Visceral Adipose Tissue Of Mice Research Articles

Different Effects of Obesity and Fasting on the Expression of Type 3 Deiodinase and Thyroid Hormone Receptors in the Liver and Visceral Adipose Tissue of C57BL/6 Male Mice.

Energy status can alter thyroid hormone signalling in different tissues. Little is known about the effect of fasting on the local thyroid hormone metabolism under high-fat diet (HFD)-induced obesity. We aimed to investigate the fasting effect on deiodinase type 3 (DIO3) and thyroid hormone receptors (TRs) expression in liver and visceral adipose tissue (VAT) of HFD-induced obese mice. The 30 male C57BL/6 mice were divided into three groups (n = 10/group): control (CON) group, obese (OB) group, and fasted obese (OBF) group. In a 14-week study, the expression levels of DIO3 and TRs in the liver and VAT of mice were measured by real-time polymerase chain reaction. Gene expression results were shown as fold changes defined by 2-ΔΔct. Comparison between groups was performed by using one-way-ANOVA or Kruskal-Wallis ANOVA test. In the liver, there was a significantly lower expression of DIO3 and higher expression of TRs in obese fasted mice compared to obese mice. Compared to the lean mice, OBF mice had significantly lower expression of DIO3 and higher expression of TRβ. In the VAT, mRNA expression of DIO3 was significantly increased in OBF and OB groups compared to the CON group. There were no significant differences in the mRNA expression of TRs between groups. Our findings suggest that fasting may be more effective in improving thyroid hormone metabolism in the liver rather than the VAT of obese mice.

IL-33 regulates adipogenesis via Wnt/β-catenin/PPAR-γ signaling pathway in preadipocytes

Interleukin-33 (IL-33), an emerging cytokine within the IL-1 family, assumes a pivotal function in the control of obesity. However, the specific mechanism of its regulation of obesity formation remains unclear. In this study, we found that the expression level of IL-33 increased in visceral adipose tissue in mice fed with a high-fat diet (HFD) compared with that in mice fed with a normal diet (ND). In vitro, we also found the expression level of IL-33 was upregulated during the adipogenesis of 3T3-L1 cells. Functional test results showed that knockdown of IL-33 in 3T3-L1 cells differentiation could promote the accumulation of lipid droplets, the content of triglyceride and the expression of adipogenic–related genes (i.e. PPAR-γ, C/EBPα, FABP4, LPL, Adipoq and CD36). In contrast, overexpression of IL-33 inhibits adipogenic differentiation. Meanwhile, the above tests were repeated after over-differentiation of 3T3-L1 cells induced by oleic acid, and the results showed that IL-33 played a more significant role in the regulation of adipogenesis. To explore the mechanism, transcriptome sequencing was performed and results showed that IL-33 regulated the PPAR signaling pathway in 3T3-L1 cells. Further, Western blot and confocal microscopy showed that the inhibition of IL-33 could promote PPAR-γ expression by inhibiting the Wnt/β-catenin signal in 3T3-L1 cells. This study demonstrated that IL-33 was an important regulator of preadipocyte differentiation and inhibited adipogenesis by regulating the Wnt/β-catenin/PPAR-γ signaling pathway, which provided a new insight for further research on IL-33 as a new intervention target for metabolic disorders.

Four-and-a-Half LIM-Domain Protein 2 (FHL2) Induces Neuropeptide Y (NPY) in Macrophages in Visceral Adipose Tissue and Promotes Diet-Induced Obesity.

