Inflammation Correlates With Markers of T‐Cell Subsets Including Regulatory T Cells in Adipose Tissue From Obese Patients

Accumulation of cytotoxic and T‐helper (T_h)1 cells together with a loss of regulatory T cells in gonadal adipose tissue was recently shown to contribute to obesity‐induced adipose tissue inflammation and insulin resistance in mice. Human data on T‐cell populations in obese adipose tissue and their potential functional relevance are very limited. We aimed to investigate abundance and proportion of T‐lymphocyte sub‐populations in human adipose tissue in obesity and potential correlations with anthropometric data, insulin resistance, and systemic and adipose tissue inflammation. Therefore, we analyzed expression of marker genes specific for pan‐T cells and T‐cell subsets in visceral and subcutaneous adipose tissue from highly obese patients (BMI >40 kg/m², n = 20) and lean to overweight control subjects matched for age and sex (BMI <30 kg/m²; n = 20). All T‐cell markers were significantly upregulated in obese adipose tissue and correlated with adipose tissue inflammation. Proportions of cytotoxic T cells and T_h1 cells were unchanged, whereas those of regulatory T cells and T_h2 were increased in visceral adipose tissue from obese compared to control subjects. Systemic and adipose tissue inflammation positively correlated with the visceral adipose abundance of cytotoxic T cells and T_h1 cells but also regulatory T cells within the obese group. Therefore, this study confirms a potential role of T cells in human obesity‐driven inflammation but does not support a loss of protective regulatory T cells to contribute to adipose tissue inflammation in obese patients as suggested by recent animal studies.     

Effect of some hormones on the rate of the triacylglycerol/fatty-acid substrate cycle in adipose tissue of the mouse in vivo

A method is described for the measurement of the rate of the triacylglycerol/fatty-acid cycle in adipose tissue of the mouse in vivo, which depends upon the incor-poration of tritium from [³H]H₂O into the glycerol and fatty-acid moieties of triacyiglycerol. The rate of the cycling is increased two-fold by feeding, an effect that is completely abolished by the β-adrenergic blocker propranolol. The β-adrenergic agonist fenoterol increased the rate of cycling five-fold in white adipose tissue and three-fold in brown adipose tissue. Cold exposure had no effect on the rate of cycling in white adipose tissue but increased the rate almost two-fold in brown adipose tissue. The increased rate of cycling during feeding, which may be due to increased sympathetic nervous activity, is consistent with the view that the role of cycling is to increase sensitivity of metabolic control systems when required.     

Cold-induced changes in brown adipose tissue thermogenic capacity of immunocompetent and immunodeficient hairless mice

Mild cold acclimation (22°C, 3 weeks) of hairless mice was shown to increase 5-fold the brown adipose tissue uncoupling protein content in immunodeficient BALB/c nu/nu mice, but by only 2.3-fold in immunocompetent BFU mice. The difference in activation of brown adipose tissue thermogenic capacity was due to a 2-fold increase in the content of brown adipose tissue in nu/nu mice only, which was paralleled by an increase in brown adipose tissue protein but not DNA content. Likewise, only in nu/nu mice the cold acclimation increased the reaction of natural killer cells in blood and peritoneal exudate with a shift from spleen to lymph nodes and increased the phagocytic index. The results indicate that the immune system may influence the defence against cold at the level of brown adipose tissue thermogenesis.Abbreviations AU arbitrary unit(s) - bw body weight - HEMA 2-hydromethyl-metacrylate copolymer - BAT brown adipose tissue - UCP uncoupling protein - ATPase mitochondrial F_oF₁-ATPsynthase - IL-1 interleukin 1 - TNF tumour necrosis factor - NK cells natural killer cells - T _a ambient temperature     

Activation of prostaglandin E2-EP4 signaling reduces chemokine production in adipose tissue

