Adipose Tissue Explants Research Articles

Adipocyte phospholipid transfer protein and lipoprotein metabolism.

Phospholipid transfer protein (PLTP) is highly expressed in adipose tissues. Thus, the effect of adipose tissue PLTP on plasma lipoprotein metabolism was examined. We crossed PLTP-Flox-ΔNeo and adipocyte protein 2 (aP2)-Cre recombinase (Cre) transgenic mice to create PLTP-Flox-ΔNeo/aP2-Cre mice that have a 90 and a 60% reduction in PLTP mRNA in adipose tissue and macrophages, respectively. PLTP ablation resulted in a significant reduction in plasma PLTP activity (22%), high-density lipoprotein-cholesterol (21%), high-density lipoprotein-phospholipid (20%), and apolipoprotein A-I (33%) levels, but had no effect on nonhigh-density lipoprotein levels in comparison with those of PLTP-Flox-ΔNeo controls. To eliminate possible effects of PLTP ablation by macrophages, we lethally irradiated PLTP-Flox-ΔNeo/aP2-Cre mice and PLTP-Flox-ΔNeo mice, and then transplanted wild-type mouse bone marrow into them to create wild-type→PLTP-Flox-ΔNeo/aP2-Cre and wild-type→PLTP-Flox-ΔNeo mice. Thus, we constructed a mouse model (wild-type→PLTP-Flox-ΔNeo/aP2-Cre) with PLTP deficiency in adipocytes but not in macrophages. These knockout mice also showed significant decreases in plasma PLTP activity (19%) and cholesterol (18%), phospholipid (17%), and apolipoprotein A-I (26%) levels. To further investigate the mechanisms behind the reduction in plasma apolipoprotein A-I and high-density lipoprotein lipids, we measured apolipoprotein A-I-mediated cholesterol efflux in adipose tissue explants and found that endogenous and exogenous PLTP significantly increased cholesterol efflux from the explants. Adipocyte PLTP plays a small but significant role in plasma PLTP activity and promotes cholesterol efflux from adipose tissues.

Age-Associated Increase in Cytokine Production During Systemic Inflammation-II: The Role of IL-1β in Age-Dependent IL-6 Upregulation in Adipose Tissue.

Expression of interleukin-6 (IL-6) upon acute inflammatory stress is significantly augmented by aging in adipose tissue, a major source of this cytokine. In the present study, we examined the mechanism of age-dependent IL-6 overproduction using visceral white adipose tissue from C57BL/6 mice. Upon treatment with lipopolysaccharide (LPS) in vitro, IL-6 was produced by adipose tissue explants, and secreted levels were significantly higher in cultures from aged (24 months) mice compared to young (4 months). Interleukin 1 beta (IL-1β) and tumor necrosis factor alpha (TNFα), two inducers of IL-6, were mainly produced by the lungs and spleen rather than adipose tissue in mice after LPS injection. Treatment of adipose explants with physiological levels of IL-1β induced significant age-dependent secretion of IL-6, while treatment with TNFα had little effect, demonstrating an augmented response of adipose tissues to IL-1β in the aged. In vitro experiments utilizing a neutralizing antibody against IL-1β and in vivo experiments utilizing IL-1-receptor-1 deficient mice, confirmed that IL-6 overproduction in the aged is regulated by autocrine/paracrine action of IL-1β which specifically occurs in aged adipose tissues. These findings indicate an elevated inflammatory potential of adipose tissue in the aged and a unique IL-1β-mediated mechanism for IL-6 overproduction, which may impact age-associated vulnerability to acute inflammatory diseases such as sepsis.

15-LOX metabolites and angiogenesis: angiostatic effect of 15(S)-HPETE involves induction of apoptosis in adipose endothelial cells.

