Adipocytes In Response Research Articles

Role of PKG‐I in differentiation of bone marrow‐derived mesenchymal stem cells (BM‐MSCs, OP9 cells) into lipid‐accumulating fat cells through RhoA phosphorylation, IRS‐1 and PI3‐kinase in insulin signaling pathway (609.18)

3T3‐L1 cells have been used as a model for obesity and insulin response studies. A major limitation in using 3T3‐L1 has been a minimum two week time period required for allowing the cells to differentiate. Recently OP9 cells (bone marrow‐derived mesenchymal stem cells) have been found to differentiate into adipocytes within two days and express late markers of adipocyte differentiation. In an earlier study we found that PKG‐Iα is necessary for OP9 cell survival, proliferation and migration. Further, we have used BM‐MSCs as a model to study the mechanism of Insulin resistance and the role of PKG‐Iα in the pathway regulation. Recent studies show that serine‐phosphorylation of IRS (insulin‐receptor‐substrate) shortens its half‐life and decreases its association with PI3‐kinase. Our laboratory has shown that two isoforms of PKG‐I (PKG‐Iα and PKG‐Iβ) differ dramatically in their cellular functions. BM‐MSCs express only PKG‐Iα. Interestingly, type‐1 and type‐2 diabetes are associated with dramatic decreases in expression and kinase‐activity of PKG‐Iα. In the present study, we investigated the ability of BM‐MSCs to differentiate into lipid‐accumulating adipocytes in response to high‐insulin and the potential involvement of other insulin‐signaling‐proteins, PKG‐I, RhoA, IRS‐1 and PI3‐kinase (PI3K). We found that when these cells differentiate into lipid‐droplet‐containing cells the process is associated with dramatic decreases (upto 50%) of protein expression levels of PKG‐I, IRS‐1, PI3K and a decrease in RhoA serine‐188‐phosphorylation (site phosphorylated and regulated by PKG‐I). These changes in PKG‐I‐catalyzed phosphorylation of RhoA and decreased levels of IRS‐1 and PI3K may play a key role in the differentiation of BM‐MSC into fat cells, making them a useful model for adipogenesis, obesity and type‐2 diabetes.

Effects of pregnancy vitamin D status on adipose tissue development and inflammation in lean, male adult mice offspring (1037.4)

The bioactive form of vitamin D, 1,25‐dihydroxyvitamin D, and its receptor have been identified for a role in preadipocyte differentiation to adipocytes through the modulation of adipogenic transcript factor genes. We sought to determine if maternal vitamin D status influences adipose tissue development or inflammation in adult offspring. Female C57BL/6J mice were randomized to either a vitamin D deficient diet or a control diet (AIN‐93G) for 21 days prior to mating and maintained on this diet throughout pregnancy until postnatal (PN) day 7, at which point both dietary groups were switched to the control diet. At PN day 75, perigonadal (PGAT) and subcutaneous (SQAT) adipose tissue were collected from adult male offspring for general histology and mRNA expression of adipogenic genes. Serum was also collected. There were no differences between groups in whole body weight, fat pad weights, or adipocyte size or number. Mice born to vitamin D deficient dams had elevated serum concentrations of the pro‐inflammatory markers interleukin‐2 and resistin. Maternal vitamin D status during pregnancy does not appear to influence the development of adipose in lean male offspring maintained on a standard diet; however, increases in circulating resistin, an adipose‐derived protein, suggests an adipocyte response.Grant Funding Source: Supported by: UM Research Investment Fund and SIRC Research Grant

Superantigen activates the gp130 receptor on adipocytes resulting in altered adipocyte metabolism

ObjectiveThe bacteria Staphylococcus aureus is part of the normal bacterial flora and produces a repertoire of enterotoxins which can cause food poisoning and toxic shock and might contribute to the pathogenesis of inflammatory diseases. These enterotoxins directly cross-link the T cell receptor with MHC class II, activating large amounts of T cells and are therefore called superantigens. It was recently discovered that the superantigen SEA binds to the cytokine receptor gp130. As obesity and type 2 diabetes are highly associated with inflammation of the adipose tissue and gp130 has been shown to play an important role in adipocytes, we wanted to investigate the effect of SEA on adipocyte signaling and function. Materials/methodsBinding of SEA to gp130 was examined using surface plasmon resonance in a cell free system. Effects of SEA on adipocyte signaling, insulin sensitivity and function were studied using western blotting and biological assays for lipolysis, lipogenesis and glucose uptake. ResultsWe demonstrate that SEA binds to gp130 with a medium affinity. Furthermore, SEA induces phosphorylation of a key downstream target, STAT3, in adipocytes. SEA also inhibits insulin-induced activation of PKB and PKB downstream signaling which was associated with reduced basal and insulin induced glucose uptake, reduced lipogenesis as well as reduced ability of insulin to inhibit lipolysis. ConclusionsSEA inhibits insulin signaling as well as insulin biological responses in adipocytes supporting that bacterial infection might contribute to the development of insulin resistance and type 2 diabetes.

