Adipose Tissue Explants Research Articles

Targeting the association of calgranulin B (S100A9) with insulin resistance and type 2 diabetes

Calgranulin B (S100A9) was recognized as a candidate type 2 diabetes (T2D) gene in the genomic profiling of muscle from a rodent model of T2D and identifying the human orthologs of genes localized in T2D susceptibility regions. Circulating and S100A9 expressions in muscle and adipose tissue, isolated fat cells, and mouse models were evaluated. A common 5'-upstream single-nucleotide polymorphism (SNP; rs3014866) for S100A9 was analyzed, as well as the effects of weight loss and treatments in vitro with recombinant S100A9. S100a9 expression was increased in muscle of diabetic mice (1.6-fold, p = 0.002), and in muscle from subjects with impaired glucose tolerance (∼4-fold, p = 0.028; n = 34). The rs3014866 SNP was associated with circulating S100A9 and the risk of T2D, having TT carriers at 28 % (p = 0.03) lower risk (n = 1,450). Indeed, increased circulating S100A9 (∼4-fold, p = 0.03; n = 206) and subcutaneous (2-fold, p = 0.01) and omental (1.4-fold, p = 0.04) S100A9 gene expressions (n = 83) in TT carriers run in parallel to decreased fasting glucose and glycated hemoglobin. Accordingly, metformin led to increased S100A9 mRNA in ex vivo-treated adipose tissue explants (n = 5/treatment). Otherwise, obese subjects showed a compensatory increase in circulating and S100A9 expressions in adipose (n = 126), as further demonstrated by decreased levels after diet- (-34 %, p = 0.002; n = 20) and surgery-induced (-58 %, p = 0.02; n = 8) weight loss. Lipopolysaccharide led to increased S100A9 in adipose from mice (n = 5/treatment) while recombinant S100A9 downregulated inflammation in adipocytes (n = 3/treatment). Current findings support the strategy of testing differentially expressed genes in mice and human orthologs associated with T2D. The increased S100A9 reported for obesity and insulin resistance may be envisioned as a compensatory mechanism for inflammation.

Dietary Restriction and Weight Loss Improves the Outcome of Acute Lymphoblastic Leukemia in Diet-Induced Obesity

Abstract 3570 Background:Obesity increases the mortality of numerous types of cancer. Furthermore, children over 10 years of age who are obese at diagnosis of acute lymphoblastic leukemia (ALL) have twice the risk of relapse. We have found that obese mice transplanted with leukemia are more likely to relapse after treatment with chemotherapy. We further found that adipose tissue can protect ALL cells from chemotherapy in culture. But it is unclear whether a weight loss intervention after leukemia diagnosis will improve outcome. Hypothesis:A weight loss intervention by dietary modification in diet induced obese mice will decrease the ability of their adipose tissue explants to protect ALL cells from chemotherapy. Methods:Diet-induced obese C57Bl/6 mice (n=7) were switched to a low-fat diet either 3 or 21 days prior to sacrifice at 20 weeks of age. Adipose tissue explants (100 ± 5 mg) from perirenal, visceral, and subcutaneous sites were collected and used in transwell co-cultures with 250 X103 8093 murine pre-B ALL cells. Explants from non-dieted obese mice and lean mice were used as controls. Viable ALL cells were quantified using trypan blue exclusion after a 3-day exposure to 15 nM daunorubicin or 5 nM of vincristine. Results:Dietary intervention significantly reduced body weights in the short (39.2 ± 2.4 grams; P= 0.014) and long (32 ± 2.9 grams; P=0.041) diet groups compared to obese mice (44.5 grams ± 2.6).Leukemia cells were partially protected from daunorubicin by both visceral (21.5±15.7×103 viable cells, p=0.01) and perirenal (28.0±20.7×103, p=0.013) fat explants from obese mice, compared to cultures without adipose tissue (9.4±8.6×103). Fat explants from mice dieted for 3 days prior to collection showed similar protection of ALL cells from daunorubicin (not shown); however, fat explants from mice dieted for 21 days did not protect ALL cells from daunorubicin (visceral: 9.4±8.0×103, p=0.009; perirenal: 7.8±6.3×103, p=0.066 vs. obese).Similar findings were obtained with vincristine. Leukemia cells were protected from vincristine significantly by visceral fat from obese mice (50.8±5.9×103, p=0.01) and marginally by perirenal and subcutaneous fat (93.8±139.1×103, p=0.06 and 113.4±143.7×103, p=0.07 respectively) compared to cultures without adipose tissue (12.4±11.7×103). While short dieting did not restore sensitivity to vincristine, long dieting of obese mice reduced explants leukemia protection to vincristine significantly in visceral (21.8±20.2×103, p=0.004) and marginally in perirenal (34.9±43.3×103, p=0.06) explants compared to the obese group. Conclusions:Dietary intervention and weight loss in diet induced obese mice decreases the ability of adipose tissue ex vivo to protect ALL cells from daunorubicin and vincristine. These results could support a potential role for dietary intervention in improving leukemia outcome in obese patients. Disclosures:No relevant conflicts of interest to declare.