Obesity is characterized by the expansion of the adipose tissue, usually accompanied by inflammation, with a prominent role of macrophages infiltrating the visceral adipose tissue (VAT). This chronic inflammation is a major driver of obesity-associated comorbidities. Four-and-a-half LIM-domain protein 2 (FHL2) is a multifunctional adaptor protein that is involved in the regulation of various biological functions and the maintenance of the homeostasis of different tissues. In this study, we aimed to gain new insights into the expression and functional role of FHL2 in VAT in diet-induced obesity. We found enhanced FHL2 expression in the VAT of mice with Western-type diet (WTD)-induced obesity and obese humans and identified macrophages as the cellular source of enhanced FHL2 expression in VAT. In mice with FHL2 deficiency (FHL2KO), WTD feeding resulted in reduced body weight gain paralleled by enhanced energy expenditure and uncoupling protein 1 (UCP1) expression, indicative of activated thermogenesis. In human VAT, FHL2 was inversely correlated with UCP1 expression. Furthermore, macrophage infiltration and the expression of the chemokine MCP-1, a known promotor of macrophage accumulation, was significantly reduced in WTD-fed FHL2KO mice compared with wild-type (wt) littermates. While FHL2 depletion did not affect the differentiation or lipid metabolism of adipocytes in vitro, FHL2 depletion in macrophages resulted in reduced expressions of MCP-1 and the neuropeptide Y (NPY). Furthermore, WTD-fed FHL2KO mice showed reduced NPY expression in VAT compared with wt littermates, and NPY expression was enhanced in VAT resident macrophages of obese individuals. Stimulation with recombinant NPY induced not only UCP1 expression and lipid accumulation but also MCP-1 expression in adipocytes. Collectively, these findings indicate that FHL2 is a positive regulator of NPY and MCP-1 expression in macrophages and herewith closely linked to the mechanism of obesity-associated lipid accumulation and inflammation in VAT. Thus, FHL2 appears as a potential novel target to interfere with the macrophage-adipocyte crosstalk in VAT for treating obesity and related metabolic disorders.

Immuno(T)herapy for age-related diseases.

During the last decade, the stimulation of T-cell function by the blockage of immunosuppressive checkpoints has experienced an outstanding impact in the treatment of cancer. Development of the chimeric antigen receptor T-cell technology has also emerged as a powerful alternative for patients suffering from oncological processes, especially those affected by hematological neoplasms. Recent evidence suggest that the use of immunotherapy could be extended to non-oncological diseases and could be especially relevant for age-associated disorders, opening exciting therapeutic options for a wide range of diseases of the elderly. Here we comment on the emergence of T-cell-based immunotherapies as feasible approaches that could revolutionize the future of GeroScience.

Alterations in mouse visceral adipose tissue mRNA expression of islet G-protein-coupled receptor ligands in obesity.

Adipose tissue mass expansion in obesity leads to alterations in expression and secretion of adipokines, some of which may alter islet function by binding to G-protein-coupled receptors (GPCRs) expressed by islets. We have therefore quantified expression of mRNAs encoding islet GPCR ligands in visceral adipose tissue retrieved from lean and diet-induced obese mice to determine alterations in islet GPCR ligand mRNAs in obesity. Epididymal adipose tissue was retrieved from C57BL/6 mice that had been maintained on a control-fat diet (10% fat) or high-fat diet (60% fat) for 16 weeks and RT-qPCR was used to quantify mRNAs encoding ligands for islet GPCRs. Of the 155 genes that encode ligands for islet GPCRs, 45 and 40 were expressed in visceral adipose tissue retrieved from lean and obese mice respectively. The remaining mRNAs were either expressed at trace level (0.0001% to 0.001% relative to Actb expression) or absent (<0.0001%). Obesity was associated with significant alterations in GPCR ligand mRNA expression in visceral adipose tissue, some of which encode for peptides with established effects on islet function (e.g. neuropeptide Y), or for GPCR ligands that have not previously been investigated for their effects on islets (e.g. (C-C motif) ligand 4; Ccl4). Mouse visceral adipose tissue showed significant alterations in expression of mRNAs encoding islet GPCR ligands in obesity. Our data point to ligands of interest for future research on adipose-islet crosstalk via secreted ligands acting at islet GPCRs. Such research may identify islet GPCRs with therapeutic potential for T2D.

Obesity-induced upregulation of chemokine Ccl4 in mouse visceral adipose tissue: effects on [beta]-cell function

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Deletion of adipose triglyceride lipase abolishes blood flow increase after β3-adrenergic stimulation in visceral adipose tissue of mice.

