Quantification of Lipid Area within Thermogenic Mouse Perivascular Adipose Tissue Using Standardized Image Analysis in FIJI

2021 ◽  
pp. 1-7
Author(s):  
Benjamin W. Tero ◽  
Bethany Fortier ◽  
Ashley N. Soucy ◽  
Ginger Paquette ◽  
Lucy Liaw
Keyword(s):  
Image Analysis ◽  
Adipose Tissue ◽  
Lipid Content ◽  
Chow Diet ◽  
Physiological Measures ◽  
Perivascular Adipose Tissue ◽  
Normal Chow ◽  
Adipocyte Cell ◽  
Hematoxylin Eosin ◽  
Reproducible Manner

Quantification of adipocyte size and number is routinely performed for white adipose tissues using existing image analysis software. However, thermogenic adipose tissue has multilocular adipocytes, making it difficult to distinguish adipocyte cell borders and to analyze lipid proportion using existing methods. We developed a simple, standardized method to quantify lipid content of mouse thermogenic adipose tissue. This method, using FIJI analysis of hematoxylin/eosin stained sections, was highly objective and highly reproducible, with ∼99% inter-rater reliability. The method was compared to direct lipid staining of adipose tissue, with comparable results. We used our method to analyze perivascular adipose tissue (PVAT) from C57BL/6 mice on a normal chow diet, compared to calorie restriction or a high fat diet, where lipid storage phenotypes are known. Results indicate that lipid content can be estimated within mouse PVAT in a quantitative and reproducible manner, and shows correlation with previously studied molecular and physiological measures.

scholarly journals Estrogens Protect against High-Fat Diet-Induced Insulin Resistance and Glucose Intolerance in Mice

Endocrinology ◽  
2009 ◽  
Vol 150 (5) ◽  
pp. 2109-2117 ◽  
Author(s):  
Elodie Riant ◽  
Aurélie Waget ◽  
Haude Cogo ◽  
Jean-François Arnal ◽  
Rémy Burcelin ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Adipose Tissue ◽  
Glucose Intolerance ◽  
High Fat Diet ◽  
Chow Diet ◽  
High Fat ◽  
Normal Chow ◽  
Visceral Adipose ◽  
Deficient Mice ◽  
Normal Chow Diet

Although corroborating data indicate that estrogens influence glucose metabolism through the activation of the estrogen receptor α (ERα), it has not been established whether this pathway could represent an effective therapeutic target to fight against metabolic disturbances induced by a high-fat diet (HFD). To this end, we first evaluated the influence of chronic 17β-estradiol (E2) administration in wild-type ovariectomized mice submitted to either a normal chow diet or a HFD. Whereas only a modest effect was observed in normal chow diet-fed mice, E2 administration exerted a protective effect against HFD-induced glucose intolerance, and this beneficial action was abolished in ERα-deficient mice. Furthermore, E2 treatment reduced HFD-induced insulin resistance by 50% during hyperinsulinemic euglycemic clamp studies and improved insulin signaling (Akt phosphorylation) in insulin-stimulated skeletal muscles. Unexpectedly, we found that E2 treatment enhanced cytokine (IL-6, TNF-α) and plasminogen activator inhibitor-1 mRNA expression induced by HFD in the liver and visceral adipose tissue. Interestingly, although the proinflammatory effect of E2 was abolished in visceral adipose tissue from chimeric mice grafted with bone marrow cells from ERα-deficient mice, the beneficial effect of the hormone on glucose tolerance was not altered, suggesting that the metabolic and inflammatory effects of estrogens can be dissociated. Eventually comparison of sham-operated with ovariectomized HFD-fed mice demonstrated that endogenous estrogens levels are sufficient to exert a full protective effect against insulin resistance and glucose intolerance. In conclusion, the regulation of the ERα pathway could represent an effective strategy to reduce the impact of high-fat diet-induced type 2 diabetes.