Ectopic expression of RBP4 impairs the insulin pathway and inguinal fat deposition in mice

A large body of evidence has linked retinol-binding protein 4 (RBP4) to systemic insulin resistance, but little is known about its function in fat deposition. This study aimed to confirm the involvement of RBP4 in inguinal fat deposition and insulin by intraperitoneal injection of adenovirus-mediated RBP4 to mice. Intraperitoneal injection of adenoviral vectors was validated as an efficient gene manipulation tool for over-expressing recombinant proteins in vivo. Ectopic expression of RBP4 decelerated inguinal fat deposition by decreasing the size of adipocytes. Moreover, the introduction of exogenous RBP4 blunted the response of inguinal adipocytes to insulin signals. These findings suggest that RBP4 impaired in vivo adipogenesis, partly through the repression of the insulin pathway.

Chemokine network during adipogenesis in 3T3-L1 cells

Obesity is recognized as a low-grade chronic inflammatory state which involves a chemokine network contributing to a variety of diseases. As a first step toward understanding the roles of the obesity-driven chemokine network, we used a 3T3-L1 cell differentiation model to identify the chemokine profiles elicited during adipogenesis and how this profile is modified by epidermal growth factor (EGF) and tumor necrosis factor-α (TNF) as a growth and proinflammatory factor, respectively. The chemokine network was monitored using PCR arrays and qRT-PCR while main signaling pathways of EGF and TNF were measured using immunoblotting. The dominant chemokines in preadipocytes were CCL5, CCL8, CXCL1, and CXCL16, and in adipocytes CCL6 and CXCL13. The following chemokines were found in both preadipocytes and adipocytes: CCL2, CCL7, CCL25, CCL27, CXCL5, CXCL12, and CX3CL1. Among chemokine receptors, CXCR7 was specific for preadipocytes and CXCR2 for adipocytes. These findings indicate the development of a CXCL12–CXCR7 axis in preadipocytes and a CXCL5–CXCR2 axis in adipocytes. In addition to induction of CCL2 and CCL7 in both preadipocytes and adipocytes, EGF enhanced specifically CXCL1 and CXCL5 in adipocytes, indicating the potentiation of CXCR2-mediated pathway in adipocytes. TNF induced CCL2, CCL7, and CXCL1 in preadipocytes but had no response in adipocytes. EGFR downstream activation was dominant in adipocytes whereas NFκB activation was dominant in preadipocytes. Taken together, the adipocyte-driven chemokine network in the 3T3-L1 cell differentiation model involves CXCR2-mediated signaling which appears more potentiated to growth factors like EGF than proinflammatory factors like TNF.

LPS response pattern of inflammatory adipokines in an in vitro 3T3-L1 murine adipocyte model

In vitro 3T3-L1 mouse cells represent a reliable model to investigate the inflammatory phenotype of adipocytes activated by bacteria-derived lipopolysaccharide (LPS). In this study we have evaluated the differential expression of adipokines in response to increasing doses of LPS and various incubation times. 3T3-L1 mouse adipocytes were treated with E. coli LPS (from 0 to 10 μg/ml) for a time course ranging from 4 to 24 h, 4 h each. A time point at 2 h was also included to highlight early activation by LPS. mRNA expression by RT-PCR on cell lysates and ELISA assays on cell culture supernatants were performed. Cells activated by increasing doses of LPS upregulated TNF-α expression in the first 2 h, but this expression slowed down within 6-8 h, while IL-6 expression was increasing. This reduction was also observed for CXCL12/SDF1α. Unlike IL-10, IL-6 expression was constantly upregulated by prolonging incubation with LPS. TNF-α and CXCL12 gene expression occurred early in the time-course and exhibited a second increase following the first 4-6 h of incubation with LPS. Optimal expression of most adipokines needed 6-8 h of a prolonged treatment with LPS at 37 °C. The chemokines MIP-1α/CCL3 and MIP-1β/CCL4 were maximally expressed within the first 8 h, then significantly reduced in the following times. IL-10 expression was upregulated by low doses of LPS and downregulated by prolonging time with the bacterial endotoxin. ELISA analysis of released products generally confirmed the result from gene expression experiments. These data, while assessing previously reported results, highlighted new evidence about the time-dependency in LPS-mediated adipokine production, thus contributing to the comprehension of the inflammatory response of adipocyte.

