Heart Fat Infiltration In Subjects With and Without Coronary Artery Disease.

CONTEXT: Fat may accumulate around the heart in epicardial adipose tissue or inside the heart as lipid droplets (LDs). OBJECTIVE: To compare myocardial steatosis between subjects with and without coronary artery disease (CAD and non-CAD) and to identify which cells contain LDs. DESIGN: Body mass index, waist circumference, glucose, insulin, homeostasis model assessment index, leptin, adiponectin, and high-sensitivity C-reactive protein were evaluated in CAD and non-CAD subjects. Biopsies were collected from right atrial myocardium. Immunohistochemistry for perilipin (PLIN) 1 and 2 was used to characterize LDs and their localization in adipocytes or myocardial cells, respectively. Cardiomyocytes apoptosis and hypoxia inducible factor 1 alpha were obtained in a subgroup of subjects. SETTING: The study took place in a hospital. PATIENTS: Male subjects consecutively undergoing elective cardiac surgery either for coronary bypass grafting (CAD, n = 23) or for valve replacement (non-CAD, n = 18). MAIN OUTCOMES AND MEASURES: The study was designed to compare myocardial steatosis between subjects with and without coronary artery disease. RESULTS: PLIN1 and PLIN2 resulted significantly higher in CAD than in non-CAD subjects, as did apoptosis. PLIN1 was positively associated with circulating leptin, high-sensitivity C-reactive protein, and apoptosis, and negatively with adiponectin. PLIN2 was positively associated with body mass index, waist circumference, and leptin and negatively with adiponectin. After taking into account the absence/presence of hypertension, diabetes, and CAD/non-CAD, adiponectin was negatively associated with PLIN1 (r(2) = 0.532); waist circumference and adiponectin were associated with PLIN2 (r(2) = 0.399). CONCLUSIONS: Myocardial steatosis is greater in CAD than non-CAD subjects, depending on both metabolically active adipocytes interspersed among cardiomyocytes and higher fat deposition inside cardiomyocytes; serum adiponectin and waist circumference are independent predictors of myocardial steatosis.

LPS response pattern of inflammatory adipokines in an in vitro 3T3-L1 murine adipocyte model.

OBJECTIVE: In vitro 3T3-L1 mouse cells represent a reliable model to investigate the inflammatory phenotype of adipocytes activated by bacteria-derived lipopolysaccharide (LPS). In this study we have evaluated the differential expression of adipokines in response to increasing doses of LPS and various incubation times. METHODS: 3T3-L1 mouse adipocytes were treated with E. coli LPS (from 0 to 10 µg/ml) for a time course ranging from 4 to 24 h, 4 h each. A time point at 2 h was also included to highlight early activation by LPS. mRNA expression by RT-PCR on cell lysates and ELISA assays on cell culture supernatants were performed. RESULTS: Cells activated by increasing doses of LPS upregulated TNF-α expression in the first 2 h, but this expression slowed down within 6-8 h, while IL-6 expression was increasing. This reduction was also observed for CXCL12/SDF1α. Unlike IL-10, IL-6 expression was constantly upregulated by prolonging incubation with LPS. TNF-α and CXCL12 gene expression occurred early in the time-course and exhibited a second increase following the first 4-6 h of incubation with LPS. Optimal expression of most adipokines needed 6-8 h of a prolonged treatment with LPS at 37 °C. The chemokines MIP-1α/CCL3 and MIP-1ß/CCL4 were maximally expressed within the first 8 h, then significantly reduced in the following times. IL-10 expression was upregulated by low doses of LPS and downregulated by prolonging time with the bacterial endotoxin. ELISA analysis of released products generally confirmed the result from gene expression experiments. CONCLUSION: These data, while assessing previously reported results, highlighted new evidence about the time-dependency in LPS-mediated adipokine production, thus contributing to the comprehension of the inflammatory response of adipocyte.

Additive effects of microRNAs and transcription factors on CCL2 production in human white adipose tissue.

Adipose tissue inflammation is present in insulin-resistant conditions. We recently proposed a network of microRNAs (miRNAs) and transcription factors (TFs) regulating the production of the proinflammatory chemokine (C-C motif) ligand-2 (CCL2) in adipose tissue. We presently extended and further validated this network and investigated if the circuits controlling CCL2 can interact in human adipocytes and macrophages. The updated subnetwork predicted that miR-126/-193b/-92a control CCL2 production by several TFs, including v-ets erythroblastosis virus E26 oncogene homolog 1 (avian) (ETS1), MYC-associated factor X (MAX), and specificity protein 12 (SP1). This was confirmed in human adipocytes by the observation that gene silencing of ETS1, MAX, or SP1 attenuated CCL2 production. Combined gene silencing of ETS1 and MAX resulted in an additive reduction in CCL2 production. Moreover, overexpression of miR-126/-193b/-92a in different pairwise combinations reduced CCL2 secretion more efficiently than either miRNA alone. However, although effects on CCL2 secretion by co-overexpression of miR-92a/-193b and miR-92a/-126 were additive in adipocytes, the combination of miR-126/-193b was primarily additive in macrophages. Signals for miR-92a and -193b converged on the nuclear factor-κB pathway. In conclusion, TF and miRNA-mediated regulation of CCL2 production is additive and partly relayed by cell-specific networks in human adipose tissue that may be important for the development of insulin resistance/type 2 diabetes.