Abstract 3672: Loss of Perivascular Adipocyte Paracrine Function Leads to Increased Vascular Tone and Glucose Intolerance in Hypertension

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Kaivan Khavandi ◽  
Adam Greenstein ◽  
Sarah Withers ◽  
Kazuhiko Sonoyama ◽  
Sarah Lewis ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Metabolic Syndrome ◽  
Adipose Tissue ◽  
Vascular Tone ◽  
Tnf Alpha ◽  
Perivascular Adipose Tissue ◽  
The Metabolic Syndrome ◽  
Adipocyte Cell ◽  
Therapeutic Opportunity

In order to investigate the contribution of perivascular adipose tissue (PVAT) to arterial function, a total of 55 small arteries harvested from 35 skin biopsies of patients with Metabolic Syndrome and matched controls were mounted as ring preparations in a wire myograph. Contractility to cumulative doses of Norepinephrine in the presence or absence of PVAT showed an anticontractile effect in arteries from healthy volunteers (p=0.009), which was lost in patients with Metabolic Syndrome. Bioassay studies confirmed that PVAT releases a hydrophilic anticontractile factor in health, which is absent in obesity. Using a soluble fragment of the human Type 1 receptor, we identified that the anticontractile factor was adiponectin, which is the sole mediator of vasodilation, acting by increasing endothelial bioavailability of nitric oxide. Significant endothelial dysfunction was observed in patients with Metabolic Syndrome (p<0.001). Quantitative image analysis of adipose tissue revealed significantly increased adipocyte cell size in patients with Metabolic Syndrome, compared with healthy controls (p<0.006). There was immunohistochemical evidence of inflammation with upregulation of TNF-alpha receptor 1 in these patients (p<0.001). Application of exogenous TNF-alpha abolished the anticontractile effect of PVAT by reducing adiponectin bioavailability. Oxidative stress also induced by cytokines TNF-alpha and IL-6 but not IL-1, reduced adiponectin production from PVAT and increased basal tone. When the obese microenvironment was replicated in vitro by inflicting hypoxia on PVAT, adiponectin activity was lost but then rescued by incubation with cytokine antagonists. Further application of the adiponectin receptor fragment abolished PVAT relaxation. We conclude that in healthy arteries, PVAT releases adiponectin which reduces vascular tone. In obesity, this is lost by a cascade of adipocyte hypertrophy, hypoxia, inflammation and oxidative stress. The resulting vasoconstriction contributes to hypertension, hypertriglyceridaemia and insulin resistance. Direct targeting of adiponectin release from PVAT therefore provides a novel therapeutic opportunity in the Metabolic Syndrome.

Abstract 374: Interleukin-19: A Novel Pro-Angiogenic and Anti-Inflammatory Adipokine

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Christine Vrakas ◽  
Sheri E Keleman ◽  
Rosario Scalia ◽  
Michael V Autieri
Keyword(s):  
Adipose Tissue ◽  
Visceral Fat ◽  
Subcutaneous Fat ◽  
Chow Diet ◽  
Vascular Cells ◽  
Regulatory Factor ◽  
Normal Chow ◽  
Gene Regulatory ◽  
Anti Inflammatory ◽  
Normal Chow Diet

Uncontrolled inflammation leads to many of the chronic diseases associated with obesity. Due to a lack of oxygen in the tissue, expanding adipose tissue becomes hypoxic and pro-inflammatory. Adipocytes release pro-angiogenic factors in an effort to restore blood flow to the tissue. Presently, little is known about the potential for endogenously expressed anti-inflammatory cytokines to attenuate inflammation and also provide pro-angiogenic effects. IL-19 is uniquely anti-inflammatory, pro-angiogenic and is both expressed by and targets various cells types. IL-19 expression in adipocytes and stromal vascular cells is increased in visceral compared to subcutaneous fat, and is also increased in visceral fat on high fat diet (HFD) compared to normal chow diet. There is no known mechanism to explain the role of IL-19 in adipose tissue expansion, and we hypothesized that IL-19 may have pro-angiogenic and anti-inflammatory properties in expanding adipose tissue. We have identified a gene regulatory factor, Interleukin Enhancer-Binding Factor 3 (ILF3) that is induced in adipocytes and stromal vascular cells by HFD and IL-19 treatment. We found that both IL-19 and VEGF induce ILF3 expression in cultured human endothelial cells (hECs). Proliferation is significantly reduced when ILF3 is knocked down using siRNA in hECs. Furthermore, when ILF3 is knocked down and hECs are stimulated with VEGF several angiogenic cytokines are also decreased. Through immunohistochemistry we found that ILF3 translocates from the nucleus to the cytoplasm in visceral fat of C57BL/6 mice fed a HFD, and remains in the nucleus when fed a normal chow diet. In summary IL-19 may be a unique HFD responsive adipokine functioning to reduce inflammation and increase angiogenesis in expanding adipose tissue. The angiogenic function of IL-19 may work through induction of the gene regulatory factor, ILF3.