Inflammation is critical in the dysregulated growth of adipose tissue and associated vascular dysfunctions. 15-Lipoxygenase metabolites, important mediators of inflammation in adipose tissue during obese conditions, may contribute to codependence of inflammation and angiogenesis in adipose tissue. We have already reported the pro-angiogenic effect of 15(S)-HETE in adipose tissue. The present study was designed to understand the effect of 15(S)-HPETE, precursor of 15(S)-HETE, on angiogenesis in adipose tissue. Results showed that 15(S)-HPETE exerts an anti-angiogenic effect in adipose tissue. This was evidenced from decreased endothelial sprouting in adipose tissue explants, inhibition of angiogenic phenotype in adipose endothelial cells, decreased production of CD31 and VEGF in endothelial cells treated with 15(S)-HPETE. Further studies to examine the molecular mechanism of anti-angiogenic effect of 15(S)-HPETE showed that it inhibited cell survival signaling molecule Akt and anti-apoptotic Bcl-2 and also activated caspase-3 in adipose endothelial cells. These observations indicate that 15(S)-HPETE exerts its angiostatic effect in adipose tissue by inducing apoptosis of endothelial cells.

Metabolic programming of adipose tissue structure and function in male rat offspring by prenatal undernutrition.

BackgroundA number of different pathways to obesity with different metabolic outcomes are recognised. Prenatal undernutrition in rats leads to increased fat deposition in adulthood. However, the form of obesity is metabolically distinct from obesity induced through other pathways (e.g. diet-induced obesity). Previous rat studies have shown that maternal undernutrition during pregnancy led to insulin hyper-secretion and obesity in offspring, but not to systemic insulin resistance. Increased muscle and liver glycogen stores indicated that glucose is taken up efficiently, reflecting an active physiological function of these energy storage tissues. It is increasingly recognised that adipose tissue plays a central role in the regulation of metabolism and pathophysiology of obesity development. The present study investigated the cell size and endocrine responsiveness of subcutaneous and visceral adipose tissue from prenatally undernourished rats. We aimed to identify whether these adipose tissue depots contribute to the altered energy metabolism observed in these offspring.MethodsAdipocyte size was measured in both subcutaneous (ScAT) and retroperitoneal adipose tissue (RpAT) in male prenatally ad libitum fed (AD) or prenatally undernourished (UN) rat offspring. Metabolic responses were investigated in adipose tissue explants stimulated by insulin and beta3 receptor agonists ex vivo. Expression of markers of insulin signalling was determined by Western blot analyses. Data were analysed by unpaired t-test or Two Way ANOVA followed by Fisher’s PLSD post-hoc test, where appropriate.ResultsAdipocytes in offspring of undernourished mothers were larger, even at a lower body weight, in both RpAT and ScAT. The insulin response of adipose tissue was reduced in ScAT, and statistically absent in RpAT of UN rats compared with control. This lack of RpAT insulin response was associated with reduced expression of insulin signalling pathway proteins. Adrenergic receptor-driven lipolysis was observed in both adipose depots; however insulin failed to express its anti-lipolytic effect in RpAT in both, AD and UN offspring.ConclusionsMetabolic dysregulation in offspring of undernourished mothers is mediated by increased adipocyte size and reduced insulin responsiveness in both ScAT and especially in RpAT. These functional and morphological changes in adipocytes were accompanied by impaired activity of the insulin signalling cascade highlighting the important role of different adipose tissue depots in the pathogenesis of metabolic disorders.

Fluvastatin causes NLRP3 inflammasome-mediated adipose insulin resistance.

