Interaction of epoxyeicosatrienoic acids and adipocyte fatty acid-binding protein in the modulation of cardiomyocyte contractility.

BACKGROUND: Adipocyte fatty acid-binding protein (FABP4) is a member of a highly conserved family of cytosolic proteins that bind with high affinity to hydrophobic ligands, such as saturated and unsaturated long-chain fatty acids and eicosanoids. Recent evidence has supported a novel role for FABP4 in linking obesity with metabolic and cardiovascular disorders. In this context, we identified FABP4 as a main bioactive factor released from human adipose tissue that directly suppresses heart contraction in vitro. As FABP4 is known to be a transport protein, it cannot be excluded that lipid ligands are involved in the cardiodepressant effect as well, acting in an additional and/or synergistic way. OBJECTIVE: We investigated a possible involvement of lipid ligands in the negative inotropic effect of adipocyte factors in vitro. RESULTS: We verified that blocking the CYP epoxygenase pathway in adipocytes attenuates the inhibitory effect of adipocyte-conditioned medium (AM) on isolated adult rat cardiomyocytes, thus suggesting the participation of epoxyeicosatrienoic acids (EETs) in the cardiodepressant activity. Analysis of AM for EETs revealed the presence of 5,6-, 8,9-, 11,12- and 14,15-EET, whereas 5,6-EET represented about 45% of the total EET concentration in AM. Incubation of isolated cardiomyocytes with EETs in similar concentrations as found in AM showed that 5,6-EET directly suppresses cardiomyocyte contractility. Furthermore, after addition of 5,6-EET to FABP4, the negative inotropic effect of FABP4 was strongly potentiated in a concentration-dependent manner. CONCLUSIONS: These data suggest that adipocytes release 5,6-EET and FABP4 into the extracellular medium and that the interaction of these factors modulates cardiac function. Therefore elevated levels of FABP4 and 5,6-EET in obese patients may contribute to the development of heart dysfunction in these subjects.

Changes in morphology and function of adrenal cortex in mice fed a high-fat diet.

BACKGROUND/OBJECTIVES: Obesity is a major risk factor for the development of type 2 diabetes and other debilitating diseases. Obesity and diabetes are intimately linked with altered levels of adrenal steroids. Elevated levels of these hormones induce insulin resistance and cause cardiovascular diseases. The mechanisms underlying obesity-related alterations in adrenal steroids are still not well understood. Here, we investigated how diet-induced obesity affects the morphology and function of the mouse adrenal cortex. METHODS: We fed animals either a high-fat diet (HFD) or a normal diet (60% kcal from fat or 10% kcal from fat, respectively) for 18 weeks. We then assessed various aspects of adrenal gland morphology and function, as well as basal plasma concentrations of steroid hormones and ACTH. RESULTS: We show that adrenal glands of mice fed a HFD release more corticosterone and aldosterone, resulting in higher plasma levels. This increase is driven by adrenal cortical hyperplasia, and by increased expression of multiple genes involved in steroidogenesis. We demonstrate that diet-induced obesity elevates Sonic hedgehog signaling in Gli1-positive progenitors, which populate the adrenal capsule and give rise to the steroidogenic cells of the adrenal cortex. Feeding animals with a HFD depletes Gli1-positive progenitors, as the adrenal cortex expands. CONCLUSIONS: This work provides insight into how diet-induced obesity changes the biology of the adrenal gland. The association of these changes with increased Shh signaling suggests possible therapeutic strategies for obesity-related steroid hormone dysfunction.

Calcium-dependent release of adipocyte fatty acid binding protein from human adipocytes.

BACKGROUND: Fatty acid binding protein 4 (FABP4) is a predominantly cytosolic protein of the adipocytes, but also abundantly present in human plasma; its plasma concentrations were linked to obesity and metabolic syndrome. Recent studies have suggested a direct extracellular effect of FABP4 in the regulation of glucose metabolism and heart function independently of its effect as a carrier protein. Interestingly, FABP4 has no secretory signal sequence; hence, the mechanisms how FABP4 is released from adipocytes are unclear. METHODS AND RESULTS: In this study we investigated the mechanisms for FABP4 secretion from human adipocytes by using isolated primary pre-adipocytes (PAs) and the human adipocyte cell strain Simpson-Golabi-Behmel syndrome. In undifferentiated PAs, FABP4 expression was barely detectable and increased continuously during differentiation. The increase in FABP4 mRNA expression was accompanied by high levels of FABP4 secretion. In differentiated human adipocytes, FABP4 secretion was not abolished by blocking the Golgi-dependent secretory pathway in vitro, supporting a non-classical secretion mechanism for FABP4. However, raising intracellular Ca(2+) levels enhanced FABP4 secretion in a concentration-dependent manner. CONCLUSION: This study shows that FABP4 is actively released from human adipocytes in vitro via a non-classical, calcium-dependent mechanism.