Autophagy Activity Is Up-Regulated in Adipose Tissue of Obese Individuals and Modulates Proinflammatory Cytokine Expression

Autophagy, an evolutionary conserved process aimed at recycling damaged organelles and protein aggregates in the cell, also modulates proinflammatory cytokine production in peripheral blood mononuclear cells. Because adipose tissue inflammation accompanied by elevated levels of proinflammatory cytokines is characteristic for the development of obesity, we hypothesized that modulation of autophagy alters adipose tissue inflammatory gene expression and secretion. We tested our hypothesis using ex vivo and in vivo studies of human and mouse adipose tissue. Levels of the autophagy marker LC3 were elevated in sc adipose tissue of obese vs. lean human subjects and positively correlated to both systemic insulin resistance and morphological characteristics of adipose tissue inflammation. Similarly, autophagic activity levels were increased in adipose tissue of obese and insulin resistant animals as compared with lean mice. Inhibition of autophagy by 3-methylalanine in human and mouse adipose tissue explants led to a significant increase in IL-1β, IL-6, and IL-8 mRNA expression and protein secretion. Noticeably, the enhancement in IL-1β, IL-6, and keratinocyte-derived chemoattractant (KC) by inhibition of autophagy was more robust in the presence of obesity. Similar results were obtained by blocking autophagy using small interfering RNA targeted to ATG7 in human Simpson-Golabi-Behmel syndrome adipocytes. Our results demonstrate that autophagy activity is up-regulated in the adipose tissue of obese individuals and inhibition of autophagy enhances proinflammatory gene expression both in adipocytes and adipose tissue explants. Autophagy may function to dampen inflammatory gene expression and thereby limit excessive inflammation in adipose tissue during obesity.

Caveolin-1 Deficiency Leads to Increased Susceptibility to Cell Death and Fibrosis in White Adipose Tissue: Characterization of a Lipodystrophic Model

Caveolin-1 (CAV1) is an important regulator of adipose tissue homeostasis. In the present study we examined the impact of CAV1 deficiency on the properties of mouse adipose tissue both in vivo and in explant cultures during conditions of metabolic stress. In CAV1−/− mice fasting caused loss of adipose tissue mass despite a lack of hormone-sensitive lipase (HSL) phosphorylation. In addition, fasting resulted in increased macrophage infiltration, enhanced deposition of collagen, and a reduction in the level of the lipid droplet protein perilipin A (PLIN1a). Explant cultures of CAV1−/− adipose tissue also showed a loss of PLIN1a during culture, enhanced secretion of IL-6, increased release of lactate dehydrogenase, and demonstrated increased susceptibility to cell death upon collagenase treatment. Attenuated PKA-mediated signaling to HSL, loss of PLIN1a and increased secretion of IL-6 were also observed in adipose tissue explants of CAV1+/+ mice with diet-induced obesity. Together these results suggest that while alterations in adipocyte lipid droplet biology support adipose tissue metabolism in the absence of PKA-mediated pro-lipolytic signaling in CAV1−/− mice, the tissue is intrinsically unstable resulting in increased susceptibility to cell death, which we suggest underlies the development of fibrosis and inflammation during periods of metabolic stress.