Statins reduce lipid levels and are widely prescribed. Statins have been associated with an increased incidence of type 2 diabetes, but the mechanisms are unclear. Activation of the NOD-like receptor family, pyrin domain containing 3 (NLRP3)/caspase-1 inflammasome, promotes insulin resistance, a precursor of type 2 diabetes. We showed that four different statins increased interleukin-1β (IL-1β) secretion from macrophages, which is characteristic of NLRP3 inflammasome activation. This effect was dose dependent, absent in NLRP3(-/-) mice, and prevented by caspase-1 inhibition or the diabetes drug glyburide. Long-term fluvastatin treatment of obese mice impaired insulin-stimulated glucose uptake in adipose tissue. Fluvastatin-induced activation of the NLRP3/caspase-1 pathway was required for the development of insulin resistance in adipose tissue explants, an effect also prevented by glyburide. Fluvastatin impaired insulin signaling in lipopolysaccharide-primed 3T3-L1 adipocytes, an effect associated with increased caspase-1 activity, but not IL-1β secretion. Our results define an NLRP3/caspase-1-mediated mechanism of statin-induced insulin resistance in adipose tissue and adipocytes, which may be a contributing factor to statin-induced development of type 2 diabetes. These results warrant scrutiny of insulin sensitivity during statin use and suggest that combination therapies with glyburide, or other inhibitors of the NLRP3 inflammasome, may be effective in preventing the adverse effects of statins.

Spatiotemporal dynamics of triglyceride storage in unilocular adipocytes

The spatiotemporal dynamics of triglyceride (TG) storage in unilocular adipocytes are not well understood. Here we applied ex vivo technology to study trafficking and metabolism of fluorescent fatty acids in adipose tissue explants. Live imaging revealed multiple cytoplasmic nodules surrounding the large central lipid droplet (cLD) of unilocular adipocytes. Each cytoplasmic nodule harbors a series of closely associated cellular organelles, including micro-lipid droplets (mLDs), mitochondria, and the endoplasmic reticulum. Exogenously added free fatty acids are rapidly adsorbed by mLDs and concurrently get esterified to TG. This process is greatly accelerated by insulin. mLDs transfer their content to the cLD, serving as intermediates that mediate packaging of newly synthesized TG in the large interior of a unilocular adipocyte. This study reveals novel cell biological features that may contribute to the mechanism of adipocyte hypertrophy.

Abstract 192: Role of adipose tissue in bladder cancer progression

Abstract Introduction: Obesity has been found to be associated with increased risk of bladder cancer, worse-cancer-specific outcomes and increased risk of recurrence. Moreover, bladder cancers that involve macroscopic extravesical invasion of the perivesical fat are highly predisposed to lymph node metastases and local recurrence. The objective of our study is to elucidate the mechanism by which adipose tissue contributes to bladder cancer progression. We hypothesize that adipose tissue in bladder cancer patients has the potential to modulate the tumor microenvironment by inducing pro-tumorigenic pathways in bladder cancer progression. Methods: Human adipose tissue fragments (bladder, urachal, subcutaneous and prostate when applicable) were collected from muscle-invasive bladder cancer (MIBC) patients undergoing cystectomy. Information on patient Body Mass Index (BMI), diabetes diagnosis, medications and gender were used in analysis. BMI status was classified into normal (under 25 kg/m2), overweight (25-29.9 kg/m2) and obese (≥30 kg/m2). Gene expression was profiled from total RNA isolated from 100mg of fat using the Illumina Human HT12 whole genome expression BeadChips. Fat conditioned media (CM) was prepared from 500 mg of the patient fat processed as primary adipose tissue explant cultures for 16 hours. Secreted factors from CM were measured using MILLIPLEX MAP 41-plex Human Cytokine/Chemokine Panel. Migratory potential was assayed by Transwell (BD falcon) migration. T24 human bladder cancer cells were placed in the upper chamber with patient fat CM or control serum free media in the lower chamber. Cells were allowed to transmigrate across the porous membrane for 18 hours. Results: At the genomic level in fat specimens from twelve cancer patients with different BMI status indicated a number of differentially expressed genes previously implicated in cancer progression. The expression of CCL3/macrophage inflammatory protein-1α (a secreted protein) and matrix metalloproteinase-9 are significantly upregulated in fat samples from obese and overweight patients relative to normal. At the protein level in CM, three factors: interleukin-8, growth regulated oncogene-alpha and monocyte chemotactic protein-1, are found in high levels across all patient tissue depots. Surprisingly, exposing T24 cells to CM from bladder fat depots increased their migratory potential. Ongoing studies are evaluating the specific factors involved in this difference. Conclusions: The identified differentially expressed genes and secreted factors provide a possible mechanism to explain the effects of obesity on tumor progression in bladder cancer. In addition to BMI, the migratory potential of T24 cells was also differentially modulated by the different fat deposit types. Ongoing studies are investigating the effect of adipose tissue-derived factors on bladder cancer to help improve outcome in advance bladder cancer patients with added complexities of obesity. Citation Format: Nisha Hariharan, Robert S. Svatek, Carolina B. Livi, Jonathan A.L. Gelfond, Teresa L. Johnson-Pais, Robin J. Leach. Role of adipose tissue in bladder cancer progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 192. doi:10.1158/1538-7445.AM2014-192

