ICAM-1 expression in adipose tissue: effects of diet-induced obesity in mice

Adipocyte death has been reported in both obese humans and rodents. However, its role in metabolic disorders, including insulin resistance, hepatic steatosis, and inflammation associated with obesity has not been studied. We now show using real-time reverse transcription-PCR arrays that adipose tissue of obese mice display a pro-apoptotic phenotype. Moreover, caspase activation and adipocyte apoptosis were markedly increased in adipose tissue from both mice with diet-induced obesity and obese humans. These changes were associated with activation of both the extrinsic, death receptor-mediated, and intrinsic, mitochondrial-mediated pathways of apoptosis. Genetic inactivation of Bid, a key pro-apoptotic molecule that serves as a link between these two cell death pathways, significantly reduced caspase activation, adipocyte apoptosis, prevented adipose tissue macrophage infiltration, and protected against the development of systemic insulin resistance and hepatic steatosis independent of body weight. These data strongly suggest that adipocyte apoptosis is a key initial event that contributes to macrophage infiltration into adipose tissue, insulin resistance, and hepatic steatosis associated with obesity in both mice and humans. Inhibition of adipocyte apoptosis may be a new therapeutic strategy for the treatment of obesity-associated metabolic complications.

Obesity-induced metabolic dysfunctions increase the risk for vascular diseases, including type II diabetes and stroke. Managing obesity is of interest to address the worldwide health problem; however, the role of genetic variability in human obesity development and specific targets for obesity-related metabolic disease have not been thoroughly studied. A SNP in the brain-derived neurotropic factor (BDNF) gene that results in the substitution of a valine with a methionine at codon 66 (Val66Met) occurs with a high frequency in humans. This study addressed the effect of genetic variability in developing obesity and the efficacy of the inhibition of cluster of differentiation 36 (CD36), a multifunctional receptor implicated in obesity and insulin resistance, in WT mice and mice with the BDNF Val66Met variant. CD36 inhibition by salvionolic acid B (SAB) in diet-induced obese WT mice reduced visceral fat accumulation and improved insulin resistance. The benefit of SAB was abrogated in CD36 knockout mice, showing the specificity of SAB. In addition, mice with the Val66Met variant in both alleles (BDNFM/M) fed a high-fat diet exhibited extreme obesity with increased CD36 gene and protein levels in macrophages. Chronic SAB treatment in BDNFM/M mice significantly decreased visceral fat accumulation and improved insulin resistance. Notably, the effect of SAB was greater in the extremely obese BDNFM/M mice compared with the WT mice. The study demonstrated a link between BDNF Val66Met and elevated CD36 expression and suggested that CD36 inhibition may be a potential strategy to improve metabolic dysfunctions and to normalize risk factors for vascular diseases in the obese population.

BACKGROUND AND OBJECTIVES: Hypercholesterolemia triggered by high-fat and high-fructose diets increases Reactive Oxygen Species production, causing oxidative stress and increasing the expression of Intercellular Adhesion Molecule-1 (ICAM-1) in endothelial cells as a form of inflammatory response. High-fiber diet could restrict lipolysis in adipose tissue, decreasing the secretion of pro-inflammatory cytokines while indirectly decreasing the expression of ICAM-1. METHODS: The research analyzed beneficial effects of high-fiber diet divided into five groups: normal (N); hypercholesterolemia (HC); HC + 1.04 g fiber/rat/day (HFD1); HC + 2.07 g fiber/rat/day (HFD2) and HC + 3.11 g fiber/rat/day (HFD3) for 6-weeks intervention on the level and expression of ICAM-1 in rats induced by high-fat and high-fructose diets. RESULTS: The administration of a high-fiber diet reduced the levels of ICAM-1 plasma hypercholesterolemia rats (HFD1, HFD2 and HFD3) when compared with the hypercholesterolemia group ( p < 0.001) without fiber administration. In addition, the administration of a high-fiber diet also decreased ICAM-1 gene expression in rat adipose tissue when compared with the hypercholesterolemia group ( p < 0.05). The decreased plasma levels of ICAM-1 were not correlated with the reduced ICAM-1 gene expression in rat adipose tissue after administration of a high-fiber diet. CONCLUSIONS: The high-fiber diet administration was able to decrease expression and level of ICAM-1 in hypercholesterolemia rats induced by high-fat and high-fructose diets.