Metagenomic sequencing of the human gut microbiome before and after bariatric surgery in obese patients with type 2 diabetes: correlation with inflammatory and metabolic parameters.

Roux-en-Y gastric bypass (RYGB) has become a prominent therapeutic option for long-term treatment of morbid obesity and type 2 diabetes mellitus (T2D). Cross talk and pathogenetic consequences of RYGB-induced profound effects on metabolism and gut microbiome are poorly understood. The aim of the present study therefore was to characterize intra-individual changes of gut microbial composition before and 3 months after RYGB by metagenomic sequencing in morbidly obese patients (body mass index (BMI)>40 kg m(-)(2)) with T2D. Subsequently, metagenomic data were correlated with clinical indices. Based on gene relative abundance profile, 1061 species, 729 genera, 44 phyla and 5127 KO (KEGG Orthology) were identified. Despite high diversity, bacteria could mostly be assigned to seven bacterial divisions. The overall metagenomic RYGB-induced shift was characterized by a reduction of Firmicutes and Bacteroidetes and an increase of Proteobacteria. Twenty-two microbial species and 11 genera were significantly altered by RYGB. Using principal component analysis, highly correlated species were assembled into two common components. Component 1 consisted of species that were mainly associated with BMI and C-reactive protein. This component was characterized by increased numbers of Proteobacterium Enterobacter cancerogenus and decreased Firmicutes Faecalibacterium prausnitzii and Coprococcus comes. Functional analysis of carbohydrate metabolism by KO revealed significant effects in 13 KOs assigned to phosphotransferase system. Spearmen's Rank correlation indicated an association of 10 species with plasma total- or low-density lipoprotein cholesterol, and 5 species with triglycerides. F. prausnitzii was directly correlated to fasting blood glucose. This is the first clinical demonstration of a profound and specific intra-individual modification of gut microbial composition by full metagenomic sequencing. A clear correlation exists of microbiome composition and gene function with an improvement in metabolic and inflammatory parameters. This will allow to develop new diagnostic and therapeutic strategies based on metagenomic sequencing of the human gut microbiome.

Adipocyte-derived factors suppress heart contraction.

BACKGROUND: Obesity is strongly associated with cardiovascular diseases including systemic hypertension, coronary artery disease and heart failure. Despite several investigations the pathophysiological mechanisms involved remain unclear. We have previously shown that adipose tissue exerts a highly potent activity with an acute depressant effect on cardiomyocytes, thus suggesting direct involvement of adipose tissue in the development of heart dysfunction. OBJECTIVE AND DESIGN: This study investigates the effects of adipocyte factors obtained from subcutaneous adipose tissue on the whole cardiac function by using isolated perfused rat hearts in a Langendorff mode. We recorded changes in coronary flow, developed isovolumetric left ventricular pressure, contraction rate and relaxation rate. RESULTS: We observed a significant decrease in heart contractility parameters as well as in coronary flow within a few seconds of incubation with adipocyte factors. The cardiodepressant effects could not be blocked by the nonselective cyclooxygenase-inhibitor indomethacin. Human adipocytes release tumor necrosis factor-α, interleukin-6 (IL-6) and IL-1ß into extracellular medium. These cytokines were tested for their potential effect but were, however, not responsible for the cardiodepressant effect observed. CONCLUSION: These data indicate that human adipocytes secrete factors with a strong acute depressant effect on cardiac force generation and coronary flow due to contraction of the coronary vessels, thus suggesting a direct role of adipose tissue in the pathogenesis of cardiac dysfunction.

Smooth-muscle contraction without smooth-muscle myosin.

Here we have used gene-targeting to eliminate expression of smooth-muscle myosin heavy chain. Elimination of this gene does not affect expression of non-muscle myosin heavy chain, and knockout individuals typically survive for three days. Prolonged activation, by KCl depolarisation, of intact bladder preparations from wild-type neonatal mice produces an initial transient state (phase 1) of high force generation and maximal shortening velocity, which is followed by a sustained state (phase 2) characterized by low force generation and maximal shortening velocity. Similar preparations from knockout neonatal mice do not undergo phase 1, but exhibit a normal phase 2. We propose that, in neonatal smooth muscle phase 1 is generated by recruitment of smooth-muscle myosin heavy chain, whereas phase 2 can be generated by activation of non-muscle myosin heavy chain. We conclude that phase 1 becomes indispensable for survival and normal growth soon after birth, particularly for functions such as homeostasis and circulation.