Dynamic changes in adipose tissue blood flow (ATBF) with nutritional status play a role in the regulation of metabolic and endocrine functions. Activation of the sympathetic nervous system via β-adrenergic receptors (β-AR) contributes to the control of postprandial enhancement of ATBF. Herein, we sought to identify the role of each β-AR subtype in the regulation of ATBF in mice. We monitored the changes in visceral epididymal ATBF (VAT BF), induced by local infusion of dobutamine, salbutamol, and CL316,243 (a selective β1-, β2-, and β3-AR agonist, respectively) into VAT of lean CD-1 mice and global adipose triglyceride lipase (ATGL) knockout (KO) mice, using laser Doppler flowmetry. Administration of CL316,243, known to promote lipolysis in adipocytes, significantly increased VAT BF of CD-1 mice to a greater extent compared to that of the vehicle, whereas administration of dobutamine or salbutamol did not produce significant differences in VAT BF. The increase in VAT BF induced by β3-AR stimulation disappeared in ATGL KO mice as opposed to their wild-type (WT) littermates, implying a role of ATGL-mediated lipolysis in the regulation of VAT BF. Different vascular reactivities occurred despite no significant differences in vessel density and adiposity between the groups. Additionally, the expression levels of the angiogenesis-related genes were significantly higher in VAT of ATGL KO mice than in that of WT, implicating an association of ATBF responsiveness with angiogenic activity in VAT. Our findings suggest a potential role of β3-AR signaling in the regulation of VAT BF via ATGL-mediated lipolysis in mice.

Alpha-lipoic acid supplementation increases the efficacy of exercise- and diet-induced obesity treatment and induces immunometabolic changes in female mice and women.

The decrease in the regulatory T cells (Tregs) population is highly involved in adipose tissue inflammation and insulin resistance in obesity. Tregs depend on fatty acids via β-oxidation for immunosuppressive function adapting their antioxidant systems to allow survival to oxidative stress. In this study, we have hypothesized that a dietary supplementation with alpha-lipoic acid (ALA), a powerful antioxidant, would improve immunometabolism when added to the classical strategy of obesity treatment. First, we showed by in vitro experiments that ALA favors the polarization of mice CD4+T cells toward Tregs. Next, we have carried out a translational study where female obese mice and women were supplemented with ALA or vehicle/placebo (mice: 2.5 gALA /kgfood ; 6weeks; women: 600 mgALA /day, 8weeks) while following a protocol including regular exercise and a change in diet. Fatty acid oxidation potential and activity of nuclear erythroid-related factor 2 (NRF2) of mouse secondary lymphoid tissues were improved by ALA supplementation. ALA reduced visceral adipose tissue (VAT) mass and preserved Tregs in VAT in mice. In women, ALA supplementation induced significant metabolic changes of circulating CD4+T cells including increased oxidative capacity and fatty acid oxidation, ameliorated their redox status, and improved the reduction of visceral fat mass. While appropriate biological markers are still required to be used in clinics to judge the effectiveness of long-term obesity treatment, further studies in female mice and women are needed to determine whether these immunometabolic changes would reduce VAT mass-associated risk for secondary health issues arising from obesity.

MicroRNA 182 is a Novel Negative Regulator of Adipogenesis by Targeting CCAAT/Enhancer-Binding Protein α.

ObjectiveRecent studies have shown that microRNAs (miRNAs/miRs) play key roles in adipogenesis. This study aimed to investigate the role and underlying mechanism of miR‐182 in adipogenesis.MethodsThis study used the 3T3‐L1 cell line and human visceral adipose tissue (VAT)‐derived adipocytes to determine the role of miR‐182 in adipogenesis. Adipose tissues from mice with high‐fat diet–induced obesity, ob/ob mice, or human individuals with obesity were used to determine the association of miR‐182 levels with obesity. A luciferase reporter assay was used to determine the target of miR‐182.ResultsThe expression level of miR‐182 was greatly downregulated during white adipogenesis and markedly lower in the VAT of mice and humans with obesity. Ectopic expression of miR‐182 in 3T3‐L1 cells and human adipocytes suppressed the formation of lipid droplets and the expression of adipogenic genes. The luciferase reporter assay showed that miR‐182 targeted the 3′‐untranslated sequence of CCAAT/enhancer‐binding protein α (C/EBPα) directly. In addition, glucocorticoids negatively regulated miR‐182 expression, which, in turn, suppressed the glucocorticoid‐induced expression of C/EBPα.ConclusionsTaken together, our studies identified miR‐182 as a novel negative regulator of adipogenesis and a potential therapeutic target for obesity.