Inflammation of adipose tissue induces metabolic derangements associated with obesity. Thus, determining ways to control or inhibit inflammation in adipose tissue is of clinical interest. The present study tested the hypothesis that in mouse adipose tissue, endogenous prostaglandin E₂ (PGE₂) negatively regulates inflammation via activation of prostaglandin E receptor 4 (EP4). PGE₂ (5–500nM) attenuated lipopolysaccharide-induced mRNA and protein expression of chemokines, including interferon-γ-inducible protein 10 and macrophage-inflammatory protein-1α in mouse adipose tissue. A selective EP4 antagonist (L161,982) reversed, and two structurally different selective EP4 agonists [CAY10580 and CAY10598] mimicked these actions of PGE₂. Adipose tissue derived from EP4-deficient mice did not display this response. These findings establish the involvement of EP4 receptors in this anti-inflammatory response. Experiments performed on adipose tissue from high-fat-fed mice demonstrated EP4-dependent attenuation of chemokine production during diet-induced obesity. The anti-inflammatory actions of EP4 became more important on a high-fat diet, in that EP4 activation suppressed a greater variety of chemokines. Furthermore, adipose tissue and systemic inflammation was enhanced in high-fat-fed EP4-deficient mice compared with wild-type littermates, and in high-fat-fed untreated C57BL/6 mice compared with mice treated with EP4 agonist. These findings provide in vivo evidence that PGE₂-EP4 signaling limits inflammation. In conclusion, PGE₂, via activation of EP4 receptors, functions as an endogenous anti-inflammatory mediator in mouse adipose tissue, and targeting EP4 may mitigate adipose tissue inflammation.     

Effect of Angiotensin II Type 2 Receptor-Interacting Protein on Adipose Tissue Function via Modulation of Macrophage Polarization

We demonstrated that angiotensin II type 2 (AT₂) receptor-interacting protein (ATIP) 1 ameliorates inflammation-mediated vascular remodeling independent of the AT₂ receptor, leading us to explore the possibility of whether ATIP1 could exert anti-inflammatory effects and play a role in other pathophysiological conditions. We examined the possible anti-inflammatory effects of ATIP1 in adipose tissue associated with amelioration of insulin resistance. In mice fed a high-cholesterol diet, adipose tissue macrophage (ATM) infiltration and M1-to-M2 ratio were decreased in ATIP1 transgenic mice (ATIP1-Tg) compared with wild-type mice (WT), with decreased expression of inflammatory cytokines such as tumor necrosis factor-α and monocyte chemoattractant protein-1 in white adipose tissue (WAT), but an increase in interleukin-10, an anti-inflammatory cytokine. Moreover, 2-[³H]deoxy-d-glucose (2-[³H]DG) uptake was significantly increased in ATIP1-Tg compared with WT. Next, we examined the roles of ATIP1 in BM-derived hematopoietic cells, employing chimeric mice produced by BM transplantation into irradiated type 2 diabetic mice with obesity, KKAy, as recipients. ATM infiltration and M1-to-M2 ratio were decreased in ATIP1 chimera (ATIP1-tg as BM donor), with improvement of insulin-mediated 2-[³H]DG uptake and amelioration of inflammation in WAT. Moreover, serum adiponectin concentration in ATIP1 chimera was significantly higher than that in WT chimera (WT as BM donor) and KKAy chimera (KKAy as BM donor). These results indicate that ATIP1 could exert anti-inflammatory effects in adipose tissue via macrophage polarization associated with improvement of insulin resistance, and ATIP1 in hematopoietic cells may contribute to these beneficial effects on adipose tissue functions in type 2 diabetes.     

UCP1 Induction during Recruitment of Brown Adipocytes in White Adipose Tissue Is Dependent on Cyclooxygenase Activity

Background

The uncoupling protein 1 (UCP1) is a hallmark of brown adipocytes and pivotal for cold- and diet-induced thermogenesis.

Methodology/Principal Findings

Here we report that cyclooxygenase (COX) activity and prostaglandin E₂ (PGE₂) are crucially involved in induction of UCP1 expression in inguinal white adipocytes, but not in classic interscapular brown adipocytes. Cold-induced expression of UCP1 in inguinal white adipocytes was repressed in COX2 knockout (KO) mice and by administration of the COX inhibitor indomethacin in wild-type mice. Indomethacin repressed β-adrenergic induction of UCP1 expression in primary inguinal adipocytes. The use of PGE₂ receptor antagonists implicated EP₄ as a main PGE₂ receptor, and injection of the stable PGE₂ analog (EP_3/4 agonist) 16,16 dm PGE₂ induced UCP1 expression in inguinal white adipose tissue. Inhibition of COX activity attenuated diet-induced UCP1 expression and increased energy efficiency and adipose tissue mass in obesity-resistant mice kept at thermoneutrality.