Long-term niacin treatment induces insulin resistance and adrenergic responsiveness in adipocytes by adaptive downregulation of phosphodiesterase 3B

The lipid-lowering effect of niacin has been attributed to the inhibition of cAMP production in adipocytes, thereby inhibiting intracellular lipolysis and release of nonesterified fatty acids (NEFA) to the circulation. However, long-term niacin treatment leads to a normalization of plasma NEFA levels and induces insulin resistance, for which the underlying mechanisms are poorly understood. The current study addressed the effects of long-term niacin treatment on insulin-mediated inhibition of adipocyte lipolysis and focused on the regulation of cAMP levels. APOE*3-Leiden.CETP transgenic mice treated with niacin for 15 wk were subjected to an insulin tolerance test and showed whole body insulin resistance. Similarly, adipocytes isolated from niacin-treated mice were insulin resistant and, interestingly, exhibited an increased response to cAMP stimulation by 8Br-cAMP, β1- and β2-adrenergic stimulation. Gene expression analysis of the insulin and β-adrenergic pathways in adipose tissue indicated that all genes were downregulated, including the gene encoding the cAMP-degrading enzyme phosphodiesterase 3B (PDE3B). In line with this, we showed that insulin induced a lower PDE3B response in adipocytes isolated from niacin-treated mice. Inhibiting PDE3B with cilostazol increased lipolytic responsiveness to cAMP stimulation in adipocytes. These data show that long-term niacin treatment leads to a downregulation of PDE3B in adipocytes, which could explain part of the observed insulin resistance and the increased responsiveness to cAMP stimulation.

Carnosic acid inhibits TLR4-MyD88 signaling pathway in LPS-stimulated 3T3-L1 adipocytes.

BACKGROUND/OBJECTIVESCarnosic acid (CA), found in rosemary (Rosemarinus officinalis) leaves, is known to exhibit anti-obesity and anti-inflammatory activities. However, whether its anti-inflammatory potency can contribute to the amelioration of obesity has not been elucidated. The aim of the current study was to investigate the effect of CA on Toll-like receptor 4 (TLR4) pathways in the presence of lipopolysaccharide (LPS) in 3T3-L1 adipocytes.MATERIALS/METHODS3T3-L1 adipocytes were treated with CA (0-20 µM) for 1 h, followed by treatment with LPS for 30 min; mRNA expression of adipokines and protein expression of TLR4-related molecules were then measured.RESULTSLPS-stimulated 3T3-L1 adipocytes showed elevated mRNA expression of tumor necrosis factor (TNF)-α, interleukin-6, and monocyte chemoattractant protein-1, and CA significantly inhibited the expression of these adipokine genes. LPS-induced up regulation of TLR4, myeloid differentiation factor 88, TNF receptor-associated factor 6, and nuclear factor-κB, as well as phosphorylated extracellular receptor-activated kinase were also suppressed by pre-treatment of 3T3-L1 adipocytes with CA.CONCLUSIONSResults of this study suggest that CA directly inhibits TLR4-MyD88-dependent signaling pathways and decreases the inflammatory response in adipocytes.

Agonist-specific participation of SOC and ARC channels and iPLA2 in the regulation of Ca2+ entry during oscillatory responses in adipocytes