Abstract 3037: Adipokines modulate human mammary stem cell self-renewal through mTOR

Abstract Rationale: Excess visceral adiposity is associated with an increased incidence of a variety of human ailments, including cancer. We tested the hypothesis that adipose-derived factors drive normal mammary stem cell self-renewal. Methods: Visceral adipose tissue explants from obese F344 rats and cultured mouse 3T3-L1 adipocytes were grown with serum-free media for 48 hours. Primary human mammary epithelial cells, MCF10A, SUM149 and MCF7 cells were exposed to rhleptin (500 ng/ml), or adipocyte conditioned media and expanded in a non-adherent sphere forming assay for 10 days. Spheres were counted and size was quantified in Image J. Adipokine concentrations were assayed with the Proteome Profiler Adipokine Array. Other cultures were pre-treated with mTOR inhibitors rapamycin (100 nM) or everolimus (100 nM) for 2 hours prior to exposure to rhleptin or adipocyte conditioned media for 2 hours. S6 protein phosphorylation was quantified from Western blots. Results: Data shown in Table 1 suggest that conditioned media from adipose tissue and from 3T3-L1 adipocytes increase human mammary stem cell self-renewal by 103% and 189%, respectively, whereas rhleptin increases stem cell self-renewal by 37%. Blockade of leptin activity with neutralizing antibodies or chimeric soluble receptors reduced the adipocyte conditioned media's stimulation of mammary epithelial cell self-renewal by 10-15%. Media conditioned by the mouse 3T3-L1 adipocytes or primary rat visceral adipose explants increased pS6 protein by 170% in MCF10A cells. mTOR inhibitors reduced rhleptin and adipocyte conditioned media stimulation of self-renewal in MCF10A and normal human mammary stem cells by 78-85%. Conclusion: Adipocyte secreted factors and rhleptin promote increased mammary epithelial cell self-renewal through a process involving the activation of mTOR. Leptin contributes to a small fraction of the activity in adipocyte conditioned media, and is sufficient but not required for mammary stem cell self-renewal. Change in stem cell self-renewalrhLeptin3T3-L1 Adipocyte Conditioned MediaRat Adipose Tissue Conditioned MediaBaseline # Spheres% Change (± SEM)P Value% Change (± SEM)P Value% Change (± SEM)P ValuePrimary human organoids19737 ± 7.005104 ± 20.006190 ± 26.001MCF-10A cells28834 ± 16.043320 ± 32.002389 ± 27.002MCF-7 cells57638 ± 9.007142 ± 15.001262 ± 40.006SUM149 cells95435 ± 6.09174 ± 20.012167 ± 21.003 Citation Format: Ray Esper, Dame Michael, Zora Djuric, William Smith, Max Wicha, Dean Brenner. Adipokines modulate human mammary stem cell self-renewal through mTOR. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3037. doi:10.1158/1538-7445.AM2014-3037