Fat-reducing effects of dehydroepiandrosterone involve upregulation of ATGL and HSL expression, and stimulation of lipolysis in adipose tissue

Dehydroepiandrosterone (DHEA) reduces body fat in rodents and humans, and increases glycerol release from isolated rat epididymal adipocytes and human visceral adipose tissue explants. It suggests that DHEA stimulates triglyceride hydrolysis in adipose tissue; however, the mechanisms underlying this action are still unclear.We examined the effects of DHEA on the expression of adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL), the key enzymes of lipolysis, in rat epididymal white adipose tissue (eWAT). Male Wistar rats were fed a diet containing 0.6% DHEA for 2weeks and eWAT was analyzed for mRNA and protein expression of ATGL and HSL, as well as mRNA expression of peroxisome proliferator-activated receptor γ 2 (PPARγ2) and its downstream target fatty acid translocase (FAT). Glycerol release from eWAT explants and serum free fatty acids (FFA) were also measured.Rats that received DHEA gained less weight, had 23% lower eWAT mass and 31% higher serum FFA levels than controls. Cultured explants of eWAT from DHEA-treated rats released 81% more glycerol than those from control rats. DHEA administration upregulated ATGL mRNA (1.62-fold, P<0.05) and protein (1.78-fold, P<0.05) expression as well as augmented HSL mRNA levels (1.36-fold, P<0.05) and Ser660 phosphorylation of HSL (2.49-fold, P<0.05). PPARγ2 and FAT mRNA levels were also increased in DHEA-treated rats (1.61-fold, P<0.05 and 2.16-fold, P<0.05; respectively). Moreover, ATGL, HSL, and FAT mRNA levels were positively correlated with PPARγ2 expression.This study demonstrates that DHEA promotes lipid mobilization in adipose tissue by increasing the expression and activity of ATGL and HSL. The effects of DHEA appear to be mediated, at least in part, via PPARγ2 activation, which in turn upregulates ATGL and HSL gene expression.

Glypican-4 enhances insulin signaling via interaction with the insulin receptor and serves as a novel adipokine.

Obesity, especially visceral obesity, is associated with insulin resistance and metabolic syndrome. We previously identified the cell surface proteoglycan glypican-4 as differentially expressed in subcutaneous versus visceral white fat depots. Here we show that glypican-4 is released from cells and adipose tissue explants of mice, and that circulating glypican-4 levels correlate with BMI and insulin sensitivity in humans. Furthermore, glypican-4 interacts with the insulin receptor, enhances insulin receptor signaling, and enhances adipocyte differentiation. Conversely, depletion of glypican-4 results in reduced activation of the insulin receptor and prevents adipocyte differentiation in vitro by inhibiting insulin-mediated C/EBPβ phosphorylation. These functions of glypican-4 are independent of its glycosylphosphatidylinositol membrane anchorage, as a nonmembrane–bound mutant of glypican-4 phenocopies the effects of native glypican-4 overexpression. In summary, glypican-4 is a novel circulating insulin sensitizing adipose-derived factor that, unlike other insulin sensitizers, acts directly on the insulin receptor to enhance signaling.

Modulation of DNA synthesis in porcine adipose tissue by growth hormone, insulin and leptin

Adipose tissue plays critical role in energy homeostasis by responding to nutritional, neural, and hormonal signals and secreting adipokines that control feeding, thermogenesis, immunity, and neuroendocrine function. DNA synthesis takes place in the adipose tissue continuously due to multiplication and differentiation of various cell populations. The present study was undertaken to analyze effect of growth hormone, insulin and leptin on DNA synthesis in porcine adipose tissue under in vitro conditions and the differential distribution of thymidine incorporation among adipose and stromal-vascular fractions. Adipose tissue explants were incubated with various hormonal combinations in the media containing 3[H] thymidine and its incorporation into cell/DNA was measured later. Growth hormone, insulin and leptin increased thymidine uptake by 4.95, 2.45 and 1.85 times over the non hormonal control tissue explants. Among the combinations GH + leptin and insulin +leptin incubated groups had the lowest and highest thymidine uptake respectively. Out of the total thymidine incorporated in the adipose tissue, 90.49% of the thymidine was taken up by the stromo- vascular cells in comparison to adipocytes (9.51%) in control group. Based on the present study it can be concluded that GH, insulin and leptin increased cellular uptake of thymidine, indicating that all these hormones promoted DNA synthesis and /or multiplication in the cells and stromal vascular fraction of adipose tissue is actively involved in DNA synthesis.