Tissue‐specific differences in inflammatory infiltrate and matrix metalloproteinase expression in adipose tissue and liver of mice with diet‐induced obesity

Cidea and Cidec play an important role in regulating triglyceride storage in liver and adipose tissue. It is not known if the Cidea and Cidec genes respond to a high fat diet (HFD) or exercise training, two interventions that alter lipid storage. The purpose of the present study was to determine the effect of a HFD and voluntary wheel running (WR) on Cidea and Cidec mRNA and protein expression in adipose tissue and liver of mice. A HFD promoted a significant increase in Cidea and Cidec mRNA levels in adipose tissue and liver. The increase in Cidea and Cidec mRNAs in adipose tissue and liver in response to a HFD was prevented by WR. Similar to the changes in Cidea mRNA, Cidea protein levels in adipose tissue significantly increased in response to a HFD, a process that was, again, prevented by WR. However, in adipose tissue the changes in Cidec mRNA did not correspond to the changes in Cidec protein levels, as a HFD decreased Cidec protein abundance. Interestingly, in adipose tissue Cidea protein expression was significantly related to body weight (R=.725), epididymal adipose tissue (EWAT) mass (R=.475) and insulin resistance (R=.706), whereas Cidec protein expression was inversely related to body weight (R=-.787), EWAT mass (R=-.706), and insulin resistance (R=-.679). Similar to adipose tissue, Cidea protein expression in liver was significantly related to body weight (R=.660), EWAT mass (R=.468), and insulin resistance (R=.599); however, unlike adipose tissue, Cidec protein levels in liver were not related to body weight or EWAT mass and only moderately associated with insulin resistance (R=-.422, P=0.051). Overall, our findings indicate that Cidea is highly associated with adiposity and insulin resistance, whereas Cidec is related to insulin sensitivity. The present study suggests that Cide proteins might play an important functional role in the development of obesity, hepatic steatosis, as well as the pathogenesis of type 2 diabetes.

During the first few hours after heart transplantation, the occurrence of graft failure is unpredictable and devastating. An explosive cascade of inflammatory events within the reperfused graft vasculature is likely to be mediated, at least in part, by the local expression of the leukocyte adhesion receptor intercellular adhesion molecule-1 (ICAM-1, CD54). Furthermore, although proinflammatory cytokines such as interleukin-1 (IL-1) are known to autoinduce their own (and ICAM-1) expression in vitro, there are no data to identify their functional in vivo cross talk in the setting of isograft transplantation. To determine the role of ICAM-1 in primary graft failure, we used an isogeneic vascularized model of heterotopic cardiac transplantation. ICAM-1 mRNA and protein increased in grafts during the early posttransplant period and were predominantly localized in the endothelium. The functional significance of this was established using donor hearts obtained from either ICAM-1-deficient (ICAM-1 -/-) or control (ICAM-1 +/+) mice. ICAM-1 +/+ grafts exhibited increased neutrophil infiltration, reduced left ventricular compliance, and poorer survival than did ICAM-1 -/- grafts. Increased ICAM-1 expression was not limited to ICAM-1 +/+ grafts but also occurred in unmanipulated recipient organs located remote from the site of surgery (but only after transplantation of ICAM-1 +/+, not ICAM-1 -/-, cardiac grafts). This expression of ICAM-1 in remote organs appeared to be triggered by IL-1alpha released from the graft, because (1) in situ hybridization revealed increased IL-1 mRNA within cells of the reperfused graft, including myocytes and endothelial cells; (2) ICAM-1 expression in remote organs coincided with a significant increase in serum levels of IL-1alpha after transplantation of ICAM-1 +/+ grafts; both remote organ ICAM-1 expression and IL-1alpha levels were blunted by implantation of ICAM-1 -/- grafts; and (3) remote organ ICAM-1 expression and neutrophil infiltration and IL-1 levels could be blocked by the administration of an IL-1 receptor antagonist. These data demonstrate an apparent positive-feedback loop in which local ICAM-1 and IL-1 expression leads to a mutual amplification of each other's expression within the reperfused graft, promulgating inflammatory events that are likely to be an important cause of primary cardiac graft failure. Because IL-1 receptor blockade reduces the IL-1-mediated autoinduction of IL-1, reduces the expression of ICAM-1 in both the graft and remote organs, and improves graft survival, it may provide a new and effective strategy to prevent the occurrence of primary cardiac graft failure.