Abstract P099: Loss Of The Brown-Like Phenotype Of Aortic PVAT Is Associated With Mitochondrial Dysfunction In Obesity-Induced Endothelial Dysfunction In Female Rats

Hypertension ◽  
2020 ◽  
Vol 76 (Suppl_1) ◽  
Author(s):  
Nicole Maddie ◽  
Maria Alicia A Carrillo-sepulveda
Keyword(s):  
Adipose Tissue ◽  
Endothelial Dysfunction ◽  
Endothelial Function ◽  
Mitochondrial Dysfunction ◽  
Uncoupling Protein ◽  
Obese Group ◽  
Female Rats ◽  
Chow Diet ◽  
Anatomical Location ◽  
Perivascular Adipose Tissue

Endothelial dysfunction is a major complication of obesity and is an early contributor to hypertension. Perivascular adipose tissue (PVAT) surrounds most blood vessels and has different phenotypic properties based on its anatomical location. Thoracic aortic PVAT from humans and rodents is a brown-like adipose tissue and plays a vasculo-protective role under physiological conditions. In obesity, aortic PVAT expands, switches from a brown-like to a white-like phenotype and contributes to endothelial dysfunction. We hypothesized that loss of the brown-like phenotype of aortic PVAT in obesity is associated with mitochondrial dysfunction, resulting in PVAT and endothelial dysfunction. Eight-week-old female Wistar rats were randomized into two experimental groups: the Lean group (n=8) received a chow diet (5% fat, 48.7% carbohydrate [3.2% sucrose], 24.1% protein) and the Obese group (n=8) received a western diet (21% fat, 50% carbohydrate [34% sucrose], 20% protein), for 20 weeks. Increased body weight (340.57 vs. 265.37g leans, p<0.05) was confirmed in the obese group. At the experimental endpoint, thoracic aortas with intact (+PVAT) or removed PVAT (-PVAT) were obtained for analysis. Endothelial function was assessed in aortic rings +PVAT or -PVAT by performing concentration-response to acetylcholine using wire myography. The aortic ring (-PVAT) from the obese group exhibited impaired endothelium-dependent vasodilation (p<0.01). This effect was heightened in aortic rings (+PVAT) (p<0.05), showing a negative effect of PVAT on endothelial function during obesity. Mitochondrial dysfunction in PVAT from the obese group was characterized by decreased mitochondrial density (30% reduction, p<0.05), detected by quantification of Mitotracker fluorescence, and increased reactive oxygen species levels (4.34-fold increase, p<0.01), as evidenced by DHE staining. These effects were accompanied by decreased uncoupling protein-1 expression in the obese group (55% reduction, p<0.01). Moreover, Oil Red O staining showed larger lipid droplets in aortic PVAT from the obese group. Our results support that obesity-induced endothelial dysfunction is associated with a loss of the brown-like phenotype and mitochondrial dysfunction in PVAT.

scholarly journals TIR-Domain-Containing Adaptor-Inducing Interferon-β (TRIF) Is Involved in Glucose Metabolism in Adipose Tissue through the Insulin/AKT Signaling Pathway