Activation of Ca 2+ entry upon cell stimulation by agonists can be accomplished by different mech� anisms, including storeoperated calcium entry (SOCE) and the action of phospholipase A2 (PLA2) prod� ucts. In adipocytes there are two relatively independent pathways for Ca 2+ mobilization from intracellular stores. In the present work we studied, whether these pathways are coupled with particular mechanisms of Ca 2+ entry into the cell. It is shown that acetylcholine (ACh) induces oscillatory responses in cytosolic Ca 2+ concentration independently of SOCE inhibition by YM�58483 or 2�APB. These oscillations were abolished by the addition of La 3+ , which inhibits both storeoperated calc ium (SOC) and ARC channels (regulated by arachidonic acid, AA). The responses to ACh were suppressed by AACOCF3 inhibiting PLA 2 of type IV and VIA (iPLA2). Oscillations evoked by fetal bovine serum (FBS) were distinguished by the baseline spiking and, in contrast, were terminated by YM�58483 and La 3+ but were not dependent on AACOCF3. The same cell could respond to ACh and FBS at their sequential addition in any order with the intermediate wash. Oscilla� tory responses of a similar (base or elevated line) form to phenylephrine decayed only gradually after the inhi� bition of phospholipase C or inositol 1,4,5�trisphosph ate receptor, and were partially attenuated by the inhib� itors YM�58483 and La 3+ without appreciable influence of AACOCF3. AA at concentrations 1-10 μM caused oscillations when added after spontaneous cessation of AChinduced oscillations or itself, with a dis� cernible effect produced at lower concentrations after ACh. Calmodulin inhibitor R24571 caused oscilla� tions, which could be suppressed by YM�58483 or AACOCF 3 suggesting activation of SOCE and iPLA 2 , respectively. Taken together, these results indicate that the mechanism of Ca 2+ entry activation depends on the signaling pathway involved by an agonist. ACh does not employ SOCE but activates PLA2 with probable participation of the form VIA, which entails the action of its product(s) on ARC channels and likely on lyso� phospholipidactivated channels. FBS acts through SOCE without participation of PLA2. These two versions can coexist in the case of phenylephrine.

Implication of the Tpl2 Kinase in Inflammatory Changes and Insulin Resistance Induced by the Interaction Between Adipocytes and Macrophages

Adipose tissue inflammation is associated with the development of insulin resistance. In obese adipose tissue, lipopolysaccharides (LPSs) and saturated fatty acids trigger inflammatory factors that mediate a paracrine loop between adipocytes and macrophages. However, the inflammatory signaling proteins underlying this cross talk remain to be identified. The mitogen-activated protein kinase kinase kinase tumor progression locus 2 (Tpl2) is activated by inflammatory stimuli, including LPS, and its expression is up-regulated in obese adipose tissue, but its role in the interaction between adipocytes and macrophages remains ill-defined. To assess the implication of Tpl2 in the cross talk between these 2 cell types, we used coculture system and conditioned medium (CM) from macrophages. Pharmacological inhibition of Tpl2 in the coculture markedly reduced lipolysis and cytokine production and prevented the decrease in adipocyte insulin signaling. Tpl2 knockdown in cocultured adipocytes reduced lipolysis but had a weak effect on cytokine production and did not prevent the alteration of insulin signaling. By contrast, Tpl2 silencing in cocultured macrophages resulted in a marked inhibition of cytokine production and prevented the alteration of adipocyte insulin signaling. Further, when Tpl2 was inhibited in LPS-activated macrophages, the produced CM did not alter adipocyte insulin signaling and did not induce an inflammatory response in adipocytes. By contrast, Tpl2 silencing in adipocytes did not prevent the deleterious effects of a CM from LPS-activated macrophages. Together, these data establish that Tpl2, mainly in macrophages, is involved in the cross talk between adipocytes and macrophages that promotes inflammatory changes and alteration of insulin signaling in adipocytes.

Human skeletal muscle fibroblasts, but not myogenic cells, readily undergo adipogenic differentiation

We characterised the adherent cell types isolated from human skeletal muscle by enzymatic digestion, and demonstrated that even at 72 hours after isolation these cultures consisted predominantly of myogenic cells (CD56(+), desmin(+)) and fibroblasts (TE-7(+), collagen VI(+), PDGFRα(+), vimentin(+), fibronectin(+)). To evaluate the behaviour of the cell types obtained, we optimised a double immuno-magnetic cell-sorting method for the separation of myogenic cells from fibroblasts. This procedure gave purities of >96% for myogenic (CD56(+), desmin(+)) cells. The CD56(-) fraction obtained from the first sort was highly enriched in TE-7(+) fibroblasts. Using quantitative analysis of immunofluorescent staining for lipid content, lineage markers and transcription factors, we tested if the purified cell populations could differentiate into adipocytes in response to treatment with either fatty acids or adipocyte-inducing medium. Both treatments caused the fibroblasts to differentiate into adipocytes, as shown by loss of intracellular TE-7, upregulation of the adipogenic transcription factors PPARγ and C/EBPα, and adoption of a lipid-laden adipocyte morphology. By contrast, myogenic cells did not undergo adipogenesis and showed differential regulation of PPARγ and C/EBPα in response to these adipogenic treatments. Our results show that human skeletal muscle fibroblasts are at least bipotent progenitors that can remain as extracellular-matrix-producing cells or differentiate into adipocytes.