BackgroundFABP4 is predominantly expressed in adipose tissue, and its circulating levels are linked with obesity and a poor atherogenic profile.ObjectiveIn patients with a wide BMI range, we analyze FABP4 expression in adipose and hepatic tissues in the settings of obesity and insulin resistance. Associations between FABP4 expression in adipose tissue and the FABP4 plasma level as well as the main adipogenic and lipolytic genes expressed in adipose tissue were also analyzed.MethodsThe expression of several lipogenic, lipolytic, PPAR family and FABP family genes was analyzed by real time PCR. FABP4 protein expression in total adipose tissues and its fractions were determined by western blot.ResultsIn obesity FABP4 expression was down-regulated (at both mRNA and protein levels), with its levels mainly predicted by ATGL and inversely by the HOMA-IR index. The BMI appeared as the only determinant of the FABP4 variation in both adipose tissue depots. FABP4 plasma levels showed a significant progressive increase according to BMI but no association was detected between FABP4 circulating levels and SAT or VAT FABP4 gene expression. The gene expression of FABP1, FABP4 and FABP5 in hepatic tissue was significantly higher in tissue from the obese IR patients compared to the non-IR group.ConclusionThe inverse pattern in FABP4 expression between adipose and hepatic tissue observed in morbid obese patients, regarding the IR context, suggests that both tissues may act in a balanced manner. These differences may help us to understand the discrepancies between circulating plasma levels and adipose tissue expression in obesity.

BackgroundSupra-nutritional doses of curcumin, derived from the spice Curcuma longa, have been proposed as a potential treatment of inflammation and metabolic disorders related to obesity. The aim of the present study was to test whether Curcuma longa extract rich in curcumin and associated with white pepper (Curcuma-P®), at doses compatible with human use, could modulate systemic inflammation in diet-induced obese mice. We questioned the potential relevance of changes in adiposity and gut microbiota in the effect of Curcuma-P® in obesity.Methodology/Principal FindingsMice were fed either a control diet (CT), a high fat (HF) diet or a HF diet containing Curcuma longa extract (0.1 % of curcumin in the HF diet) associated with white pepper (0.01 %) for four weeks. Curcumin has been usually combined with white pepper, which contain piperine, in order to improve its bioavailability. This combination did not significantly modify body weight gain, glycemia, insulinemia, serum lipids and intestinal inflammatory markers. Tetrahydrocurcumin, but not curcumin accumulated in the subcutaneous adipose tissue. Importantly, the co-supplementation in curcuma extract and white pepper decreased HF-induced pro-inflammatory cytokines expression in the subcutaneous adipose tissue, an effect independent of adiposity, immune cells recruitment, angiogenesis, or modulation of gut bacteria controlling inflammation.Conclusions/SignificanceThese findings support that nutritional doses of Curcuma longa, associated with white pepper, is able to decrease inflammatory cytokines expression in the adipose tissue and this effect could be rather linked to a direct effect of bioactive metabolites reaching the adipose tissue, than from changes in the gut microbiota composition.