2020 ◽  
Vol 2020 ◽  
pp. 1-9
Author(s):  
Junling Yang ◽  
Ken-Ichiro Fukuchi
Keyword(s):  
Insulin Resistance ◽  
Adipose Tissue ◽  
Epididymal Adipose Tissue ◽  
Chow Diet ◽  
Low Grade ◽  
Interferon Β ◽  
Tlr Signaling ◽  
Normal Chow ◽  
Low Grade Inflammation

Obesity significantly increases the risk of developing type 2 diabetes mellitus and other metabolic diseases. Obesity is associated with chronic low-grade inflammation in white adipose tissues, which is thought to play an essential role in developing insulin resistance. Many lines of evidence indicate that toll-like receptors (TLRs) and their downstream signaling pathways are involved in development of chronic low-grade inflammation and insulin resistance, which are associated with obesity. Mice lacking molecules positively involved in the TLR signaling pathways are generally protected from high-fat diet-induced inflammation and insulin resistance. In this study, we have determined the effects of genetic deficiency of toll/interleukin-1 receptor-domain-containing adaptor-inducing interferon-β (TRIF) on food intake, bodyweight, glucose metabolism, adipose tissue macrophage polarization, and insulin signaling in normal chow diet-fed mice to investigate the role of the TRIF-dependent TLR signaling in adipose tissue metabolism and inflammation. TRIF deficiency (TRIF−/−) increased food intake and bodyweight. The significant increase in bodyweight in TRIF−/− mice was discernible as early as 24 weeks of age and sustained thereafter. TRIF−/− mice showed impaired glucose tolerance in glucose tolerance tests, but their insulin tolerance tests were similar to those in TRIF+/+ mice. Although no difference was found in the epididymal adipose mass between the two groups, the percentage of CD206+ M2 macrophages in epididymal adipose tissue decreased in TRIF−/− mice compared with those in TRIF+/+ mice. Furthermore, activation of epididymal adipose AKT in response to insulin stimulation was remarkably diminished in TRIF−/− mice compared with TRIF+/+ mice. Our results indicate that the TRIF-dependent TLR signaling contributes to maintaining insulin/AKT signaling and M2 macrophages in epididymal adipose tissue under a normal chow diet and provide new evidence that TLR4-targeted therapies for type 2 diabetes require caution.

scholarly journals The Role of Brown Adipose Tissue in the Development and Treatment of Nonalcoholic Steatohepatitis: An Exploratory Gene Expression Study in Mice

2020 ◽  
Vol 52 (12) ◽  
pp. 869-876
Author(s):  
Toon J.I. De Munck ◽  
Pan Xu ◽  
Brechtje L.J. Vanderfeesten ◽  
Montserrat Elizalde ◽  
Ad A.M. Masclee ◽  
...  
Keyword(s):  
Gene Expression ◽  
Adipose Tissue ◽  
Lipid Metabolism ◽  
Energy Expenditure ◽  
Brown Adipose Tissue ◽  
Nonalcoholic Steatohepatitis ◽  
Dietary Intervention ◽  
Chow Diet ◽  
Brown Adipose ◽  
Normal Chow

AbstractBrown adipose tissue (BAT) might be a beneficial mediator in the development and treatment of nonalcoholic steatohepatitis (NASH). We aim to evaluate the gene expression of BAT activity-related genes during the development and the dietary and surgical treatment of NASH. BAT was collected from male C57BL/6J mice that received a high fat-high sucrose diet (HF-HSD) or a normal chow diet (NCD) for 4 and 20 weeks (n=8–9 per dietary group and timepoint) and from mice that underwent dietary intervention (return to NCD) (n=8), roux-en-y gastric bypass (RYGB) (n=6), or sham procedure (n=6) after 12 weeks HF-HSD. Expression of BAT genes involved in lipid metabolism (Cd36 and Cpt1b; p<0.05) and energy expenditure (Ucp1 and Ucp3; p<0.05) were significantly increased after 4 weeks HF-HSD compared with NCD, whereas in the occurrence of NASH after 20 weeks HF-HSD no difference was observed. We observed no differences in gene expression regarding lipid metabolism or energy expenditure at 8 weeks after dietary intervention (no NASH) compared with HF-HSD mice (NASH), nor in mice that underwent RYGB compared with SHAM. However, dietary intervention and RYGB both decreased the BAT gene expression of inflammatory cytokines (Il1b, Tnf-α and MCP-1; p<0.05). Gene expression of the batokine neuregulin 4 was significantly decreased after 20 weeks HF-HSD (p<0.05) compared with NCD, but was restored by dietary intervention and RYGB (p<0.05). In conclusion, BAT is hallmarked by dynamic alterations in the gene expression profile during the development of NASH and can be modulated by dietary intervention and bariatric surgery.

