of PGC-1 correlated with the mRNA levels of IRS-1, GLUT-4, and UCP-1 in adipose tissue. Furthermore, the adipose tissue expression of PGC-1 and IRS-1 correlated with insulin action in vivo. In contrast, no differential expression of PGC-1, GLUT-4, or IRS-1 was found in the skeletal muscle of insulin-resistant vs insulin-sensitive subjects. The findings suggest that PGC-1 may be involved in the differential gene expression and regulation between adipose tissue and skeletal muscle. The combined reduction of PGC-1 and insulin signaling molecules in adipose tissue implicates adipose tissue dysfunction which, in turn, can impair the systemic insulin response in the insulin-resistant subjects.     

RASSF6 Expression in Adipocytes Is Down-Regulated by Interaction with Macrophages

Macrophage infiltration into adipose tissue is associated with obesity and the crosstalk between adipocytes and infiltrated macrophages has been investigated as an important pathological phenomenon during adipose tissue inflammation. Here, we sought to identify adipocyte mRNAs that are regulated by interaction with infiltrated macrophages in vivo. An anti-inflammatory vitamin, vitamin B6, suppressed macrophage infiltration into white adipose tissue and altered mRNA expression. We identified >3500 genes whose expression is significantly altered during the development of obesity in db/db mice, and compared them to the adipose tissue mRNA expression profile of mice supplemented with vitamin B6. We identified PTX3 and MMP3 as candidate genes regulated by macrophage infiltration. PTX3 and MMP3 mRNA expression in 3T3-L1 adipocytes was up-regulated by activated RAW264.7 cells and these mRNA levels were positively correlated with macrophage number in adipose tissue in vivo. Next, we screened adipose genes down-regulated by the interaction with macrophages, and isolated RASSF6 (Ras association domain family 6). RASSF6 mRNA in adipocytes was decreased by culture medium conditioned by activated RAW264.7 cells, and RASSF6 mRNA level was negatively correlated with macrophage number in adipose tissue, suggesting that adipocyte RASSF6 mRNA expression is down-regulated by infiltrated macrophages in vivo. Finally, this study also showed that decreased RASSF6 expression up-regulates mRNA expression of several genes, such as CD44 and high mobility group protein HMGA2. These data provide novel insights into the biological significance of interactions between adipocytes and macrophages in adipose tissue during the development of obesity.     

Lysophosphatidylserine regulates blood glucose by enhancing glucose transport in myotubes and adipocytes

Lysophosphatidylserine (LPS) is known to have diverse cellular effects, but although LPS is present in many biological fluids, its in vivo effects have not been elucidated. In the present study, we investigated the effects of LPS on glucose metabolism in vivo, and how skeletal muscle cells respond to LPS stimulation. LPS enhanced glucose uptake in a dose- and time-dependent manner in L6 GLUT4myc myotubes, and this effect of LPS on glucose uptake was mediated by a Gα_i and PI 3-kinase dependent signal pathway. LPS increased the level of GLUT4 on the cell surface of L6 GLUT4myc myotubes, and enhanced glucose uptake in 3T3-L1 adipocytes. In line with its cellular functions, LPS lowered blood glucose levels in normal mice, while leaving insulin secretion unaffected. LPS also had a glucose-lowering effect in STZ-treated type 1 diabetic mice and in obese db/db type 2 diabetic mice. This study shows that LPS-stimulated glucose transport both in skeletal muscle cells and adipocytes, and significantly lowered blood glucose levels both in type 1 and 2 diabetic mice. Our results suggest that LPS is involved in the regulation of glucose homeostasis in skeletal muscle and adipose tissue.