Human adipose tissue from normal and tumoral breast regulates the behavior of mammary epithelial cells

Stromal-epithelial interactions mediate both breast development and breast cancer progression. In the present work, we evaluated the effects of conditioned media (CMs) of human adipose tissue explants from normal (hATN) and tumor (hATT) breast on proliferation, adhesion, migration and metalloproteases activity on tumor (MCF-7 and IBH-7) and non-tumor (MCF-10A) human breast epithelial cell lines. Human adipose tissues were obtained from patients and the conditioned medium from hATN and hATT collected after 24 h of incubation. MCF-10A, MCF-7 and IBH-7 cells were grown and incubated with CMs and proliferation and adhesion, as well as migration ability and metalloprotease activity, of epithelial cells after exposing cell cultures to hATN- or hATT-CMs were quantified. The statistical significance between different experimental conditions was evaluated by one-way ANOVA. Tukey's post hoc tests were performed. Tumor and non-tumor breast epithelial cells significantly increased their proliferation activity after 24 h of treatment with hATT-CMs compared to control-CMs. Furthermore, cellular adhesion of these two tumor cell lines was significantly lower with hATT-CMs than with hATN-CMs. Therefore, hATT-CMs seem to induce significantly lower expression or less activity of the components involved in cellular adhesion than hATN-CMs. In addition, hATT-CMs induced pro-MMP-9 and MMP-9 activity and increased the migration of MCF-7 and IBH-7 cells compared to hATN-CMs. We conclude that the microenvironment of the tumor interacts in a dynamic way with the mutated epithelium. This evidence leads to the possibility to modify the tumor behavior/phenotype through the regulation or modification of its microenvironment. We developed a model in which we obtained CMs from adipose tissue explants completely, either from normal or tumor breast. In this way, we studied the contribution of soluble factors independently of the possible effects of direct cell contact.

Relative Hypoadiponectinemia, Insulin Resistance, and Increased Visceral Fat in Euthyroid Prepubertal Girls With Low‐Normal Serum Free Thyroxine

A lower activity of the thyroid axis within the clinical reference range is related to a dysmetabolic phenotype in adult populations. We posited that such an association is already present as early as in prepubertal childhood. Serum thyroid stimulating hormone (TSH) and free T4, body fat (bioelectric impedance), insulin resistance (homeostasis model assessment of insulin resistance (HOMA(IR))), total and high molecular weight (HMW)-adiponectin and serum lipids were assessed in 234 euthyroid prepubertal children (113 boys and 121 girls) attending primary care clinics. Visceral fat (abdominal ultrasound) was measured in a subset of these subjects (n = 147; 74 boys and 73 girls). Explants of visceral adipose tissue from an additional six prepubertal children (three boys and three girls) were used to study the regulation of total and HMW-adiponectin by thyroid hormone. Serum free T4 was in girls independently associated with HMW-adiponectin, HOMA(IR) and visceral fat, so that circulating HMW-adiponectin decreased by 30% (β = 0.305 P < 0.005, R(2) = 0.13) and HOMA(IR) and visceral fat increased, respectively, by 90% (β = -0.255 P < 0.01, R(2) = 0.05) and 30% (β = -0.369, P < 0.005, R(2) = 0.12) from the highest to the lowest tertile of serum free T4. Nonsignificant differences in these parameters were found in boys. Treatment of visceral fat explants with thyroid hormone increased total and HMW-adiponectin by 70% and 53%, respectively, above control values (P < 0.01). In conclusion, a dysmetabolic phenotype, consisting of relative hypoadiponectinemia, insulin resistance and increased visceral fat, is associated with low-normal serum free thyroxine in euthyroid prepubertal girls. These associations may be partly explained by a positive regulation of HMW-adiponectin secretion by thyroid hormone.

Identification and Functional Characterization of Protein Kinase A Phosphorylation Sites in the Major Lipolytic Protein, Adipose Triglyceride Lipase

Catecholamine-stimulated lipolysis occurs by activating adenylate cyclase and raising cAMP levels, thereby increasing protein kinase A (PKA) activity. This results in phosphorylation and modulated activity of several key lipolytic proteins. Adipose triglyceride lipase (ATGL) is the primary lipase for the initial step in triacylglycerol hydrolysis, and ATGL activity is increased during stimulated lipolysis. Here, we demonstrate that murine ATGL is phosphorylated by PKA at several serine residues in vitro and identify Ser(406) as a functionally important site. ATGL null adipocytes expressing ATGL S406A (nonphosphorylatable) had reduced stimulated lipolysis. Studies in mice demonstrated increased ATGL Ser(406) phosphorylation during fasting and moderate intensity exercise, conditions associated with elevated lipolytic rates. ATGL Ser(404) (corresponding to murine Ser(406)) phosphorylation was increased by β-adrenergic stimulation but not 5'AMP-activated protein kinase activation in human subcutaneous adipose tissue explants, which correlated with lipolysis rates. Our studies suggest that β-adrenergic activation can result in PKA-mediated phosphorylation of ATGL Ser(406), to moderately increase ATGL-mediated lipolysis.