Conclusions/Significance

Our findings provide evidence that induction of UCP1 expression in white adipose tissue, but not in classic interscapular brown adipose tissue is dependent on cyclooxygenase activity. Our results indicate that cyclooxygenase-dependent induction of UCP1 expression in white adipose tissues is important for diet-induced thermogenesis providing support for a surprising role of COX activity in the control of energy balance and obesity development.     

Endurance training induces depot-specific changes in IL-10/TNF-α ratio in rat adipose tissue

White adipose tissue (WAT) is the source of pro- and anti-inflammatory cytokines and recently, it has been recognized as an important source of interleukin 10 (IL-10). Acute physical exercise is known to induce an anti-inflammatory cytokine profile, however, the effect of chronic physical exercise on the production of IL-10 by WAT has never been examined. We assessed IL-10 and TNF-α concentration in WAT of rats engaged in endurance training. Animals were randomly assigned to either a sedentary control group (S, n = 7) or an endurance trained group (T, n = 8). Trained rats ran on a treadmill 5 days/wk for 8 wk (55–65% VO_2max). Detection of IL-10 and TNF-α protein and mRNA expression, as well as the gene expression of PPAR-γ, and immunocytochemistry to detect mononuclear phagocytes were carried out. A reduction in absolute retroperitoneal adipose tissue (RPAT) weight in T (44%; p < 0.01), when compared with S was observed. IL-10 concentration was increased (1.5-fold, p < 0.05), to a higher extent than that of TNF-α (66%, p < 0.05) in the mesenteric adipose tissue (MEAT) of the trained group, while no change related to training was observed in RPAT. In MEAT, IL-10/TNF-α ratio was increased in T, when compared with S (30%; p < 0.05). PPAR-γ gene expression was increased in T (1.1-fold; p < 0.01), when compared with S in the same adipose depot. No monocyte infiltration was found. In conclusion, exercise training induced increased IL-10 expression in the mesenteric depot, resulting in a modified IL-10/TNF-α ratio. We also conclude that WAT presents a depot-specific response to endurance training regarding the studied aspects.     

An in vivo microdialysis coupled with liquid chromatography/tandem mass spectrometry study of cortisol metabolism in monkey adipose tissue

It is postulated that elevated tissue concentrations of cortisol may be associated with the development of metabolic syndrome, obesity, and type 2 diabetes. The 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) enzyme regenerates cortisol from inactive cortisone in tissues such as liver and adipose. To better understand the pivotal role of 11β-HSD1 in disease development, an in vivo microdialysis assay coupled with liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis using stable isotope-labeled (SIL) cortisone as a substrate was developed. This assay overcomes the limitations of existing methodologies that suffer from radioactivity exposure and analytical assay sensitivity and specificity concerns. Analyte extraction efficiencies (E_d) were evaluated by retrodialysis. The conversion of SIL-cortisone to SIL-cortisol in rhesus monkey adipose tissue was studied. Solutions containing 100, 500, and 1000 ng/mL SIL-cortisone were locally delivered through an implanted 30-mm microdialysis probe in adipose tissue. At the delivery rate of 1.0 and 0.5 μL/min, E_d values for SIL-cortisone were between 58.7 ± 5.6% (n = 4) and 72.7 ± 1.3% (n = 4), whereas at 0.3 μL/min E_d reached nearly 100%. The presence of 11β-HSD1 activities in adipose tissue was demonstrated by production of SIL-cortisol during SIL-cortisone infusion. This methodology could be applied to cortisol metabolism studies in tissues of other mammalian species.     

Stimulation of cyclic AMP and lipolysis in adipose tissue of normal and obese Avy/a mice by LY79771, a phenethanolamine,and stereoisomers

The stimulation of cyclic AMP and lipolysis by LY79771, a phenethanolamine antiobesity compound, and its 3 stereoisomers in adipose tissue of obese viable yellow mice and normal mice were studied. Both activities were stereo-specific with LY79771, the R,S isomer, and LY79730, the R,R isomer, being more potent than LY103085, the S,S isomer, and LY103672, the S,R isomer. Propranolol, a nonspecific β-antagonist, completely inhibited the elevation of cyclic AMP and lipolysis whereas atenolol, a specific β₁ antagonist, inhibited the elevation of cyclic AMP but did not completely inhibit lipolysis. These findings indicate that the elevation of cyclic AMP was mediated by the β₁- receptor whereas the stimulation of lipolysis was mediated by both the β₁ and β₂ receptors. The adipose tissue of the obese viable yellow mice responded to these compounds less than that of the normal mice.     