Early growth response 1 (EGR-1) works as a master regulator that plays a key role in triggering inflammation-induced tissue injury after ischemia and reperfusion. This study tested the hypothesis that postconditioning (Postcon) or anti-inflammatory compound, curcumin, ameliorates inflammatory responses and further reduces infarct size by normalizing EGR-1 expression during reperfusion. In the control group, male Sprague-Dawley rats were subjected to 30-min ischemia and 180-min reperfusion. Postcon with four cycles of 10-s/10-s reperfusion/ischemia was applied at the onset of reperfusion. Curcumin (150 mg/kg per day) was fed 5 days before ischemia. Relative to the control, Postcon reduced expression of EGR-1 mRNA and protein, as further identified by less EGR-1 immunoreactivity in myocardial nuclei and microvessels during reperfusion. Along with EGR-1 downregulation, levels of plasma and myocardial tumor necrosis factor α and interleukin 6 (IL-6) were significantly decreased. Upregulated P-selectin and intercellular adhesion molecule 1 mRNA and protein as well as their immunoreactivity at area at risk myocardium were significantly attenuated. Neutrophil extravasation identified by myeloperoxidase immunohistochemical staining was inhibited. Infarct size, determined with triphenyltetrazolium chloride staining, was smaller in the Postcon group than that in the control. The protection achieved with pretreatment with curcumin was comparable to the benefits gained by Postcon in all end points measured. In H9C2 rat cardiomyoblast cell line, EGR-1 siRNA downregulated hydrogen peroxide-induced EGR-1 mRNA expression and subsequently reduced tumor necrosis factor α mRNA level. These results suggest that EGR-1 seems to play a critical role in myocardial reperfusion injury because downregulation of EGR-1 either by Postcon or the use of pharmacological intervention reduces infarct size, most likely through an inhibition of inflammation-mediated processes.

Intercellular adhesion molecule-1 (ICAM-1) mediates two important functional aspects of tumor biology, namely enhancement of tumor metastasis and mediation of host defense mechanisms such as lymphocyte-mediated tumor cytotoxicity. Since ICAM-1 is expressed by most renal cell carcinomas (RCC), the regulation of ICAM-1 expression is important in understanding the biological behavior of RCC. We report an investigation on ICAM-1 expression and molecular regulation by cytokines and protein kinase C activator on RCC cell lines. Of the various cytokines, tumor necrosis factor alpha (TNF alpha), interferon-gamma (IFN gamma), and phorbol myristate acetate (PMA) strongly upregulated ICAM-1 protein expression on RCC. The kinetics of ICAM-1 message induction was studied by Northern analysis of total RNA extracted from RCC and normal kidney proximal tubular (NKPT) cells. Time course studies showed that ICAM-1 mRNA was upregulated by INF gamma, TNF alpha, and PMA, plateaued after 2 h, and remained increased for up to 24 h. Although ICAM-1 mRNA in NKPT cells was upregulated by these cytokines, their messages returned to basal levels after 24 h. ICAM-1 mRNA stability assays showed that both unstimulated and stimulated RCC cells had very stable ICAM-1 mRNA up to 24 h. In order to investigate whether increased gene transcription contributes to ICAM-1 upregulation, RCC cells were treated with TNF alpha, IFN gamma, or PMA with or without simultaneous addition of actinomycin D. ICAM-1 message induction-blocking studies suggested that primary upregulation of ICAM-1 mRNA may be caused by transcriptional upregulation. These results suggest that long-lasting ICAM-1 message upregulation in response to cytokines or PMA may be due to transcriptional upregulation in the early phase and stabilization of ICAM-1 message in the later phase (after 4 h). These observations suggest that RCC may lack the normal downregulatory mechanisms which control ICAM-1 expression and may explain the high frequency of ICAM-1 expression observed on primary human RCC.