Refining the adipose progenitor cell landscape in healthy and obese visceral adipose tissue using single-cell gene expression profiling.

Obesity is a serious health concern and is associated with a reduced quality of life and a number of chronic diseases, including diabetes, heart disease, stroke, and cancer. With obesity rates on the rise worldwide, adipose tissue biology has become a top biomedical research priority. Despite steady growth in obesity-related research, more investigation into the basic biology of adipose tissue is needed to drive innovative solutions aiming to curtail the obesity epidemic. Adipose progenitor cells (APCs) play a central role in adipose tissue homeostasis and coordinate adipose tissue expansion and remodeling. Although APCs are well studied, defining and characterizing APC subsets remains ambiguous because of ill-defined cellular heterogeneity within this cellular compartment. In this study, we used single-cell RNA sequencing to create a cellular atlas of APC heterogeneity in mouse visceral adipose tissue. Our analysis identified two distinct populations of adipose tissue-derived stem cells (ASCs) and three distinct populations of preadipocytes (PAs). We identified novel cell surface markers that, when used in combination with traditional ASC and preadipocyte markers, could discriminate between these APC subpopulations by flow cytometry. Prospective isolation and molecular characterization of these APC subpopulations confirmed single-cell RNA sequencing gene expression signatures, and ex vivo culture revealed differential expansion/differentiation capabilities. Obese visceral adipose tissue featured relative expansion of less mature ASC and PA subpopulations, and expression analyses revealed major obesity-associated signaling alterations within each APC subpopulation. Taken together, our study highlights cellular and transcriptional heterogeneity within the APC pool, provides new tools to prospectively isolate and study these novel subpopulations, and underscores the importance of considering APC diversity when studying the etiology of obesity.

ICOS signaling in visceral adipose tissue regulatory T cell homeostasis and function

Abstract Obesity and insulin resistance/type 2 diabetes are linked by chronic inflammation in visceral adipose tissue (VAT). Foxp3+ regulatory T cells (TR) are critical for maintaining immune tolerance and are recently appreciated for their role in tissue repair and homeostasis. Indeed, a unique population of TR reside in the VAT of mice and humans, and these TR regulate adipose inflammation and preserve insulin sensitivity and glucose tolerance. ICOS is a costimulatory molecule expressed at high levels by TR. Surprisingly, the abundance of TR in VAT is elevated by nearly 2-fold in IcosKO mice compared to WT controls, and IcosKO TR preferentially reconstitute the VAT of WT:IcosKO mixed bone marrow chimeras. TR in the VAT of IcosKO mice express high levels of VAT-TR markers, like ST2, KLRG1, and IL-10, compared to WT mice, suggesting a potent ability to prevent insulin resistance and diet-induced metabolic syndrome. Ongoing studies aim to determine whether IcosKO mice are more resistant to diet-induced metabolic syndrome by assessing insulin sensitivity/glucose tolerance, indices of inflammation, and VAT immune cell phenotype and function, and to mechanistically elucidate how ICOS signaling controls the accumulation and phenotype of VAT-TR. These studies will yield important information on the molecular pathways involved in regulating the onset of metabolic syndrome and provide new potential therapeutic targets.

Influence of long term administration of tofogliflozin on chronic inflammation of visceral adipose tissue in mice with obesity induced by a high-fat diet.