Fatty acids induce leukotriene C4 synthesis in macrophages in a fatty acid binding protein-dependent manner

Obesity results in increased macrophage recruitment to adipose tissue that promotes a chronic low-grade inflammatory state linked to increased fatty acid efflux from adipocytes. Activated macrophages produce a variety of pro-inflammatory lipids such as leukotriene C₄ (LTC₄) and 5-, 12-, and 15-hydroxyeicosatetraenoic acid (HETE) suggesting the hypothesis that fatty acids may stimulate eicosanoid synthesis. To assess if eicosanoid production increases with obesity, adipose tissue of leptin deficient ob/ob mice was analyzed. In ob/ob mice, LTC₄ and 12-HETE levels increased in the visceral (but not subcutaneous) adipose depot while the 5-HETE levels decreased and 15-HETE abundance was unchanged. Since macrophages produce the majority of inflammatory molecules in adipose tissue, treatment of RAW264.7 or primary peritoneal macrophages with free fatty acids led to increased secretion of LTC₄ and 5-HETE, but not 12- or 15-HETE. Fatty acid binding proteins (FABPs) facilitate the intracellular trafficking of fatty acids and other hydrophobic ligands and in vitro stabilize the LTC₄ precursor leukotriene A₄ (LTA₄) from non-enzymatic hydrolysis. Consistent with a role for FABPs in LTC₄ synthesis, treatment of macrophages with HTS01037, a specific FABP inhibitor, resulted in a marked decrease in both basal and fatty acid-stimulated LTC₄ secretion but no change in 5-HETE production or 5-lipoxygenase expression. These results indicate that the products of adipocyte lipolysis may stimulate the 5-lipoxygenase pathway leading to FABP-dependent production of LTC₄ and contribute to the insulin resistant state.     

Generation of hydrogen peroxide by brown adipose tissue mitochondria

This is the first report on the generation of H₂O₂ by brown adipose tissue mitochondria. Flavin dehydrogenase-linked substrates like succinate, glycerol-1-phosphate, and fatty acyl CoA were good substrates for the reaction, while NAD⁺-linked substrates were less effective. In cold-acclimated animals the activity showed a substantial increase (2.5-fold). TheK _m andV _max of the reaction were considerably lower than those of the respective dehydrogenase. Metal ions, particularly Cu²⁺ and Fe²⁺ were potent inhibitors of the reaction. Nucleoside diphosphates, which were inhibitors by themselves, potentiated the inhibitory action of Fe²⁺ ions. In most of the properties, the H₂O₂ generator of brown adipose tissue mitochondria resembled that of liver mitochondria.     

Glycogen synthase: the existence of a single demonstrable from in bovine adipose tissue.

Glycogen synthase from bovine adipose tissue has been kinetically characterized. Glucose 6-phosphate increased enzyme activity 50-fold with an activation constant (A_0.5) of 2.6 mm. Mg²⁺ reversibly decreased this A_0.5 to 0.75 mm without changing the amount of stimulation by glucose 6-phosphate. Mg²⁺ did not alter the apparent K_m for UDP-glucose (0.13 mm). The pH optimum was broad and centered at pH 7.6. The glucose 6-phosphate activation of the enzyme was reversible and competitively inhibited by ATP (K_i = 0.6 mm) and P_i(K_i = 2.0 mm). The use of exogenous sources of glycogen synthase and glycogen synthase phosphatase suggests that (i) adipose tissue glycogen synthase phosphatase activity in fed mature steers is low or undetectable, and (ii) endogenous bovine adipose tissue glycogen synthase can be activated to other glucose 6-phosphate-dependent forms by addition of adipose tissue extracts from fasted steers or fed rats.     