Inflammatory profile in subcutaneous and epicardial adipose tissue in men with and without diabetes.

In recent years, evidence has emerged indicating that insulin resistance and diabetes mellitus type 2 are associated with inflammation of adipose tissue (AT). Interest has been focused on epicardial AT (EAT) because of its possible involvement with atherosclerosis and cardiovascular diseases. The aim of this study was to characterize adipocyte size and inflammatory profile in subcutaneous (SAT) and EAT among subjects with or without diabetes. Biopsies were collected from SAT and EAT in 34 men undergoing elective cardiac surgery. Weight, height, body mass index, waist circumference, as well as serum levels of glucose, insulin, lipids, adiponectin, and leptin were determined in all subjects. Adiponectin, MCP-1, and CD68 mRNA levels present within cells from AT biopsies were determined by real-time polymerase chain reaction. Adipocyte size was determined by optic microscopy and morphometry. Regarding the experimental group as a whole, gene-expression levels within EAT were significantly lower for adiponectin and higher, albeit not significantly, for MCP-1, when compared with that of SAT. In addition, adipocytes in EAT were significantly smaller than those in SAT. Subjects with diabetes showed lower adiponectin gene-expression levels in both SAT and EAT when compared with subjects without diabetes. By contrast, MCP-1 and CD68 gene-expression levels were higher in both tissue types of diabetic subjects. Adipocyte size in EAT was significantly larger in diabetic subjects than in nondiabetic subjects. Our data revealed a predominantly inflammatory profile in both SAT and EAT in subjects with diabetes in comparison with those without diabetes.

Myosteatosis and myofibrosis: relationship with aging, inflammation and insulin resistance.

The mechanisms impairing muscle quality and leading to myofibrosis (MF) and myosteatosis (MS) are incompletely known. In biopsies of paraspinous muscle (PM) of 16 elderly men undergoing elective vertebral surgery, we histologically determined the area of MF and MS expressed as muscle quality index (MQI), in order to investigate the relation between them, as well as the main predictors of muscle quality. Total PM area and intermuscular adipose tissue (IMAT) were evaluated by MRI and body composition by DXA. Circulating fasting glucose, insulin, hs-CRP, leptin, adiponectin and IL-6 were measured and HOMA index calculated. Quantification of gene expression in PM and in subcutaneous adipose tissue (SAT) overlying the muscle was performed by rt-PCR. The degree of MS and MF was significantly and positively related to each other and positively associated with BMI, waist, FM and FM% as well as with IMAT. The area of PM was negatively related with MF even after adjustment for weight. Leptin was positively associated with MF and MS, whereas hs-CRP to MF. In backward regression analyses, larger waist and smaller PM area explained 90% of MF variance, whereas leptin about 80% of MS variance. IL-6 expression in SAT was significantly higher in participants with higher MQI values. In PM biopsies we found significantly higher expression of SOCS-3 and a trend toward higher expression of myostatin with greater degrees of MQI. MS and MF are related phenomena that concur to alter muscle quality and both should be considered in further studies on the evolution of sarcopenia.

Sarcopenia, cachexia and congestive heart failure in the elderly.

Skeletal muscle abnormalities and loss are frequently present in patients with mild or moderate cardiac heart failure (CHF) and may contribute to fatigue and dyspnea. These muscle abnormalities may be associated with age related body composition changes, such as sarcopenia. Muscle damage has also been observed in subjects with cardiac cahexia, a serious CHF complication, associated with poor prognosis independently of functional disease severity, age, and measures of exercise capacity and cardiac function. Loss of muscle mass is a feature of cachexia, whereas most sarcopenic subjects are not cachectic. Individuals with no weight loss, no anorexia, and no measurable systemic inflammatory response may be sarcopenic. Patients with severe CHF show multiple marked histological abnormalities of skeletal muscle, such as muscle fiber atrophy. These abnormalities are different in sarcopenia and cachexia. The majority of mechanisms involved in sarcopenia play a role even in the determination of cachexia and they are amplified in cachexia where they may induce both muscle damage as well as other abnormalities, such as fat and weight loss, through activation of lypolisis or anorexia. To distinguish cachexia and sarcopenia in CHF patients, even if not easy, should be clinically relevant, because no specific treatment is available for cachectic patients whereas treatment options are possible for sarcopenia.