Gene expression signature in adipose tissue of acromegaly patients

To study the effect of chronic excess growth hormone on adipose tissue, we performed RNA sequencing in adipose tissue biopsies from patients with acromegaly (n = 7) or nonfunctioning pituitary adenomas (n = 11). The patients underwent clinical and metabolic profiling including assessment of HOMA-IR. Explants of adipose tissue were assayed ex vivo for lipolysis and ceramide levels. Patients with acromegaly had higher glucose, higher insulin levels and higher HOMA-IR score. We observed several previously reported transcriptional changes (IGF1, IGFBP3, CISH, SOCS2) that are known to be induced by GH/IGF-1 in liver but are also induced in adipose tissue. We also identified several novel transcriptional changes, some of which may be important for GH/IGF responses (PTPN3 and PTPN4) and the effects of acromegaly on growth and proliferation. Several differentially expressed transcripts may be important in GH/IGF-1-induced metabolic changes. Specifically, induction of LPL, ABHD5, and NRIP1 can contribute to enhanced lipolysis and may explain the elevated adipose tissue lipolysis in acromegalic patients. Higher expression of TCF7L2 and the fatty acid desaturases FADS1, FADS2 and SCD could contribute to insulin resistance. Ceramides were not different between the two groups. In summary, we have identified the acromegaly gene expression signature in human adipose tissue. The significance of altered expression of specific transcripts will enhance our understanding of the metabolic and proliferative changes associated with acromegaly.

Phospholipase C-Related Catalytically Inactive Protein (PRIP) Regulates Lipolysis in Adipose Tissue by Modulating the Phosphorylation of Hormone-Sensitive Lipase

Phosphorylation of hormone-sensitive lipase (HSL) and perilipin by protein kinase A (PKA) promotes the hydrolysis of lipids in adipocytes. Although activation of lipolysis by PKA has been well studied, inactivation via protein phosphatases is poorly understood. Here, we investigated whether phospholipase C-related catalytically inactive protein (PRIP), a binding partner for protein phosphatase 1 and protein phosphatase 2A (PP2A), is involved in lipolysis by regulating phosphatase activity. PRIP knockout (PRIP-KO) mice displayed reduced body-fat mass as compared with wild-type mice fed with standard chow ad libitum. Most other organs appeared normal, suggesting that mutant mice had aberrant fat metabolism in adipocytes. HSL in PRIP-KO adipose tissue was highly phosphorylated compared to that in wild-type mice. Starvation of wild-type mice or stimulation of adipose tissue explants with the catabolic hormone, adrenaline, translocated both PRIP and PP2A from the cytosol to lipid droplets, but the translocation of PP2A was significantly reduced in PRIP-KO adipocytes. Consistently, the phosphatase activity associated with lipid droplet fraction in PRIP-KO adipocytes was significantly reduced and was independent of adrenaline stimulation. Lipolysis activity, as assessed by measurement of non-esterified fatty acids and glycerol, was higher in PRIP-KO adipocytes. When wild-type adipocytes were treated with a phosphatase inhibitor, they showed a high lipolysis activity at the similar level to PRIP-KO adipocytes. Collectively, these results suggest that PRIP promotes the translocation of phosphatases to lipid droplets to trigger the dephosphorylation of HSL and perilipin A, thus reducing PKA-mediated lipolysis.

AB0106 Assessment of the Relation between the Secretory Activity of Abdominal Adipose Tissue and Clinical Activity of Rheumatoid Arthritis

BackgroundAdipose tissue has the ability to release a wide range of mediators involved in the inflammatory responseObjectivesTo investigate spontaneous adipocytokine secretion by subcutaneous abdominal adipose tissue (SAAT) explants obtained from...

AB0101 Articular Adipose Tissue from Osteoarthritis Patients is A Source of Cytokines and Adipokines

BackgroundRecently rheumatoid adipose tissue has been suggested to play an important role in chronic inflammatory joint diseases. Its ability to synthesize cytokines and adipokines has been characterized also in our...

Bacterial peptidoglycan stimulates adipocyte lipolysis via NOD1.