We previously found that senescence of cluster of differentiation 4 (CD4) T cells is accelerated in the visceral adipose tissue (VAT) of mice with diet-induced obesity (DIO) due to a high-fat diet (HFD), and that these senescent-associated T cells cause chronic inflammation of visceral adipose tissue through secretion of osteopontin, provoking systemic insulin resistance. In this study, we examined whether the development of chronic inflammation and senescence-associated T cells in VAT of DIO mice was improved by long-term weight loss after switching to normal chow (NC) or by administration of a sodium glucose cotransporter 2 inhibitor (tofogliflozin). Wild-type mice were fed an HFD for 26 weeks from 4 weeks old. At 30 weeks of age, half of these DIO mice were switched to NC with or without 0.005% tofogliflozin for 38 weeks. The other mice remained on the HFD with or without 0.005% tofogliflozin for 38 weeks. When DIO mice were switched to NC, their weight decreased to that of mice kept on NC since weaning. After 38 weeks (68 weeks of age), chronic inflammation of the VAT subsided with disappearance of senescence-associated T cells. In the HFD groups, the carbohydrate intake per mouse was half or less of that in the NC group, and urinary glucose excretion by the effect of tofogliflozin was lower in the HFD mice than in the NC mice. Mice that remained on the HFD showed no improvement in chronic inflammation in VAT, possibly because urinary glucose excretion was not sufficiently promoted by tofogliflozin due to the low carbohydrate intake. Thus, no improvement in glucose metabolism or weight loss was observed in these mice.

Metabolic memory of dietary restriction ameliorates DNA damage and adipocyte size in mouse visceral adipose tissue.

Dietary restriction (DR) is thought to exert its beneficial effects on healthspan at least partially by a senolytic and senostatic action, i.e. by reducing frequencies of cells with markers of DNA damage and senescence in multiple tissues. Due to its importance in metabolic and inflammation regulation, fat is a prime tissue for health span determination as well as a prime target for DR. We aimed to determine here whether the beneficial effects of DR would be retained over a subsequent period of ad libitum (AL) feeding. Male mice were kept under either 40% DR or AL feeding regimes from 3 to 12 months of age and then either switched back to the opposite feeding regimen or kept in the same state for another 3 months. Visceral adipose tissue from 4 to 5 mice per group for all conditions was analysed for markers of senescence (adipocyte size, γH2A.X, p16, p21) and inflammation (e.g. IL-6, TNFα, IL-1β) using immuno-staining or qPCR. Macrophages were detected by immunohistochemistry. We found that both 9 and 12 months DR (long term) as well as 3 month (short term, mid-life onset) DR reduced the number of cells harbouring DNA damage and adipocyte size (area and perimeter) in visceral adipocytes with similar efficiency. Importantly, beneficial health markers induced by DR such as small adipocyte size and low DNA damage were maintained for at least 3 month after termination of DR, demonstrating that the previously identified 'metabolic memory' of the DR state in male mice extends to senescence markers in visceral fat.

Abstract 2227: Fgf2 from visceral adipose tissue stimulates neoplastic transformation of nonmalignant epithelial cells

Abstract Background: Adiposity plays a crucial role in the pathogenesis and prognosis of different types of cancers. Epidemiological evidence suggests visceral adipose tissue (VAT) and high-fat diets (HFD) are associated with increased cancer risk however the mechanism is not understood. The aim of this study was to explore the factors in VAT that stimulate neoplastic transformation. Methods: We modeled visceral adiposity-stimulated neoplastic transformation using our novel ex vivo system of VAT-condition medium stimulated epithelial cell transformation (measured by growth in soft agar) and our in vivo murine lipectomy model of ultraviolet light B (UVB)-induced, VAT promoted skin tumor formation. FgfR1(-/-) stable cells were generated by using CRISPR-Cas9 technology and were used to investigate the role of fibroblast growth factor-2 (FGF2) and FGFR1 dependent signaling in neoplastic transformation, both in vitro and in vivo. Results: Only the VAT of obese mice fed a HFD [not VAT from low-fat diet (LFD) fed mice] stimulated neoplastic transformation of skin epithelial cells. Furthermore, human VAT stimulated both skin and mammary epithelial cell transformation. The differences in VAT activity between LFD and HFD fed mice and human donors were associated with the levels of FGF2. Circulating levels of FGF2 were associated with non-melanoma tumor formation in vivo. Human and mouse VAT failed to stimulate transformation in FgfR1(-/-) cells and do not form tumors when injected in Nude mice in vivo. Conclusion: Collectively, our data show FGF2 released from VAT and its interaction with FGFR1 is a novel and potential direct path of VAT-enhanced tumorigenesis. Blocking the FGFR1 induced signaling in VAT of abdominally obese individuals may be an important cancer prevention strategy as well as an adjuvant therapy for improving outcomes following cancer diagnosis. Citation Format: Debrup Chakraborty, Vanessa Benham, Elena Y. Demireva, Blair Bullard, Jamie J. Bernard. Fgf2 from visceral adipose tissue stimulates neoplastic transformation of nonmalignant epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2227. doi:10.1158/1538-7445.AM2017-2227