NPC1 in human white adipose tissue and obesity.

BACKGROUND: Genetic studies have implicated the NPC1 gene (Niemann Pick type C1) in susceptibility to obesity. METHODS: To assess the potential function of NPC1 in obesity, we determined its expression in abdominal white adipose tissue (WAT) in relation to obesity. NPC1 mRNA was measured by RT-qPCR in lean and obese individuals, paired samples of subcutaneous (sc) and omental (om) WAT, before and after weight loss, in isolated adipocytes and intact adipose pieces, and in primary adipocyte cultures during adipocyte differentiation. NPC1 protein was examined in isolated adipocytes. RESULTS: NPC1 mRNA was significantly increased in obese individuals in scWAT and omWAT and downregulated by weight loss. NPC1 mRNA was enriched in isolated fat cells of WAT, in scWAT versus omWAT but not modified during adipocyte differentiation. NPC1 protein mirrored expression of mRNA in lean and obese individuals. CONCLUSIONS: NPC1 is highly expressed in human WAT adipocytes with increased levels in obese. These results suggest that NPC1 may play a role in adipocyte processes underlying obesity.

Adiponectin gene expression and adipocyte diameter: a comparison between epicardial and subcutaneous adipose tissue in men.

INTRODUCTION: Interest has recently focused on epicardial fat, but little is known about epicardial adipocyte size and its relation with insulin resistance and adipokines. METHODS: Biopsies were collected from subcutaneous, epicardial-, and peritoneal fat from 21 males undergoing elective cardiac surgery either for coronary artery bypass grafting (n=11) or for valve replacement (n=10). We assessed epicardial adipocyte size, comparing it with that from subcutaneous fat and peritoneal fat. The adipocyte size was determined by using collagenase digestion of adipose tissue, separation of adipocytes by centrifugation, methylene blue staining of the nuclei, and measurement of the cell diameter. Patient's weight, height, body mass index, waist, as well as glucose, insulin, homeostatic model assessment index, adiponectin, and leptin serum levels were determined. Adiponectin mRNA levels were determined by real-time polymerase chain reaction on subcutaneous fat and epicardial fat biopsies. RESULTS: Adipocytes in epicardial fat were significantly smaller than those in subcutaneous and peritoneal fat. The adipocyte size in epicardial fat correlated positively with insulin resistance and serum leptin, and correlated negatively with serum and mRNA expression of adiponectin. Adiponectin mRNA expression in epicardial fat was significantly lower than in subcutaneous fat. Adipocyte size in epicardial fat was significantly smaller in valve-replacement patients than in coronary artery bypass graft patients. Adiponectin gene expression was lower in the latter than in the former, although not significantly. CONCLUSIONS: Adipocytes in epicardial fat are smaller than those in peritoneal and subcutaneous fat. Adipocyte size, both in epicardial and in subcutaneous fat, is positively related with insulin resistance, shows negative association with local adiponectin gene expression, and is decreased in subjects with coronary artery disease. Adiponectin gene expression is significantly lower in epicardial- than in subcutaneous fat.

Mapping of the fibroblast growth factors in human white adipose tissue.

CONTEXT: Fibroblast growth factors (FGFs) regulate the development of white adipose tissue (WAT). However, the secretion and cellular origin of individual FGFs in WAT as well as the influence of obesity are unknown. OBJECTIVE: Our objective was to map FGFs in human sc WAT, the cellular source, and association with obesity. DESIGN: Secretion, mRNA, and circulatory levels of FGFs in human abdominal sc WAT from nonobese and obese donors were examined by microarray, real-time quantitative PCR, and ELISA. The activity of FGFs in cultured human adipocytes was determined by phosphorylation assays. RESULTS: Expression of five FGFs (FGF1, FGF2, FGF7, FGF9, and FGF18) and FGF homologous factor (FHF2) was identified in WAT. Only FGF1 was released in a time-dependent manner from sc WAT, and fat cells were the major source of FGF1 secretion. FGF1 expression increased and FGF2 decreased during adipocyte differentiation. Furthermore, FGF1 was not secreted into the circulation. Although FGF1 levels were 2-fold increased in obesity, they were unaltered by weight reduction. Only FGF1 and FGF2 induced a marked concentration-dependent phosphorylation of p44/42 in cultured human adipocytes. CONCLUSIONS: Of the investigated FGFs, only FGF1 is secreted from sc WAT and predominantly so from the adipocyte fraction. The activity in adipocyte cultures and lack of secretion into the circulation suggest that FGF1 acts as an auto- or paracrine factor. FGF1 levels are increased in obesity but unaffected by weight reduction, suggesting a primary defect in obese individuals. In conclusion, FGF1 may play a superior role among the FGFs in sc WAT and obesity development.