Obesity is associated with inflammation that can drive metabolic defects such as hyperlipidemia and insulin resistance. Specific metabolites can contribute to inflammation, but nutrient intake and obesity are also associated with altered bacterial load in metabolic tissues (i.e. metabolic endotoxemia). These bacterial cues can contribute to obesity-induced inflammation. The specific bacterial components and host receptors that underpin altered metabolic responses are emerging. We previously showed that Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) activation with bacterial peptidoglycan (PGN) caused insulin resistance in mice. We now show that PGN induces cell-autonomous lipolysis in adipocytes via NOD1. Specific bacterial PGN motifs stimulated lipolysis in white adipose tissue (WAT) explants from WT, but not NOD1−/− mice. NOD1-activating PGN stimulated mitogen activated protein kinases (MAPK),protein kinase A (PKA), and NF-κB in 3T3-L1 adipocytes. The NOD1-mediated lipolysis response was partially reduced by inhibition of ERK1/2 or PKA alone, but not c-Jun N-terminal kinase (JNK). NOD1-stimulated lipolysis was partially dependent on NF-κB and was completely suppressed by inhibiting ERK1/2 and PKA simultaneously or hormone sensitive lipase (HSL). Our results demonstrate that bacterial PGN stimulates lipolysis in adipocytes by engaging a stress kinase, PKA, NF-κB-dependent lipolytic program. Bacterial NOD1 activation is positioned as a component of metabolic endotoxemia that can contribute to hyperlipidemia, systemic inflammation and insulin resistance by acting directly on adipocytes.

Agonists of the G protein-coupled receptor 109A-mediated pathway promote antilipolysis by reducing serine residue 563 phosphorylation of hormone-sensitive lipase in bovine adipose tissue explants

A balanced lipolytic regulation in adipose tissues based on fine-tuning of prolipolytic and antilipolytic pathways is of vital importance to maintain the metabolic health in dairy cows. Antilipolytic pathways, such as the G protein-coupled receptor 109A (GPR109A)-mediated pathway and the insulin signaling pathway in bovine adipose tissues may be involved in prohibiting excessive lipomobilization by reducing triglycerol hydrolysis. This study aimed to evaluate the in vitro antilipolytic potential of the mentioned pathways in bovine adipose tissue explants. Therefore, subcutaneous and retroperitoneal adipose tissue samples (approximately 100mg) of German Holstein cows were treated for 90min ex vivo with nicotinic acid (2, 8, or 32μM), nicotinamide (2, 8, or 32μM), β-hydroxybutyrate (0.2, 1, or 5mM), or insulin (12mU/L), with a concurrent lipolytic challenge provoked with 1μM isoproterenol. Lipolytic and antilipolytic responses of the adipose tissues were assessed by measuring free glycerol and nonesterified fatty acid release. To identify molecular components of the investigated antilipolytic pathways, protein abundance of GPR109A and the extent of hormone-sensitive lipase (HSL) phosphorylation at serine residue 563 were detected by Western blotting. Treatment with nicotinic acid or β-hydroxybutyrate decreased the lipolytic response in adipose tissue explants and concurrently reduced the extent of HSL phosphorylation, but treatment with nicotinamide or insulin did not. Subcutaneous adipose tissue constitutively expressed more GPR109A protein, but no other depot-specific differences were observed. This study provides evidence that the GPR109A-mediated pathway is functionally existent in bovine adipose tissues, and confirms that HSL phosphorylation at serine residue 563 is also important in antilipolytic regulation in vitro. This antilipolytic pathway may be involved in a balanced lipid mobilization in the dairy cow.

Resistin in Dairy Cows: Plasma Concentrations during Early Lactation, Expression and Potential Role in Adipose Tissue