Lipoxin A4attenuates adipose inflammation

Aging and adiposity are associated with chronic low-grade inflammation, which underlies the development of obesity-associated complications, including type 2 diabetes mellitus (T2DM). The mechanisms underlying adipose inflammation may include macrophage infiltration and activation, which, in turn, affect insulin sensitivity of adipocytes. There is a growing appreciation that specific lipid mediators (including lipoxins, resolvins, and protectins) can promote the resolution of inflammation. Here, we investigated the effect of lipoxin A4 (LXA4), the predominant endogenously generated lipoxin, on adipose tissue inflammation. Using adipose tissue explants from perigonadal depots of aging female C57BL/6J mice (Animalia, Chordata, Mus musculus) as a model of age-associated adipose inflammation, we report that LXA4 (1 nM) attenuates adipose inflammation, decreasing IL-6 and increasing IL-10 expression (P<0.05). The altered cytokine milieu correlated with increased GLUT-4 and IRS-1 expression, suggesting improved insulin sensitivity. Further investigations revealed the ability of LXA4 to rescue macrophage-induced desensitization to insulin-stimulated signaling and glucose uptake in cultured adipocytes, using vehicle-stimulated cells as controls. This was associated with preservation of Akt activation and reduced secretion of proinflammatory cytokines, including TNF-α. We therefore propose that LXA4 may represent a potentially useful and novel therapeutic strategy to subvert adipose inflammation and insulin resistance, key components of T2DM.

Inflamed tumor-associated adipose tissue is a depot for macrophages that stimulate tumor growth and angiogenesis

Tumor-associated stroma is typified by a persistent, non-resolving inflammatory response that enhances tumor angiogenesis, growth and metastasis. Inflammation in tumors is instigated by heterotypic interactions between malignant tumor cells, vascular endothelium, fibroblasts, immune and inflammatory cells. We found that tumor-associated adipocytes also contribute to inflammation. We have analyzed peritumoral adipose tissue in a syngeneic mouse melanoma model. Compared to control adipose tissue, adipose tissue juxtaposed to implanted tumors exhibited reduced adipocyte size, extensive fibrosis, increased angiogenesis and a dense macrophage infiltrate. A mouse cytokine protein array revealed up-regulation of inflammatory mediators including IL-6, CXCL1, MCP-1, MIP-2 and TIMP-1 in peritumoral versus counterpart adipose tissues. CD11b(+) macrophages contributed strongly to the inflammatory activity. These macrophages were isolated from peritumoral adipose tissue and found to over-express ARG1, NOS2, CD301, CD163, MCP-1 and VEGF, which are indicative of both M1 and M2 polarization. Tumors implanted at a site distant from subcutaneous, anterior adipose tissue were strongly growth-delayed, had fewer blood vessels and were less populated by CD11b(+) macrophages. In contrast to normal adipose tissue, micro-dissected peritumoral adipose tissue explants launched numerous vascular sprouts when cultured in an ex vivo model. Thus, inflamed tumor-associated adipose tissue fuels the growth of malignant cells by acting as a proximate source for vascular endothelium and activated pro-inflammatory cells, in particular macrophages.