The Role of MIZ-1 in MYC-Dependent Tumorigenesis

A hallmark of MYC-transformed cells is their aberrant response to antimitogenic signals. Key examples include the inability of MYC-transformed cells to arrest proliferation in response to antimitogenic signals such as TGF-β or DNA damage and their inability to differentiate into adipocytes in response to hormonal stimuli. Given the plethora of antimitogenic signals to which a tumor cell is exposed, it is likely that the ability to confer resistance to these signals is central to the transforming properties of MYC in vivo. At the same time, the inability of MYC-transformed cells to halt cell-cycle progression on stress may establish a dependence on mutations that impair or disable apoptosis. We propose that the interaction of MYC with the zinc finger protein MIZ-1 mediates resistance to antimitogenic signals. In contrast to other interactions of MYC, there is currently little evidence that MIZ-1 associates with MYC in normal, unperturbed cells. The functional interaction of both proteins becomes apparent at oncogenic expression levels of MYC and association with MIZ-1 mediates both oncogenic functions of MYC as well as tumor-suppressive responses to oncogenic levels of MYC.

Taurine chloramine modulates the expression of adipokines through inhibition of the STAT-3 signaling pathway in differentiated human adipocytes

To examine the possible role of taurine chloramine (TauCl) in modulating the expression of adipokines in adipose tissue associated with obesity, we evaluated the effect of TauCl in human differentiated adipocytes in response to IL-1β. To study the physiological effects of TauCl on adipokine expression, differentiated adipocytes were treated with IL-1β in the presence or absence of TauCl at concentrations ranging from 200 to 600 μM for 7 days. Cell culture supernatants and total RNA were analyzed by ELISA and real-time PCR, respectively, to determine protein and mRNA levels of adipokines, including adiponectin, leptin, IL-6, and IL-8. Levels of proteins involved in relevant signaling pathways were investigated by western blotting. Stimulation with IL-1β significantly decreased levels of adiponectin and leptin in adipocytes, but increased levels of IL-6 and IL-8 in a dose-dependent manner. Treatment with TauCl significantly reversed the modulation of adipokine expression by inhibiting STAT-3 signaling in IL-1β-stimulated adipocytes, independent of MAPK signaling. TauCl treatment more significantly modulated the expression of adipokines in adipocytes stimulated with IL-1β than that of non-stimulated adipocytes, suggesting that TauCl plays a significant role in modulating the expression of adipokines under inflammatory conditions. In conclusion, TauCl and other taurine derivatives that inhibit the STAT-3 signaling pathway can modulate expression of adipokines and thus may be useful as therapeutic agents for obesity-related diseases.

10E,12Z-conjugated linoleic acid impairs adipocyte triglyceride storage by enhancing fatty acid oxidation, lipolysis, and mitochondrial reactive oxygen species

Conjugated linoleic acid (CLA) is a naturally occurring dietary trans fatty acid found in food from ruminant sources. One specific CLA isomer, 10E,12Z-CLA, has been associated with health benefits, such as reduced adiposity, while simultaneously promoting deleterious effects, such as systemic inflammation, insulin resistance, and dyslipidemia. The precise mechanisms by which 10E,12Z-CLA exerts these effects remain unknown. Despite potential health consequences, CLA continues to be advertised as a natural weight loss supplement, warranting further studies on its effects on lipid metabolism. We hypothesized that 10E,12Z-CLA impairs lipid storage in adipose tissue by altering the lipid metabolism of white adipocytes. We demonstrate that 10E,12Z-CLA reduced triglyceride storage due to enhanced fatty acid oxidation and lipolysis, coupled with diminished glucose uptake and utilization in cultured adipocytes. This switch to lipid utilization was accompanied by a potent proinflammatory response, including the generation of cytokines, monocyte chemotactic factors, and mitochondrial superoxide. Disrupting fatty acid oxidation restored glucose utilization and attenuated the inflammatory response to 10E,12Z-CLA, suggesting that fatty acid oxidation is critical in promoting this phenotype. With further investigation into the biochemical pathways involved in adipocyte responses to 10E,12Z-CLA, we can discern more information about its safety and efficacy in promoting weight loss.