Expression and function of endocannabinoid receptors in the human adrenal cortex.

Endogenous cannabinoids are important signaling molecules in neuroendocrine control of homeostatic and reproductive functions including stress response and energy metabolism. The hypothalamic paraventricular and supraoptic nuclei have been shown to release endocannabinoids, which act as retrograde messengers to modulate the synaptic release of glutamate during stress response. This study endeavors to elucidate possible interaction of the endocannabinoid system with the regulation of adrenocortical function at the adrenal level. Human adrenocortical NCI-H295R cells and normal human adrenal glands were used to study the possible effects of anandamide and cannabinoid receptor 1 (CB1) antagonist SR141716A on aldosterone and cortisol secretion. Our data indicate the expression of CB1 in human adrenal cortex and adrenocortical NCI-H295R cells; CB2 was not expressed. Furthermore, anandamide inhibited basal release and stimulated release of adrenocortical steroids (corticosterone and aldosterone); this effect was reversed by CB1 antagonist (SR141716A). Therefore, the endocannabinoid system at the level of the adrenal, can directly influence adrenocortical steroidogenesis.

Evidence for hemispheric specialization in the marmoset (Callithrix penicillata) based on lateralization of behavioral/neurochemical correlations.

A correlative study between behavioral, neurochemical and hormonal measures was conducted on male black tufted-ear marmoset monkeys (Callithrix penicillata). Behavioral analysis was performed in order to examine the effects of confrontation with a natural predator (taxidermized oncilla cat, Felis tigrina). The subjects were subjected to four trials without predator, six confrontation trials with predator present, and four trials with the predator removed. Handedness was analyzed by the frequency with which they performed scratching, grooming and hanging behaviors with the left or right hands. The animals' brains were subjected to ex vivo neurochemical analysis of several structures from both hemispheres. The content of monoamines, acetycholine and metabolites were analyzed by HPLC-ED. Plasma levels of cortisol and adrenocorticotrophic hormone (ACTH) were analyzed by chemoluminescence immunoassay. Testosterone plasma concentration was determined by radioimmunoassay. Higher levels of dopamine and acetylcholine were detected in the right caudate/putamen, in comparison to the left. For the remaining areas, similar levels were observed in both hemispheres. A hand preference between and within the behaviors scored was not detected. However, correlative analyses revealed complex interactions between the behavioral and neurochemical measures, particularly in the left hemisphere. Lateralized correlations were found in relation to brain site, type of behavior, neurochemical parameter and treatment condition, thus providing evidence for functional brain asymmetries in this species. Interhemispheric comparisons of neurochemical/behavioral correlations appear to be a promising approach towards delineating hemispheric specialization of functions in this, and perhaps, other species.

Adipocyte fatty acid-binding protein levels are associated with left ventricular diastolic dysfunction in morbidly obese subjects.

OBJECTIVES: This study aimed to examine the association of adipocyte fatty acid-binding protein (FABP4) levels with left ventricular diastolic dysfunction (LVDD) in obese subjects with varying degrees of the metabolic syndrome (MetS). METHODS: Fifty morbidly obese subjects with LVDD were selected at random and matched by age (±5 years) and sex with 50 morbidly obese with normal left ventricular (LV) function. In addition, 24 healthy lean subjects were included as controls. RESULTS: Median FABP4 levels (interquartile range) in obese subjects with LVDD were significantly higher (42 ng ml(-1) (32-53)) than in obese with normal LV function (24 ng ml(-1) (36-43), P=0.036), and in normal weight controls (13 ng ml(-1) (10-20), P<0.0001). Increasing FABP4 tertiles were significantly associated with parameters of LVDD, the number of LVDD components, physical performance and epicardial fat thickness. In multivariate regression analysis adjusting for age, sex and adiposity, FABP4 levels remained significantly associated with parameters of diastolic function. The association of FABP4 levels with LVDD was mainly observed in subjects with metabolic complications, but not in metabolically healthy obese. CONCLUSIONS: FABP4 levels are significantly associated with LVDD in obese subjects, when the MetS is present. Thus, FABP4 may be a link between obesity and cardiometabolic disorders.

Lipoprotein apheresis reduces adipocyte fatty acid-binding protein serum levels.