Abstract 221: B-1b Cells Produce IgM to Malondialdehyde-Modified Low Density Lipoprotein in Perivascular Adipose Tissue in Response to Immunization And Attenuate Diet Induced Atherosclerosis

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Prasad Srikakulapu ◽  
Aditi Upadhye ◽  
Sam M Rosenfeld ◽  
Kaivon Kouhestani ◽  
Melissa Marshall ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
B Cell ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Dominant Negative ◽  
Chow Diet ◽  
Low Density ◽  
Perivascular Adipose Tissue ◽  
Cell Numbers ◽  
Helix Loop Helix

Background: B-1b cells are capable of long-lasting IgM memory and secrete more IgM, particularly malondialdehyde-modified low density lipoprotein (MDA-LDL) specific IgM than B-1a cells after transfer into hyperlipidemic Rag1 -/- mice. Id3 is a basic helix-loop-helix protein and dominant negative inhibitor of E proteins. B cell specific Id3 deficiency (Id3 BKO ) increases B-1b cell numbers systemically and provides atheroprotection. Adipose tissue is a source of B-1b-derived IgM and regulates inflammatory cytokine production from M1 macrophages locally. Perivascular adipose tissue (PVAT) has been implicated in regulation of atherosclerosis. However, the effect of B cell specific Id3 deficiency on B-1 cell responses in PVAT is not yet known. Also, whether these B-1b cells respond to MDA-LDL immunization is unknown. Hypothesis: B-1b cells are present in PVAT and produce MDA-LDL specific IgM in PVAT, and immunization with MDA-LDL can enhance B-1b-mediated atheroprotection. Methods and Results: Flow Cytometry and Enzyme-Linked ImmunoSpot (ELISPOT) analysis of PVAT of normal chow diet fed young mice demonstrated that ApoE.Id3 BKO mice have significantly higher numbers of B-1b cells and IgM secreting cells but not B-2 and B-1a cell numbers in PVAT compared to ApoE.Id3 WT mice. ELISPOT demonstrated that the % of MDA-LDL specific IgM of total IgM secreting cells was significantly greater in PVAT, but not in spleen or bone marrow, of ApoE.Id3 BKO mice compared to ApoE.Id3 WT mice, suggesting that modified lipids such as MDA-LDL in plaques and PVAT may stimulate local B-1b cells to secrete MDA-LDL specific IgM. Adoptive transfer of B-1b cells into ApoE.Rag1 -/- mice followed by MDA-LDL+PPS3 (pneumococcal polysaccharide) immunization, increased plasma IgM to MDA-LDL after 2 weeks and significantly attenuated atherosclerosis after 16 weeks of Western diet compared to PBS injected mice and B-1b cells transferred mice with PBS immunization. Conclusion: B-1b cells produce IgM to MDA-LDL in PVAT. MDA-LDL immunization increased IgM to MDA-LDL after two weeks and attenuated diet-induced atherosclerosis. Taken together, results suggest that B-1b cells may regulate atherosclerosis through both local and systemic IgM production.

scholarly journals Pleiotrophin Deficiency Induces Browning of Periovarian Adipose Tissue and Protects against High-Fat Diet-Induced Hepatic Steatosis