Human periprostatic adipose tissue promotes prostate cancer aggressiveness in vitro

BackgroundObesity is associated with prostate cancer aggressiveness and mortality. The contribution of periprostatic adipose tissue, which is often infiltrated by malignant cells, to cancer progression is largely unknown. Thus, this study aimed to determine if periprostatic adipose tissue is linked with aggressive tumor biology in prostate cancer.MethodsSupernatants of whole adipose tissue (explants) or stromal vascular fraction (SVF) from paired fat samples of periprostatic (PP) and pre-peritoneal visceral (VIS) anatomic origin from different donors were prepared and analyzed for matrix metalloproteinases (MMPs) 2 and 9 activity. The effects of those conditioned media (CM) on growth and migration of hormone-refractory (PC-3) and hormone-sensitive (LNCaP) prostate cancer cells were measured.ResultsWe show here that PP adipose tissue of overweight men has higher MMP9 activity in comparison with normal subjects. The observed increased activities of both MMP2 and MMP9 in PP whole adipose tissue explants, likely reveal the contribution of adipocytes plus stromal-vascular fraction (SVF) as opposed to SVF alone. MMP2 activity was higher for PP when compared to VIS adipose tissue. When PC-3 cells were stimulated with CM from PP adipose tissue explants, increased proliferative and migratory capacities were observed, but not in the presence of SVF. Conversely, when LNCaP cells were stimulated with PP explants CM, we found enhanced motility despite the inhibition of proliferation, whereas CM derived from SVF increased both cell proliferation and motility. Explants culture and using adipose tissue of PP origin are most effective in promoting proliferation and migration of PC-3 cells, as respectively compared with SVF culture and using adipose tissue of VIS origin. In LNCaP cells, while explants CM cause increased migration compared to SVF, the use of PP adipose tissue to generate CM result in the increase of both cellular proliferation and migration.ConclusionsOur findings suggest that the PP depot has the potential to modulate extra-prostatic tumor cells' microenvironment through increased MMPs activity and to promote prostate cancer cell survival and migration. Adipocyte-derived factors likely have a relevant proliferative and motile role.

Systemic inflammation and insulin sensitivity in obese IFN-γ knockout mice

Adipose tissue macrophages are important mediators of inflammation and insulin resistance in obesity. IFN-γ is a central regulator of macrophage function. The role of IFN-γ in regulating systemic inflammation and insulin resistance in obesity is unknown. We studied obese IFN-γ knockout mice to identify the role of IFN-γ in regulating inflammation and insulin sensitivity in obesity. IFN-γ-knockout C57Bl/6 mice and wild-type control litter mates were maintained on normal chow or a high fat diet for 13 weeks and then underwent insulin sensitivity testing then sacrifice and tissue collection. Flow cytometry, intracellular cytokine staining, and QRTPCR were used to define tissue lymphocyte phenotype and cytokine expression profiles. Adipocyte size was determined from whole adipose tissue explants examined under immunofluorescence microscopy. Diet-induced obesity induced systemic inflammation and insulin resistance, along with a pan-leukocyte adipose tissue infiltrate that includes macrophages, T-cells, and NK cells. Obese IFN-γ-knockout animals, compared with obese wild-type control animals, demonstrate modest improvements in insulin sensitivity, decreased adipocyte size, and an M2-shift in ATM phenotype and cytokine expression. These data suggest a role for IFN-γ in the regulation of inflammation and glucose homeostasis in obesity though multiple potential mechanisms, including effects on adipogenesis, cytokine expression, and macrophage phenotype.

Thel-α-Lysophosphatidylinositol/GPR55System and Its Potential Role in Human Obesity

GPR55 is a putative cannabinoid receptor, and l-α-lysophosphatidylinositol (LPI) is its only known endogenous ligand. We investigated 1) whether GPR55 is expressed in fat and liver; 2) the correlation of both GPR55 and LPI with several metabolic parameters; and 3) the actions of LPI on human adipocytes. We analyzed CB1, CB2, and GPR55 gene expression and circulating LPI levels in two independent cohorts of obese and lean subjects, with both normal or impaired glucose tolerance and type 2 diabetes. Ex vivo experiments were used to measure intracellular calcium and lipid accumulation. GPR55 levels were augmented in the adipose tissue of obese subjects and further so in obese patients with type 2 diabetes when compared with nonobese subjects. Visceral adipose tissue GPR55 correlated positively with weight, BMI, and percent fat mass, particularly in women. Hepatic GPR55 gene expression was similar in obese and type 2 diabetic subjects. Circulating LPI levels were increased in obese patients and correlated with fat percentage and BMI in women. LPI increased the expression of lipogenic genes in visceral adipose tissue explants and intracellular calcium in differentiated visceral adipocytes. These findings indicate that the LPI/GPR55 system is positively associated with obesity in humans.