In obesity, decreases in adiponectin and increases in proinflammatory adipokines are associated with heart disease. Because adipocytes express mineralocorticoid receptor (MR) and MR blockade reduces cardiovascular inflammation and injury, we tested the hypothesis that MR blockade reduces inflammation and expression of proinflammatory cytokines in adipose tissue and increases adiponectin expression in adipose tissue and hearts of obese mice. We determined the effect of MR blockade (eplerenone, 100 mg/kg per day for 16 weeks) on gene expression in retroperitoneal adipose and heart tissue from obese, diabetic db/db mice (n=8) compared with untreated obese, diabetic db/db mice (n=10) and lean, nondiabetic db/+ littermates (n=11). Expression of tumor necrosis factor-alpha, monocyte chemoattractant protein-1, plasminogen activator inhibitor type 1, and macrophage protein CD68 increased, and expression of adiponectin and peroxisome proliferator-activated receptor-gamma decreased in retroperitoneal adipose tissue from obese versus lean mice. In addition, adiponectin expression in heart was reduced in obese versus lean mice. MR blockade prevented these obesity-related changes in gene expression. Furthermore, treatment of undifferentiated preadipocytes with aldosterone (10(-8) mol/L for 24 hours) increased mRNA levels of tumor necrosis factor-alpha and monocyte chemoattractant protein-1 and reduced mRNA and protein levels of peroxisome proliferator-activated receptor-gamma and adiponectin, supporting a direct aldosterone effect on gene expression. MR blockade reduced expression of proinflammatory and prothrombotic factors in adipose tissue and increased expression of adiponectin in heart and adipose tissue of obese, diabetic mice. These effects on adiponectin and adipokine gene expression may represent a novel mechanism for the cardioprotective effects of MR blockade.

Recently irisin (encoded by Fndc5 gene) has been reported to stimulate browning and uncoupling protein 1 expression in sc adipose tissue of mice. The objective of the study was to investigate FNDC5 gene expression in human muscle and adipose tissue and circulating irisin according to obesity, insulin sensitivity, and type 2 diabetes. Adipose tissue FNDC5 gene expression and circulating irisin (ELISA) were analyzed in 2 different cohorts (n = 125 and n = 76); muscle FNDC5 expression was also evaluated in a subcohort of 34 subjects. In vitro studies in human preadipocytes and adipocytes and in induced browning of 3T3-L1 cells (by means of retinoblastoma 1 silencing) were also performed. In both sc and visceral adipose tissue, FNDC5 gene expression decreased significantly in association with obesity and was positively associated with brown adipose tissue markers, lipogenic, insulin pathway-related, mitochondrial, and alternative macrophage gene markers and negatively associated with LEP, TNFα, and FSP27 (a known repressor of brown genes). Circulating irisin and irisin levels in adipose tissue were significantly associated with FNDC5 gene expression in adipose tissue. In muscle, the FNDC5 gene was 200-fold more expressed than in adipose tissue, and its expression was associated with body mass index, PGC1α, and other mitochondrial genes. In obese participants, FNDC5 gene expression in muscle was significantly decreased in association with type 2 diabetes. Interestingly, muscle FNDC5 gene expression was significantly associated with FNDC5 and UCP1 gene expression in visceral adipose tissue. In men, circulating irisin levels were negatively associated with obesity and insulin resistance. Irisin was secreted from human adipocytes into the media, and the induction of browning in 3T3-L1 cells led to increased secreted irisin levels. Decreased circulating irisin concentration and FNDC5 gene expression in adipose tissue and muscle from obese and type 2 diabetic subjects suggests a loss of brown-like characteristics and a potential target for therapy.