Structure/Function Relationships of Adipose Phospholipase A2 Containing a Cys-His-His Catalytic Triad

Adipose phospholipase A₂ (AdPLA or Group XVI PLA₂) plays an important role in the onset of obesity by suppressing adipose tissue lipolysis. As a consequence, AdPLA-deficient mice are resistant to obesity induced by a high fat diet or leptin deficiency. It has been proposed that AdPLA mediates its antilipolytic effects by catalyzing the release of arachidonic acid. Based on sequence homology, AdPLA is part of a small family of acyltransferases and phospholipases related to lecithin:retinol acyltransferase (LRAT). To better understand the enzymatic mechanism of AdPLA and LRAT-related proteins, we solved the crystal structure of AdPLA. Our model indicates that AdPLA bears structural similarity to proteins from the NlpC/P60 family of cysteine proteases, having its secondary structure elements configured in a circular permutation of the classic papain fold. Using both structural and biochemical evidence, we demonstrate that the enzymatic activity of AdPLA is mediated by a distinctive Cys-His-His catalytic triad and that the C-terminal transmembrane domain of AdPLA is required for the interfacial catalysis. Analysis of the enzymatic activity of AdPLA toward synthetic and natural substrates indicates that AdPLA displays PLA₁ in addition to PLA₂ activity. Thus, our results provide insight into the enzymatic mechanism and biochemical properties of AdPLA and LRAT-related proteins and lead us to propose an alternate mechanism for AdPLA in promoting adipose tissue lipolysis that is not contingent on the release of arachidonic acid and that is compatible with its combined PLA₁/A₂ activity.     

Chronic administration of iron and copper potentiates adipogenic effect of high fat diet in Wistar rats

The primary objective of this research project is explore a possible adipogenic effect of iron and/or copper in albino Wistar rats kept on standard (STD) and high-fat (HFD) diets. The female Wistar rats in the study were divided into eight experimental groups (n = 6). Rats maintained on STD and HFD received 3 mg/l FeSO₄·7H₂O, 4.88 mg/l CuSO₄ and a combination of 1.5 mg/l FeSO₄·7H₂O and 2.44 mg/l CuSO₄ with drinking water. Control groups were kept on STD and HFD and received pure water without metal salts. Consumption of iron and copper in the groups of rats maintained on an STD did not produce a significant increase in weight, adipose tissue content or body mass index. However, the adipocyte size and infiltration were increased in the adipose tissue of STD-fed rats receiving a mixture of iron and copper with drinking water. The rats fed iron and copper and, especially, their combination on a HFD background had a significantly higher weight gain, adipose tissue content, morphometric parameters values and adipocyte size compared to STD- and HFD-fed controls. Iron and copper consumption produced their accumulation in the rats’ adipose tissue. Moreover, the studied metals reduced adipose tissue concentration of chromium and vanadium. The lipoprotein profile and serum oxidative stress biomarkers were affected in the rats receiving the metals and STD. Hyperglycemia was observed in the rats receiving the studied metals on HFD-background. Based on the analysis of the test subjects, the study suggests that iron and copper administration, especially combined, may potentiate adipogenic effect of HFD.     

Associations between Ultrasound Measures of Abdominal Fat Distribution and Indices of Glucose Metabolism in a Population at High Risk of Type 2 Diabetes: The ADDITION-PRO Study

Aims

Visceral adipose tissue measured by CT or MRI is strongly associated with an adverse metabolic risk profile. We assessed whether similar associations can be found with ultrasonography, by quantifying the strength of the relationship between different measures of obesity and indices of glucose metabolism in a population at high risk of type 2 diabetes.

Methods

A cross-sectional analysis of 1342 participants of the ADDITION-PRO study. We measured visceral adipose tissue and subcutaneous adipose tissue with ultrasonography, anthropometrics and body fat percentage by bioelectrical impedance. Indices of glucose metabolism were derived from a three point oral glucose tolerance test. Linear regression of obesity measures on indices of glucose metabolism was performed.