Chronic Alcohol Exposure Stimulates Adipose Tissue Lipolysis in Mice: Role of Reverse Triglyceride Transport in the Pathogenesis of Alcoholic Steatosis

Alcohol consumption induces liver steatosis; therefore, this study investigated the possible role of adipose tissue dysfunction in the pathogenesis of alcoholic steatosis. Mice were pair-fed an alcohol or control liquid diet for 8 weeks to evaluate the alcohol effects on lipid metabolism at the adipose tissue-liver axis. Chronic alcohol exposure reduced adipose tissue mass and adipocyte size. Fatty acid release from adipose tissue explants was significantly increased in alcohol-fed mice in association with the activation of adipose triglyceride lipase and hormone-sensitive lipase. Alcohol exposure induced insulin intolerance and inactivated adipose protein phosphatase 1 in association with the up-regulation of phosphatase and tensin homolog (PTEN) and suppressor of cytokine signaling 3 (SOCS3). Alcohol exposure up-regulated fatty acid transport proteins and caused lipid accumulation in the liver. To define the mechanistic link between adipose triglyceride loss and hepatic triglyceride gain, mice were first administered heavy water for 5 weeks to label adipose triglycerides with deuterium, and then pair-fed alcohol or control diet for 2 weeks. Deposition of deuterium-labeled adipose triglycerides in the liver was analyzed using Fourier transform ion cyclotron mass spectrometry. Alcohol exposure increased more than a dozen deuterium-labeled triglyceride molecules in the liver by up to 6.3-fold. These data demonstrate for the first time that adipose triglycerides due to alcohol-induced hyperlipolysis are reverse transported and deposited in the liver.

Tumor Cell-educated Periprostatic Adipose Tissue Acquires an Aggressive Cancer-promoting Secretory Profile

Background/Aims: The microenvironment produces important factors that are crucial to prostate cancer (PCa) progression. However, the extent to which the cancer cells stimulate periprostatic adipose tissue (PPAT) to produce these proteins is largely unknown. Our purpose was to determine whether PCa cell-derived factors influence PPAT metabolic activity. Methods: Primary cultures of human PPAT samples from PCa patients (adipose tissue organotypic explants and primary stromal vascular fraction, SVF) were stimulated with conditioned medium (CM) collected from prostate carcinoma (PC3) cells. Cultures without CM were used as control. We used multiplex analysis and ELISA for protein quantification, qPCR to determine mitochondrial DNA (mtDNA) copy number and zymography for matrix metalloproteinase activity, in order to evaluate the response of adipose tissue explants and SVFs to PC3 CM. Results: Stimulation of PPAT explants with PCa PC3 CM induced adipokines associated with cancer progression (osteopontin, tumoral necrosis factor alpha and interleukin-6) and reduced the expression of the protective adipokine adiponectin. Notably, osteopontin protein expression was 13-fold upregulated. Matrix metalloproteinase 9 activity and mitochondrial DNA copy number were higher after stimulation with cancer CM. Stromovascular cells from PPAT in culture were not influenced by tumor-derived factors. Conclusion: The modulation of adipokine expression by tumor CM indicates the pervasive extent to which tumor cells command PPAT to produce factors favorable to their aggressiveness.

The impact of obesity on secretion of adiponectin multimeric isoforms differs in visceral and subcutaneous adipose tissue

Hypoadiponectinemia observed in obesity is associated with insulin resistance, diabetes and atherosclerosis. The aim of the present study was to investigate secretion of adiponectin and its multimeric isoforms by explants derived from subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) in obese and non-obese subjects. Paired samples of SAT and VAT and blood samples were obtained from 23 subjects (10 non-obese and 13 obese) undergoing elective abdominal surgery. Total adiponectin quantities and adiponectin isoforms were measured in conditioned media of explants derived from SAT and VAT using enzyme-linked immunosorbent assay and non-denaturing western blot, respectively. Total adiponectin plasma levels were lower in obese than in non-obese subjects (P<0.05). Secretion of total adiponectin in adipose tissue (AT) explants was lower in obese than in non-obese subjects in SAT (P<0.05) but not in VAT. In both, SAT and VAT, the most abundant isoform released into conditioned media was the high-molecular weight (HMW) form. Its relative proportion in relation to total adiponectin was higher in conditioned media of explants from both fat depots when compared with plasma (P<0.001). The proportion of secreted HMW vs total adiponectin was higher in VAT than in SAT explants in the group of non-obese individuals (49.3±3.1% in VAT vs 40.6±2.8% in SAT; P<0.01), whereas no difference between the two depots was found in obese subjects (46.2±3.0 % in VAT vs 46.0±2.4 % in SAT). Obesity is associated with the decrease of total adiponectin secretion in SAT. The profile of adiponectin isoforms secreted by SAT and VAT explants differs from that in plasma. Secretion of total adiponectin and HMW isoform of adiponectin are different in obese and non-obese subjects in relation to AT depot.