Obesity is associated with a variety of disorders and is a significant health problem in developed countries. One factor controlling the level of adiposity is the differentiation of cells into adipocytes. Adipocyte differentiation requires expression of peroxisome proliferator-activated receptor γ (PPARγ), which is activated by ligands to regulate expression of genes involved in adipocyte differentiation. Although 15-deoxy-Δ(12,14)-prostaglandin (PG) J(2) (15d-PGJ(2)) has long been known to be a potent activator of PPARγ, the importance of its synthesis in adipose tissue in vivo is not clear. The current study utilized mice deficient in cyclooxygenase-2 (COX-2) to examine the role of COX-2-derived PGs as in vivo modulators of adiposity. As compared with strain- and age-matched wild-type controls, the genetic deficiency of COX-2 resulted in a significant reduction in total body weight and percent body fat. Although there were no significant differences in food consumption between groups, COX-2-deficient mice showed increased metabolic activity. Epididymal adipose tissue from wild-type mice produced a significantly greater level of 15d-PGJ(2), as compared with adipose tissue isolated from mice deficient in COX-2. Furthermore, production of the precursor required for 15d-PGJ(2) formation, PGD(2), was also significantly reduced in COX-2-deficient adipose tissue. The expression of markers for differentiated adipocytes was significantly reduced in adipose tissue from COX-2-deficient mice, whereas preadipocyte marker expression was increased. Macrophage-dependent inflammation was also significantly reduced in adipose tissue of COX-2-deficient mice. These findings suggest that reduced adiposity in COX-2-deficient mice results from attenuated PPARγ ligand production and adipocyte differentiation.

To examine the effect of a high-fat diet on gene expression in adipose tissues and to determine induction kinetics of adipose monocyte chemoattractant protein-1 and -3 (MCP-1 and MCP-3) in diet-induced obesity (DIO) and the effect of a lack of MCP-1 signaling on DIO susceptibility and macrophage recruitment into adipose tissue. Obese and lean adipose tissues were profiled for expression changes. The time-course of MCP-1 and MCP-3 expression was examined by reverse transcriptase-polymerase chain reaction. Plasma MCP-1 levels were determined by enzyme-linked immunosorbent assay (ELISA). Chemokine receptor-2 (CCR2) knockout mice were placed on the high-fat diet to determine DIO susceptibility. Macrophage infiltration in adipose tissue was examined by immunohistochemistry with F4/80 antibody. DIO elevated adipose expression of many inflammatory genes, including MCP-1 and MCP-3. Adipose MCP-1 and MCP-3 mRNA levels increased within 7 days of starting a high-fat diet, with elevation of plasma MCP-1 detected after 4 weeks on the diet. The induction of MCP-1 and MCP-3 expression preceded that of tumor necrosis factor-alpha. The elevated plasma MCP-1 concentration in obese mice was partially reversed by treatment with AM251. No change in DIO susceptibility and macrophage accumulation in adipose tissue were observed in CCR2 knockout mice, which lack the MCP-1 receptor CCR2. A high-fat diet elevated adipose expression of inflammatory genes, including early induction of MCP-1 and MCP-3, supporting the view that obese adipose tissues contribute to systemic inflammation. However, despite increased MCP-1 in obesity, disruption of MCP-1 signaling did not confer resistance to DIO in mice or reduce adipose tissue macrophage infiltration.

Estrogen receptors (ERs) are expressed in adipose tissue and skeletal muscle, with potential implications for glucose metabolism and insulin signaling. Previous studies examining the role of ERs in glucose metabolism have primarily used knockout mouse models of ERα and ERβ, and it is unknown whether ER expression is altered in response to an obesity-inducing high-fat diet (HFD). The purpose of the current study was to determine whether modulation of glucose metabolism in response to a HFD in intact and ovariectomized (OVX) female rats is associated with alterations in ER expression. Our results demonstrate that a 6-wk HFD (60% calories from fat) in female rats induces whole body glucose intolerance with tissue-specific effects isolated to the adipose tissue, and no observed differences in insulin-stimulated glucose uptake, GLUT4, or ERα protein expression levels in skeletal muscle. In chow-fed rats, OVX resulted in decreased ERα with a trend toward decreased GLUT4 expression in adipose tissue. Sham-treated and OVX rats fed a HFD demonstrated a decrease in ERα and GLUT4 in adipose tissue. The HFD also increased activation of stress kinases (c-jun NH₂-terminal kinase and inhibitor of κB kinase β) in the sham-treated rats and decreased expression of the protective heat shock protein 72 (HSP72) in both sham-treated and OVX rats. Our findings suggest that decreased glucose metabolism and increased inflammation in adipose tissue with a HFD in female rats could stem from a significant decrease in ERα expression.