Intermittent hypoxia in obstructive sleep apnoea mediates insulin resistance through adipose tissue inflammation.

Obstructive sleep apnoea (OSA) is increasingly associated with insulin resistance. The underlying pathophysiology remains unclear but intermittent hypoxia (IH)-mediated inflammation and subsequent dysfunction of the adipose tissue has been hypothesised to play a key role.We tested this hypothesis employing a comprehensive translational approach using a murine IH model of lean and diet-induced obese mice, an innovative IH system for cell cultures and a tightly controlled patient cohort.IH led to the development of insulin resistance in mice, corrected for the degree of obesity, and reduced insulin-mediated glucose uptake in 3T3-L1 adipocytes, associated with inhibition of the insulin-signalling pathway and downregulation of insulin-receptor substrate-1 mRNA. Providing mechanistic insight, IH induced a pro-inflammatory phenotype of visceral adipose tissue in mice with pro-inflammatory M1 macrophage polarisation correlating with the severity of insulin resistance. Complimentary in vitro analysis demonstrated that IH led to M1 polarisation of THP1-derived macrophages. In subjects without comorbidities (n=186), OSA was independently associated with insulin resistance. Furthermore, we found an independent correlation of OSA severity with the M1 macrophage inflammatory marker sCD163.This study provides evidence that IH induces a pro-inflammatory phenotype of the adipose tissue, which may be a crucial link between OSA and the development of insulin resistance.

Abstract IA18: Obesity, inflammation, and pancreatic cancer

Abstract There is epidemiologic evidence that obesity increases the risk of cancers including pancreatic cancer. Several underlying mechanisms, e.g. inflammation and insulin resistance, are proposed. However, the driving mechanisms in pancreatic cancer are poorly understood. We developed a model of diet-induced obesity and pancreatic cancer development in a state-of-the-art mouse model, which resembles important clinical features of human obesity, e.g. weight gain and metabolic disturbances. Offspring of Pdx-1-Cre (or p48-cre) and LSL-KrasG12D crosses were allocated to either a diet high in fats and calories (HFCD; ~4,535 kcal/kg; 40% of calories from fats) or control diet (CD; ~3,725 kcal/kg; 12% of calories from fats) for various time periods. Compared to control animals, mice fed the HFCD significantly gained more weight and developed hyperinsulinemia, hyperglycemia, hyperleptinemia, and elevated levels of IGF-1. Mice fed the HFCD had a substantial increase in visceral fat as measured by micro-CT. The white visceral adipose tissue in mice fed the HFCD showed signs of marked inflammation, which was more pronounced in the peri-pancreatic fat depot. In addition, the pancreas of HFCD-fed animals showed robust signs of inflammation with increased numbers of infiltrating inflammatory cells. Detailed flow cytometry analysis showed a marked elevation of infiltrating natural killer cells, myeloid-derived suppressor cells, T-regulatory cells, and tumor-infiltrating macrophages into the pancreas of mice fed the HFCD. Multiplex analysis demonstrated a significant change in the pancreatic cytokine profile in HFCD-fed mice compared to CD-fed animals. Importantly, the HFCD led to an acceleration of PanIN development in conditional KrasG12D mice. These results demonstrate that a diet high in fats and calories leads to obesity and metabolic disturbances similar to humans and accelerates early pancreatic neoplasia in the conditional KrasG12D mouse model. Based on the notion that obesity is associated with an increase in inflammatory mediators and insulin-resistance and our previous finding that non-steroidal anti-inflammatory drugs inhibit the progression of PanIN lesions in the conditional KrasG12D mouse model, we investigated a potential crosstalk between the PGE2/EP/cAMP and IGF-1/Akt/mTORC1 signaling pathways in pancreatic cancer. In multiple cell lines PGE2 increased intracellular cAMP levels, indicating the activation of Gsα-coupled EP receptors (EP2 and/or EP4). PGE2 dose- and time-dependently increased the phosphorylation of S6 ribosomal protein downstream of mTORC1, suggesting a crosstalk between PGE2/cAMP and mTORC1. In addition, the effect of PGE2 on phospho-S6rp was mimicked by Forskolin, a pharmacological cAMP stimulator. Importantly, PGE2 enhanced the effect of IGF-1 on S6rp phosphorylation, supporting the existence of a positive reinforcement. Interestingly, PGE2 and forskolin had no effect on Akt phosphorylation, suggesting a link downstream of Akt. In the presence of the PKA inhibitor H89, baseline phosphorylation of S6rp and PGE2-activated S6rp phosphorylation were reduced, indicating a role of PKA in the crosstalk. In summary, these data provide evidence of a crosstalk between the PGE2/EP/cAMP and IGF-1/Akt/mTORC1 signaling pathways. Since both pathways are over-activated in obesity-associated cancers, this interaction may be of great importance in elucidating the tumor-promoting effects of obesity in pancreatic cancer. Citation Format: Guido Eibl. Obesity, inflammation, and pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr IA18.