MMP9 expression in oesophageal adenocarcinoma is upregulated with visceral obesity and is associated with poor tumour differentiation

Overweight and obesity is linked to increased incidence and mortality of many cancer types. Of all cancers, oesophageal adenocarcinoma (OAC) displays one of the strongest epidemiological links with obesity, accounting for up to 40% of cases, but molecular pathways driving this association remain largely unknown. This study aimed to elucidate mechanisms underpinning the association of obesity and cancer, and to determine if visceral obesity is associated with aggressive tumour biology in OAC. Following co-culture with visceral adipose tissue explants, expression of genes involved in tumour cell invasion and metastasis (matrix metalloproteinase (MMP)2 and MMP9) were upregulated between 10-fold (MMP2) and 5000-fold (MMP9), and expression of tumour suppressor p53 was downregulated 2-fold in OAC cell lines. Western blotting confirmed these results at the protein level, while zymographic analysis detected increased activity of MMPs in OAC cell lines following co-culture with adipose tissue explants. When OAC cell lines were cultured with adipose tissue conditioned media (ACM) from visceral adipose tissue, increased proliferative, migratory and invasive capacity of tumour cells was observed. In OAC patient tumour biopsies, elevated gene expression of MMP9 was associated with visceral obesity, measured by visceral fat area, while increased gene expression of MMP9 and decreased gene expression of tumour suppressor p53 was associated with poor tumour differentiation. These novel data highlight an important role for visceral obesity in upregulation of pro-tumour pathways contributing to aggressive tumour biology, and may ultimately lead to development of stratified treatment for viscerally obese OAC patients.

Zilpaterol hydrochloride alters abundance of β-adrenergic receptors in bovine muscle cells but has little effect on de novo fatty acid biosynthesis in bovine subcutaneous adipose tissue explants

We predicted that zilpaterol hydrochloride (ZH), a β-adrenergic receptor (AR) agonist, would depress mRNA and protein abundance of β-AR in bovine satellite cells. We also predicted that ZH would decrease total lipid synthesis in bovine adipose tissue. Bovine satellite cells isolated from the semimembranosus muscle were plated on tissue culture plates coated with reduced growth factor matrigel or collagen. Real-time quantitative PCR was used to measure specific gene expression after 48 h of ZH exposure in proliferating satellite cells and fused myoblasts. There was no effect of ZH dose on [(3)H]thymidine incorporation into DNA in proliferating myoblasts. Zilpaterol hydrochloride at 1 µM decreased (P < 0.05) β1-AR mRNA, and 0.01 and 1 µM ZH decreased (P < 0.05) β2-AR and β3-AR mRNA in myoblasts. The expression of IGF-I mRNA tended to increase (P = 0.07) with 1 µM ZH. There was no effect (P > 0.10) of ZH on the β-AR or IGF-I gene expression in fused myotube cultures at 192 h or on fusion percentage. The β2-AR antagonist ICI-118, 551 at 0.1 µM attenuated (P < 0.05) the effect of 0.1 µM ZH to reduce expression of β1- and β2-AR mRNA. The combination of 0.01 µM ZH and 0.1 µM ICI-118, 551 caused an increase (P < 0.05) in β1-AR gene expression. There was no effect (P > 0.10) of ICI-118, 551 or ZH on β3-AR or IGF-I. Western blot analysis revealed that the protein content of β2-AR in ZH-treated myotube cultures decreased (P < 0.05) relative to control. Total lipid synthesis from acetate was increased by ZH in bovine subcutaneous adipose tissue explants in the absence of theophylline but was decreased by ZH when theophylline was included in the incubation medium. These data indicate that ZH alters mRNA and protein concentrations of β-AR in satellite cell cultures, which in turn could affect responsiveness of cells to prolonged ZH exposure in vivo. Similar to other β-adrenergic agonists, ZH had only modest effects on lipid metabolism in adipose tissue explants.