Staphylococcal Superantigens Stimulate Immortalized Human Adipocytes to Produce Chemokines

BackgroundHuman adipocytes may have significant functions in wound healing and the development of diabetes through production of pro-inflammatory cytokines after stimulation by gram-negative bacterial endotoxin. Diabetic foot ulcers are most often associated with staphylococcal infections. Adipocyte responses in the area of the wound may play a role in persistence and pathology. We studied the effect of staphylococcal superantigens (SAgs) on immortalized human adipocytes, alone and in the presence of bacterial endotoxin or staphylococcal α-toxin.Methodology/Principal FindingsPrimary non-diabetic and diabetic human preadipocytes were immortalized by the reverse transcriptase component of telomerase (TERT) and the E6/E7 genes of human papillomavirus. The immortal cells were demonstrated to have properties of non-immortalized pre-adipocytes and could be differentiated into mature and functional adipocytes. Differentiated adipocytes exposed to staphylococcal SAgs produced robust levels of cytokines IL-6 and IL-8, but there were no significant differences in levels between the non-diabetic and diabetic cells. Cytokine production was increased by co-incubation of adipocytes with SAgs and endotoxin together. In contrast, α-toxin alone was cytotoxic at high concentrations, but, at sub-cytotoxic doses, did not stimulate production of IL-6 and IL-8.Conclusions/SignificanceEndotoxin has been proposed to contribute to diabetes through enhanced insulin resistance after chronic exposure and stimulation of adipocytes to produce cytokines. Our data indicate staphylococcal SAgs TSST-1 and SEB alone and in combination with bacterial endotoxin also stimulate adipocytes to produce cytokines and thus may contribute to the inflammatory response found in chronic diabetic ulcers and in the systemic inflammation that is associated with the development and persistence of diabetes. The immortal human pre-adipocytes reported here will be useful for studies to understand further the mechanism by which toxins are involved in wound healing and the development and clinical manifestations of obesity and diabetes.

Loss of menin mediated by endothelial cells treated with CoPP is associated with increased maturation of adipocytes

Oxidative stress is caused by an increase in reactive oxygen species (ROS) relative to the antioxidant defense system. An increase in ROS is known to decrease vascular function, increase inflammatory cytokines, and promote adipocyte hypertrophy. A known regulator of the oxidative stress response is the heat shock protein, heme-oxygenase 1 (HO-1), which is induced by cobalt protoporphyrin IX (CoPP). Menin was recently found to promote the sustained expression of heat shock proteins and is implicated in the regulation of oxidative stress. In this study, we investigated how changes in menin expression affected adipogenesis via the interaction between endothelial cells and adipocytes in response to CoPP treatment during oxidative stress. Using angiotensin II (Ang II) to induce oxidative stress in endothelial cells and adipocytes, we observed the induction of various cytokines including EGF, VEGF, angiogenin, IL-6, and MCP-1. Preadipocytes cultured in endothelial cell conditioned media treated with Ang II showed no changes in differentiation markers. Preadipocytes treated with the endothelial cell-conditioned media pretreated with CoPP resulted in an increase in the number of adipocytes, which expressed higher levels of adipocyte differentiation markers in direct correlation with the complete downregulation of the stress response regulator, menin. This change was not detected in adipocytes directly treated with CoPP alone. Therefore, we concluded that loss of menin is associated with the maturation of adipocytes induced by conditioned media from endothelial cells treated with CoPP.

Activation of nucleotide oligomerization domain containing protein 1 induces lipolysis through NF-κB and the lipolytic PKA activation in 3T3-L1 adipocytes