Absorption and Biodistribution of Wheat Gluten Radiolabeled with Technetium (99mTc) in Blood, Liver and Visceral Adipose Tissue of Mice Fed on Gluten‐supplemented Diets

In our previous results, we showed that wheat gluten (WG) intake increases weight gain and adiposity through reduction in oxidation of fatty acids and increase in lipid stores. However, it is not clear if gluten or its peptides reach systemic circulation and peripheral organs to exert such effects. There are few studies addressed to the effect of WG on intestinal permeability in non-coeliac individuals. Thus, we studied the WG absorption and biodistribution to assess if gluten reaches organs. First we labeled WG with technetium (99mTc-GLU) and confirmed its stability. Mice were divided into Control gluten (CtG) – fed chow diet+WG (4.5%) and Obese gluten (ObG) high-fat diet+WG. After 8 weeks, mice received 99mTc-GLU by gavage. Scintigraphic images of gastrointestinal tract showed that most of WG absorption occurred 4h after gavage. 99mTc-gluten was detected in blood, liver and visceral adipose tissue (VAT) 30min after gavage. Kinetics of absorption and biodistribution was similar in both groups. We concluded that WG is mainly metabolized in liver and its action on VAT may be direct, as fractions of radio labeled protein were found in this tissue. Financial support: FAPEMIG, CNPq, CAPES

Impact of toll-like-receptor-9 (TLR9) deficiency on visceral adipose tissue adipokine expression during chronic DSS-induced colitis in mice.