Resistin is an adipokine that has been implicated in energy metabolism regulation in rodents but has been little studied in dairy cows. We determined plasma resistin concentrations in early lactation in dairy cows and investigated the levels of resistin mRNA and protein in adipose tissue and the phosphorylation of several components of insulin signaling pathways one week post partum (1 WPP) and at five months of gestation (5 MG). We detected resistin in mature bovine adipocytes and investigated the effect of recombinant bovine resistin on lipolysis in bovine adipose tissue explants. ELISA showed that plasma resistin concentration was low before calving, subsequently increasing and reaching a peak at 1 WPP, decreasing steadily thereafter to reach pre-calving levels at 6 WPP. Plasma resistin concentration was significantly positively correlated with plasma non esterified fatty acid (NEFA) levels and negatively with milk yield, dry matter intake and energy balance between WPP1 to WPP22. We showed, by quantitative RT-PCR and western blotting, that resistin mRNA and protein levels in adipose tissue were higher at WPP1 than at 5 MG. The level of phosphorylation of several early and downstream insulin signaling components (IRβ, IRS-1, IRS-2, Akt, MAPK ERK1/2, P70S6K and S6) in adipose tissue was also lower at 1 WPP than at 5 MG. Finally, we showed that recombinant bovine resistin increased the release of glycerol and mRNA levels for ATGL (adipose triglyceride lipase) and HSL (hormone-sensitive lipase) in adipose tissue explants. Overall, resistin levels were high in the plasma and adipose tissue and were positively correlated with NEFA levels after calving. Resistin is expressed in bovine mature adipocytes and promotes lipid mobilization in adipose explants in vitro.

Effect of growth hormone on the differentiation of bovine preadipocytes into adipocytes and the role of the signal transducer and activator of transcription 5b1

We evaluated the effect of GH on the differentiation of primary bovine preadipocytes into adipocytes. Bovine preadipocytes, derived from adipose tissue explants, were induced to differentiate into adipocytes in the presence or absence of recombinant bovine GH. The differentiation status of adipocytes was assessed by Oil Red O staining and by measuring the activity of glycerol-3-phosphate dehydrogenase (G3PDH) and the rate of acetate incorporation. Fewer preadipocytes became adipocytes in the presence of GH than in the absence of GH; adipocytes formed in the presence of GH had lower G3PDH activity and lower rate of acetate incorporation than those formed without GH treatment (P < 0.05). These data suggest an inhibitory effect of GH on the differentiation of bovine preadipocytes into adipocytes. Growth hormone decreased the expression of C/EBPα and PPARγ mRNA in bovine adipocytes (P < 0.05). Because C/EBPα and PPARγ are the master regulators of adipocyte differentiation, this data suggests that GH might inhibit the differentiation of bovine preadipocytes into adipocytes by inhibiting the expression of C/EBPα and/or PPARγ. Because the signal transducer and activator of transcription 5 (STAT5) is a major component of signaling from the GH receptor, we next determined the potential role of STAT5 in GH inhibition of bovine adipocyte differentiation. Overexpression of a constitutively active form of STAT5b (STAT5bCA) in bovine preadipocytes through adenoviral transduction mimicked the effects of GH on the formation of lipid-containing adipocytes, G3PDH activity, and acetate incorporation rate. Overexpression of STAT5bCA was associated with decreased expression of C/EBPα mRNA (P < 0.05) but not that of PPARγ mRNA in bovine adipocytes. These results support a role of STAT5b in mediating GH inhibition of C/EBPα expression but not that of PPARγ expression in bovine preadipocytes. Overall, the present study suggests that GH may inhibit adipose growth in cattle in part by inhibiting adipogenesis and that GH inhibits the differentiation of bovine preadipocytes to adipocytes through STAT5b-dependent inhibition of C/EBPα expression and STAT5b-independent inhibition of PPARγ expression.