Obesity is associated with chronic inflammation. Toll-like receptors (TLR) and NOD-like receptors (NLR) are two families of pattern recognition receptors that play important roles in the immune response and inflammation in adipocytes. Activation of TLR4 has been shown to stimulate lipolysis from adipose tissue or adipocytes. However, effects of activation of nucleotide-oligomerization domain containing protein 1 (NOD1), one of the prominent members of NLRs, on adipocyte lipolysis have not been studied. Here we report that NOD1 activation by the synthetic ligands (Tri-DAP and C12-iEDAP) stimulated lipolysis in 3T3-L1 adipocytes in a time- and dose-dependent manner. C12-iEDAP-induced lipolysis was attenuated with NOD1 siRNA knockdown, demonstrating the specificity of the effects. Moreover, inhibition of the protein kinase A (PKA)/hormone sensitive lipase (HSL) and NF-κB pathways by the pharmacological inhibitors attenuated the lipolytic effects of C12-iEDAP. Furthermore, we show NOD1 activation induced PKA activation independent of cAMP production and inhibition of NF-κB pathways attenuated phosphorylation of selected PKA lipolytic targets (phosphorylation of Perilipin Ser 517 and HSL Ser 563). Taken together, our results demonstrate a novel role of NOD1 activation, via NF-κB/PKA lipolytic activation, in inducing lipolysis in adipocytes and suggest that NOD1 activation may contribute to dyslipidemia in obesity.

LXR is a negative regulator of glucose uptake in human adipocytes

Obesity increases the risk of developing type 2 diabetes mellitus, characterised by impaired insulin-mediated glucose uptake in peripheral tissues. Liver X receptor (LXR) is a positive regulator of adipocyte glucose transport in murine models and a possible target for diabetes treatment. However, the levels of LXRα are increased in obese adipose tissue in humans. We aimed to investigate the transcriptome of LXR and the role of LXR in the regulation of glucose uptake in primary human adipocytes. The insulin responsiveness of human adipocytes differentiated in vitro was characterised, adipocytes were treated with the LXR agonist GW3965 and global transcriptome profiling was determined by microarray, followed by quantitative RT-PCR (qRT-PCR), western blot and ELISA. Basal and insulin-stimulated glucose uptake was measured and the effect on plasma membrane translocation of glucose transporter 4 (GLUT4) was assayed. LXR activation resulted in transcriptional suppression of several insulin signalling genes, such as AKT2, SORBS1 and CAV1, but caused only minor changes (<15%) in microRNA expression. Activation of LXR impaired the plasma membrane translocation of GLUT4, but not the expression of its gene, SLC2A4. LXR activation also diminished insulin-stimulated glucose transport and lipogenesis in adipocytes obtained from overweight individuals. Furthermore, AKT2 expression was reduced in obese adipose tissue, and AKT2 and SORBS1 expression was inversely correlated with BMI and HOMA index. In contrast to murine models, LXR downregulates insulin-stimulated glucose uptake in human adipocytes from overweight individuals. This could be due to suppression of Akt2, c-Cbl-associated protein and caveolin-1. These findings challenge the idea of LXR as a drug target in the treatment of diabetes.

Identification of Sucrose Non-Fermenting–Related Kinase (SNRK) as a Suppressor of Adipocyte Inflammation

In this study, the role of sucrose non-fermenting–related kinase (SNRK) in white adipocyte biology was investigated. SNRK is abundantly expressed in adipose tissue, and the expression level is decreased in obese mice. SNRK expression is repressed by inflammatory signals but increased by insulin sensitizer in cultured adipocytes. In vivo, adipose tissue SNRK expression can be decreased by lipid injection but enhanced by macrophage ablation. Knocking down SNRK in cultured adipocytes activates both JNK and IKKβ pathways as well as promotes lipolysis. Insulin-stimulated Akt phosphorylation and glucose uptake are impaired in SNRK knockdown adipocytes. Phosphoproteomic analysis with SNRK knockdown adipocytes revealed significantly decreased phosphorylation of 49 proteins by 25% or more, which are involved in various aspects of adipocyte function with a clear indication of attenuated mTORC1 signaling. Phosphorylation of 43 proteins is significantly increased by onefold or higher, among which several proteins are known to be involved in inflammatory pathways. The inflammatory responses in SNRK knockdown adipocytes can be partially attributable to defective mTORC1 signaling, since rapamycin treatment activates IKKβ and induces lipolysis in adipocytes. In summary, SNRK may act as a suppressor of adipocyte inflammation and its presence is necessary for maintaining normal adipocyte function.

AB0104 Serum adipokine (adiponectin, leptin, visfatin, and resistin) levels in patients with rheumatoid arthritis are differentially modulated by disease activity but may not contribute to treatment resistance

BackgroundEven though it is well known that adipokines play important roles in the pathogenesis of RA, the in vivo role of adipokines and their association with disease activity is poorly...