Studies postulate an involvement of adipokines in inflammatory gastrointestinal diseases. Leptin-deficient ob/ob mice as well as TLR9-deficient mice have a more moderate course of chronic DSS-induced colitis (DSS-CC) and adipocytes do express functional TLR9 molecules. Adipokine mRNA expression in visceral adipose tissue of mice before and after the induction of DSS-CC was investigated. Experiments were performed in both TLR9(wt/wt) and TLR9(-/-) mice. In vitro, the effect of TLR9 blocking peptide on leptin and visfatin protein secretion was studied in 3T3-L1 adipocytes. Induction of DSS-CC led to an upregulation of leptin mRNA expression in TLR9(wt/wt) mice, while TLR9(-/-) animals showed a significant reduction of leptin expression even below baseline. While visfatin expression remained unchanged in TLR9(wt/wt) animals, TLR9(-/-) mice exhibited a significant induction during DSS-CC. CTRP-3 expression was reduced after colitis induction only in TLR9(-/-) animals. Of note, IL-6 expression levels remained unchanged, while CXCL1/KC and cyclophilin A expression was reduced in DSS-CC. Inhibition of TLR9 signaling by using TLR9 blocking peptide led to reduced leptin protein secretion into cell culture supernatants in 3T3-L1 adipocytes, while visfatin protein secretion was enhanced. DSS-CC leads to differential adipokine expression profiles in the visceral fat pad in TLR9(wt/wt) vs. TLR9(-/-) mice. In vitro, inhibition of TLR9 signaling induces visfatin secretion while inhibiting leptin secretion in adipocytes. Thus, visceral adipokines are regulated by intact TLR9 signaling pathway and a specific interplay between the leptin- and the TLR9-pathways might be of pathophysiological importance in chronic intestinal inflammation.

Effect of high-fat or high-glucose diet on obesity and visceral adipose tissue in mice.

To investigate the effect of high-fat or high-glucose diet on obesity and visceral adipose tissue in C57BL/6 mice. Four-week-old C57BL/6 mice were allocated into normal diet group,high-fat diet group,and high-glucose diet group according to the random number table until 20 weeks old. Body weight,epididymal adipose tissue weight,blood leptin,fat infiltration in liver,M1/M2 macrophage subtypes,and monocyte chemoattractant protein-1 mRNA in epididymal adipose tissues were measured. Compared with normal diet group,body weight,epididymal adipose tissue weight,and leptin concentration in high fat diet group at 20 weeks were significantly increased (P < 0.05),and oil red O staining showed more prominent adipocyte infiltration in liver in high-fat diet group than those in normal diet and high-glucose diet group. However,no apparent differences were seen in high-glucose diet group at 20 weeks in terms of body weight,epididymal adipose tissue weight and leptin concentration. In high-fat diet group,the macrophages infiltration in epididymal adipose tissue increased with time and the percentage of M2 macrophage decreased in high-fat diet group than that in high-glucose diet group(P<0.05). Compared with normal diet group,monocyte chemoattractant protein-1 mRNA expression increased significantly in high-fat diet group(P<0.05). In high-glucose group,however,no significant differences were discerned (P > 0.05). High-fat diet,rather than 60% high glucose diet,will lead to obesity and macrophage infiltration in adipose tissues.

Indole-3-carbinol prevents diet-induced obesity through modulation of multiple genes related to adipogenesis, thermogenesis or inflammation in the visceral adipose tissue of mice

Indole-3-carbinol (I3C) is a compound found in high concentrations in Brassica family vegetables, including broccoli, cauliflower and cabbage, and is regarded as a promising chemopreventive agent against various cancers. This study assesses the protective effect of I3C against diet-induced obesity in mice. Mice were randomly grouped to receive either a normal diet, high-fat (40% energy as fat) diet (HFD) or I3C-supplemented diet (1 g/kg diet) for 10 weeks. I3C supplementation significantly ameliorated HFD-induced increases in body weight gain, visceral fat pad weights and plasma lipid levels. The visceral adipose tissue mRNA levels of uncoupling proteins 1 and 3, crucial factors of thermogenesis, and their regulators such as sirtuin 1, peroxisome proliferator-activated receptor (PPAR) α and PPARγ coactivator 1α, which were down-regulated by HFD, were normalized by supplementation with I3C. In contrast, I3C supplementation significantly decreased expression levels of a key adipogenic transcription factor, PPARγ2, and its target genes, such as leptin and adipocyte protein 2, in the visceral adipose tissue of mice maintained on the HFD. Furthermore, HFD-induced up-regulation in mRNA levels of inflammatory cytokines (tumor necrosis factor α, interferon β and interleukin 6) was significantly ameliorated by I3C. These findings suggest that I3C has a potential benefit in preventing obesity and metabolic disorders, and the action for I3C in vivo may involve multiple mechanisms including decreased adipogenesis and inflammation, along with activated thermogenesis.