Balanced duo of anti-inflammatory SFRP5 and proinflammatory WNT5A in children

Secreted frizzled-related protein 5 (SFRP5) is an adipokine protecting against obesity-related insulin resistance and diabetes. SFRP5 binds to wingless type mouse mammary tumor virus (MMTV) integration site family member 5A (WNT5A) to improve insulin sensitivity. We performed the first study of SFRP5 and WNT5A simultaneously in children. Prepubertal children (n = 342) were assessed for circulating SFRP5 (all subjects) and circulating WNT5A (210 subjects), and associations were sought with metabolic markers. In conditioned media of adipose tissue explants from 12 additional children, SFRP5 and WNT5A were studied further. The concentrations of SFRP5 and WNT5A correlated positively in serum and in conditioned media (all P < 0.001). Lower level of circulating SFRP5 (lowest quartile) was associated with higher BMI (15% increase, P < 0.0001) and lower level of high-molecular-weight adiponectin (26% decrease, P = 0.002). Circulating WNT5A related closely with insulin resistance assessed by the homeostasis model assessment for insulin resistance and hepatic markers (alanine transaminase and gamma glutamyl transpeptidase), particularly in children with lower circulating SFRP5 levels (all P < 0.004). SFRP5 and WNT5A comprise a balanced duo that may regulate metabolic homeostasis in prepubertal children.

Insulin regulates the novel adipokine adipolin/CTRP12: in vivo and ex vivo effects

There has been intense interest in the adipokines of the C1q complement/TNF-related protein (CTRP) superfamily. Adipolin (CTRP12) has been described as a novel adipokine, abundantly expressed in adipose tissue with insulin-sensitising and anti-inflammatory effects. We wanted to investigate the effects of acute and chronic hyperinsulinaemia on circulating adipolin concentrations (ELISA) via a prolonged insulin-glucose infusion in humans. We also examined the effects of insulin and the insulin sensitiser, rosiglitazone, on adipolin concentrations (western blotting) in human adipose tissue explants. We found that hyperinsulinaemic induction in healthy lean human subjects significantly increased circulating levels of adipolin (P<0.05 and P<0.01). Furthermore, in subcutaneous adipose tissue explants, insulin significantly increased adipolin protein expression and secretion (P<0.05 and P<0.01). This effect was attenuated by the phosphatidylinositol 3-kinase inhibitor, LY294002 (P<0.05). Moreover, the insulin-sensitising peroxisome proliferator-activated receptor γ (PPARγ) agonist, rosiglitazone, significantly increased adipolin protein expression and secretion in subcutaneous adipose tissue explants (P<0.05 and P<0.01). This effect was inhibited by the PPARγ antagonist, GW9662 (P<0.05). Our data provide novel insights into adipolin physiology in human subjects.

A5.7 Articular adipose tissue from OA patients is an important source of cytokines and adipokines

Background and ObjectivesRecently rheumatoid adipose tissue has been suggested to play an important role in chronic inflammatory joint diseases. Its ability to synthesize cytokines and adipokines has been characterised also...

Identification of a novel GPR81-selective agonist that suppresses lipolysis in mice without cutaneous flushing

GPR81, which exhibits a high degree of homology with GPR109a, has been recently identified as a lactate receptor. Similar to GPR109a, the activation of GPR81 by lactate suppresses lipolysis, suggesting that GPR81 may be a potential drug target for treating dyslipidemia. In addition, the fact that GPR81 is expressed only in adipocytes, whereas GPR109a is expressed in various tissues and cells, including Langerhans cells, which are considered responsible for flushing, indicates that targeting GPR81 could lead to the development of antidyslipidemia agents with a reduced risk of this side effect. However, the pharmacological role of GPR81 remains largely unclear, mainly because of the lack of potent and selective surrogate GPR81 agonists suitable for in vivo studies. In the present study, we showed that lactate-induced suppression of lipolysis in explants of white adipose tissue (WAT) depends on the presence of GPR81. We also performed high-throughput screening (HTS) and identified four novel chemical clusters as GPR81 agonists. Chemical optimization of aminothiazole derivatives led to the discovery of a lead compound with improved potency. The compound inhibited lipolysis in differentiated 3T3-L1 adipocytes. Finally, intraperitoneal administration of this compound suppressed lipolysis in mice at doses that did not cause cutaneous flushing. This is the first description of a 50nM GPR81 selective agonist with in vivo efficacy, without the side effect, i.e., flushing. These results suggest that GPR81 is an attractive drug target for treating dyslipidemia without the risk of flushing.