2021 ◽  
Vol 22 (17) ◽  
pp. 9261
Author(s):  
Agata Zuccaro ◽  
Begoña Zapatería ◽  
María Gracia Sánchez-Alonso ◽  
María Haro ◽  
María Limones ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Adipose Tissue ◽  
Lipid Metabolism ◽  
Lipid Content ◽  
High Fat Diet ◽  
Brown Adipocyte ◽  
Chow Diet ◽  
High Fat ◽  
Hepatic Lipid ◽  
Beige Adipocytes

(1) Background: Pleiotrophin preserves insulin sensitivity, regulates adipose tissue lipid turnover and plasticity, energy metabolism and thermogenesis. The aim of this study was to determine the role of pleiotrophin in hepatic lipid metabolism and in the metabolic crosstalk between the liver and brown and white adipose tissue (AT) in a high-fat diet-induced (HFD) obesity mice model. (2) Methods: We analyzed circulating variables, lipid metabolism (hepatic lipid content and mRNA expression), brown AT thermogenesis (UCP-1 expression) and periovarian AT browning (brown adipocyte markers mRNA and immunodetection) in Ptn−/− mice either fed with standard-chow diet or with HFD and in their corresponding Ptn+/+ counterparts. (3) Results: HFD-Ptn−/− mice are protected against the development of HFD-induced insulin resistance, had lower liver lipid content and lower expression of the key enzymes involved in triacylglycerides and fatty acid synthesis in liver. HFD-Ptn−/− mice showed higher UCP-1 expression in brown AT. Moreover, Ptn deletion increased the expression of specific markers of brown/beige adipocytes and was associated with the immunodetection of UCP-1 enriched multilocular adipocytes in periovarian AT. (4) Conclusions: Ptn deletion protects against the development of HFD-induced insulin resistance and liver steatosis, by increasing UCP-1 expression in brown AT and promoting periovarian AT browning.

scholarly journals Restoring Perivascular Adipose Tissue Function in Obesity Using Exercise

2021 ◽  
Author(s):  
Sophie N Saxton ◽  
Lauren K Toms ◽  
Robert G Aldous ◽  
Sarah B Withers ◽  
Jacqueline Ohanian ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Contractile Function ◽  
Ex Vivo ◽  
Vascular Complications ◽  
Electrical Field Stimulation ◽  
Organic Cation Transporter ◽  
Nervous Stimulation ◽  
Perivascular Adipose Tissue ◽  
Contractile Effect ◽  
Organic Cation Transporter 3

AbstractPurposePerivascular adipose tissue (PVAT) exerts an anti-contractile effect which is vital in regulating vascular tone. This effect is mediated via sympathetic nervous stimulation of PVAT by a mechanism which involves noradrenaline uptake through organic cation transporter 3 (OCT3) and β3-adrenoceptor-mediated adiponectin release. In obesity, autonomic dysfunction occurs, which may result in a loss of PVAT function and subsequent vascular disease. Accordingly, we have investigated abnormalities in obese PVAT, and the potential for exercise in restoring function.MethodsVascular contractility to electrical field stimulation (EFS) was assessed ex vivo in the presence of pharmacological tools in ±PVAT vessels from obese and exercised obese mice. Immunohistochemistry was used to detect changes in expression of β3-adrenoceptors, OCT3 and tumour necrosis factor-α (TNFα) in PVAT.ResultsHigh fat feeding induced hypertension, hyperglycaemia, and hyperinsulinaemia, which was reversed using exercise, independent of weight loss. Obesity induced a loss of the PVAT anti-contractile effect, which could not be restored via β3-adrenoceptor activation. Moreover, adiponectin no longer exerts vasodilation. Additionally, exercise reversed PVAT dysfunction in obesity by reducing inflammation of PVAT and increasing β3-adrenoceptor and OCT3 expression, which were downregulated in obesity. Furthermore, the vasodilator effects of adiponectin were restored.ConclusionLoss of neutrally mediated PVAT anti-contractile function in obesity will contribute to the development of hypertension and type II diabetes. Exercise training will restore function and treat the vascular complications of obesity.

Sign in / Sign up

Export Citation Format

Share Document