BACKGROUND AND METHODS: Adipocyte fatty acid-binding protein (FABP4) is a member of the intracellular lipid-binding protein family highly expressed in adipocytes and macrophages. Recent studies indicate a key role for circulating FABP4 in the pathogenesis of atherosclerosis and type 2 diabetes. We described an additional role for FABP4 in the development of cardiac dysfunction in obesity. Therefore, FABP4 seems to be a target in the prevention and treatment of metabolic and cardiovascular disorders in obesity with high potential for future therapeutic applications. However, a safe pharmacological therapy is not yet available. Lipoprotein apheresis is an established therapy for severe and otherwise untreatable hypercholesterolemia which increases life expectancy in patients at high-risk for cardiovascular events. We therefore investigated the acute effect of lipoprotein apheresis on FABP4 serum levels in 64 high-risk patients (25 women, 39 men) under regular apheresis treatment. RESULTS: FABP4 levels were significantly reduced by 23.2 ± 1.8% by apheresis treatment. Although women had higher FABP4 levels than men (53.5 ± 8.3 ng/ml vs. 30.7 ± 4.3 ng/ml), reduction rate after lipoprotein apheresis was similar in both genders. Among the apheresis methods investigated, immunoadsorption of lipoproteins was most effective in lowering circulating FABP4. CONCLUSION: These data suggest that the reduction of FABP4 serum levels may contribute to the preventive effect of lipoprotein apheresis on cardiovascular events.

Human adipocytes induce an ERK1/2 MAP kinases-mediated upregulation of steroidogenic acute regulatory protein (StAR) and an angiotensin II-sensitization in human adrenocortical cells.

OBJECTIVES: Hypertension is a major complication of overweight with frequently elevated aldosterone levels in obese patients. Our previous work suggests a direct stimulation of adrenal aldosterone secretion by adipocytes. Owing to aldosterone's important role in maintaining blood pressure homeostasis, its regulation in obesity is of major importance. One objective was to determine the signaling mechanisms involved in adipocyte-induced aldosterone secretion. In addition to a direct stimulation, a sensitization toward angiotensin II (AngII) might be involved. The second objective was to determine a possible adipokines-induced sensitization of human adrenocortical cells to AngII. DESIGN: Human subcutaneous adipocytes and adrenocortical cells, and the adrenocortical cell line NCI-H295R were used. Adrenocortical cells were screened for signal transduction protein expression and phosphorylation. Subsequently, steroidogenic acute regulatory protein (StAR), cAMP response element-binding protein (CREB), cAMP and phosphorylated extracellular regulated kinase were analyzed by Western blot, enzyme-linked immunosorbent assay, quantitative PCR, reporter gene assay and confocal microscopy to investigate their role in adipocyte-mediated aldosterone secretion. RESULTS: AngII-mediated aldosterone secretion was largely increased by preincubating H295R cells with adipocyte secretory products. StAR mRNA and StAR protein were upregulated in a time-dependent way. This steroidogenic effect was independent of the cAMP-protein kinase A (PKA) pathway as cellular cAMP was unaltered and inhibition of PKA by H89 failed to reduce aldosterone secretion. However, CREB reporter gene activity was moderately elevated. Upregulation of StAR was accompanied by ERK1/2 MAP kinase activation and nuclear translocation of the kinases. Inhibition of MAP kinase by UO126 abolished adipokine-stimulated aldosterone secretion from primary human adrenocortical and H295R cells, and inhibited StAR gene activity. Adipokines stimulated steroidogenesis also in primary human adrenocortical cells, supporting a role in human physiology and/or pathology. CONCLUSIONS: Adipokines induce aldosterone secretion from human adrenocortical cells and sensitization of the cells to stimulation by AngII, possibly mediated via ERK1/2-dependent upregulation of StAR activity. This stimulation of aldosterone secretion could be one link between overweight and inappropriately elevated aldosterone levels.

Adipocyte-derived products induce the transcription of the StAR promoter and stimulate aldosterone and cortisol secretion from adrenocortical cells through the Wnt-signaling pathway.