Results

Mean age was 66.2 years, BMI 26.9kg/m², subcutaneous adipose tissue 2.5cm and visceral adipose tissue 8.0cm. All measures of obesity were positively associated with indicators of glycaemia and inversely associated with indicators of insulin sensitivity. Associations were of equivalent magnitude except for subcutaneous adipose tissue and the visceral/subcutaneous adipose tissue ratio, which showed weaker associations. One standard deviation difference in BMI, visceral adipose tissue, waist circumference, waist/height ratio and body fat percentage corresponded approximately to 0.2mmol/l higher fasting glucose, 0.7mmol/l higher 2-hr glucose, 0.06-0.1% higher HbA1c, 30 % lower HOMA index of insulin sensitivity, 20% lower Gutt’s index of insulin sensitivity, and 100 unit higher Stumvoll’s index of beta-cell function. After adjustment for waist circumference visceral adipose tissue was still significantly associated with glucose intolerance and insulin resistance, whereas there was a trend towards inverse or no associations with subcutaneous adipose tissue. After adjustment, a 1cm increase in visceral adipose tissue was associated with ~5% lower insulin sensitivity (p≤0.0004) and ~0.18mmol/l higher 2-hr glucose (p≤0.001).

Conclusion

Visceral and subcutaneous adipose tissue assessed by ultrasonography are significantly associated with glucose metabolism, even after adjustment for other measures of obesity.     

Comparative study on human and bovine AT-SC isolation methods

Mammalian adipose tissue derived stem cells (AT-SC) have a tremendous potential in regenerative medicine for tissue engineering and somatic nuclear transfer (SNT). The isolation methods of human and bovine adipose tissue derived stem cells are compared in this paper to determine the feasibility and optimum method of isolation. The optimum isolation method will reduce the processing time, efforts and money as isolation is the first crucial and important step in stem cells research. Human abdominal subcutaneous adipose tissue and bovine abdominal subcutaneous adipose tissue are digested in three collagenase type 1 concentration 0.075%, 0.3% and 0.6% agitated at 1 h and 2 h under 37 °C in 5% CO₂ incubator. The cultures are then morphologically characterised. Human adipose tissue stem cells are found to be best isolated using abdominal subcutaneous depot, using 0.075% collagenase type 1 agitated at 1 h under 37 °C in CO₂ incubator. While bovine adipose tissue derived stem cells are best isolated using abdominal subcutaneous depot, using 0.6% collagenase type 1 agitated at 2 h under 37 °C in CO₂ incubator.     

Regulation of pancreatic islet-cell plasma membrane Ca2+ + Mg2+-ATPase by Calmodulin

The carboxyl group reagent dicyclohexylcarbodiimide inhibits the electrogenic entry of Cl^? and NO₃^? into rat liver mitochondria at alkaline pH. The inhibition is time dependent and 50% inhibition is obtained by the addition of 3–4 nmol DCCD/mg protein. The blockage of the pH-dependent anion-conducting pore appears to be unrelated to the other known actions of DCCD on rat liver mitochondria but seems similar to its effect on the uncoupling protein of brown adipose tissue.     

Hydrogen sulfide from adipose tissue is a novel insulin resistance regulator

Recent data suggested that endogenous hydrogen sulfide (H₂S) contributes to the pathogenesis of diabetes. Here, we identified that cystathionine gamma lyase (CSE) was expressed in adipose tissue in rats and endogenously generated H₂S. The CSE/H₂S system exists in both rat adipocytes and pre-adipocytes. This system was up-regulated with aging, although a high level of glucose down-regulated the system in a concentration- and time-dependent manner. H₂S inhibited the basal and insulin-stimulated glucose uptake of mature adipocytes, whereas administration of CSE inhibitors enhanced the glucose uptake of adipocytes. The PI3K but not K_ATP channel pathway is involved in the inhibitory effect of H₂S on glucose uptake. Finally, in fructose-induced diabetes in rats, we confirmed the up-regulated CSE/H₂S system in adipose tissue, which was negatively correlated with glucose uptake in this tissue. Our findings suggest that H₂S might be a novel insulin resistance regulator.     

Ectopic recombination in the central and peripheral nervous system by aP2/FABP4-Cre mice: Implications for metabolism research

aP2-Cre mice have amply been used to generate conditional adipose selective inactivation of important signaling molecules. We show that the efficiency of Cre mediated recombination in adipocytes and adipose selectivity is not always guaranteed. In particular, Cre activity was found in ganglia of the peripheral nervous system (PNS), in adrenal medulla and in neurons throughout the central nervous system (CNS). Because these tissues have an important impact on adipose tissue, care should be taken when using aP2-Cre mice to define the role of the targeted genes in adipose tissue function.