CONTEXT: Obesity is associated with hypersecretion of cortisol and aldosterone and a high prevalence of arterial hypertension. At the cellular level, a direct effect of adipocytes on the expression of the steroidogenic acute regulatory (StAR) protein, a regulator of cortisol and aldosterone synthesis, and on aldosterone and cortisol secretion has been shown. However, the molecular mechanisms mediating this effect are not known. OBJECTIVE: Wnt-signaling molecules are secreted by adipocytes and regulate the activity of SF-1, a key transcription factor in adrenal steroidogenesis. Therefore, we investigated whether adipocytes stimulate adrenal steroidogenesis through the activation of Wnt-signaling. RESULTS: Using immunohistochemistry, we detected the expression of frizzled and beta-catenin in the adult human adrenal cortex. Transient transfection of a Wnt-dependent reporter-gene into adrenal NCI-H295R cells showed an induction of Wnt-mediated transcription to 308% after treatment with human fat cell-conditioned medium (FCCM). This finding was paralleled by an induction of StAR promoter activity (420%) by FCCM. The induction of StAR promoter activity by FCCM was inhibited by 49% when Wnt-signaling was blocked by the soluble Wnt-antagonist secreted Frizzled-Related-Protein-1 (sFRP-1). Overexpression of a constitutively active mutant of beta-catenin induced the transcription of the StAR promoter (440%). beta-Catenin and FCCM induced SF-1-mediated transcription at a SF-1-driven reporter gene (420 and 402%, respectively). Furthermore, the secretion of aldosterone and cortisol by NCI-H295R cells induced by FCCM was significantly inhibited by the Wnt-antagonist sFRP-1. CONCLUSION: These data indicate that the Wnt-signaling pathway is one of the mechanisms mediating the effects of fat cells on adrenal StAR transcription and aldosterone and cortisol secretion.

Metabolic syndrome and the endocrine stress system.

Obesity constitutes one of the most serious public health problems, with rapidly increasing prevalence in western societies. Consequently, metabolic syndrome, a condition strongly associated with obesity, has become an epidemic problem. Recent studies have implicated chronic alterations to the stress system as playing a major role in the metabolic syndrome's pathophysiology. This brief review discusses the role of stress and hypothalamic-pituitary-adrenal axis dysfunction in the development of metabolic syndrome as well as new insights into the crosstalk between adipose tissue and endocrine stress system.

Increased aldosterone/renin quotient in obese hypertensive women: a novel role for low-density lipoproteins?

Obesity, especially visceral obesity, is strongly associated with arterial hypertension. Indeed, obesity hypertension has to be considered as the most common form of essential hypertension. However, the exact nature of the relationship between obesity and increased blood pressure remains poorly understood. Involvement of renin-independent mechanisms has been suggested in adrenal stimulation of aldosterone secretion in obese patients. This investigation examined the plasma levels of renin, aldosterone, insulin, and HDL and LDL in obese hypertensive and obese normotensive women. The group of hypertensive obese women showed significantly reduced plasma levels of renin and increased aldosterone/renin quotient (ARQ) compared to obese normotensive women. Plasma aldosterone levels were not significantly different between hypertensive and normotensive obese women. In addition, plasma levels of LDL-cholesterol in the hypertensive obese group were significantly increased in comparison to the obese normotensive group. No differences were observed in HDL-cholesterol or total cholesterol/HDL-C ratios between the two groups. We therefore examined the effect of LDL on angiotensin II-stimulated aldosterone release from human adrenocortical H295R cells. Treatment of adrenocortical cells with LDL led to a sensitization towards stimulation by angiotensin II, dramatically increasing angiotensin II-induced aldosterone production, so the increased aldosterone/renin ratio observed in the hypertensive group may be due to the enhanced LDL levels in these patients and/or other adipocyte-derived mineralocorticoid-stimulating factors.

Mechanisms of obesity-related hypertension.

Obesity has become an epidemic problem in western societies, contributing to metabolic diseases, hypertension and cardiovascular disease. Although the importance of obesity as a cause of hypertension is well established, the molecular basis of the relationship between obesity and increased blood pressure remains poorly understood. This brief review examines the association between obesity and hypertension along with the mechanisms proposed to explain this association, while presenting evidence of a direct causal effect of adipose tissue in the development of hypertension through the involvement of the adrenal cortex.

Human adipocytes secrete mineralocorticoid-releasing factors.

Obesity has become an epidemic problem in western societies, contributing to metabolic diseases, hypertension, and cardiovascular disease. Overweight and obesity are frequently associated with increased plasma levels of aldosterone. Recent evidence suggests that human fat is a highly active endocrine tissue. Therefore, we tested the hypothesis that adipocyte secretory products directly stimulate adrenocortical aldosterone secretion. Secretory products from isolated human adipocytes strongly stimulated steroidogenesis in human adrenocortical cells (NCI-H295R) with a predominant effect on mineralocorticoid secretion. Aldosterone secretion increased 7-fold during 24 h of incubation. This stimulation was comparable to maximal stimulation of these cells with forskolin (2 x 10(-5) M). On the molecular level, there was a 10-fold increase in the expression of steroid acute regulatory peptide mRNA. This effect was independent of adipose angiotensin II as revealed by the stimulatory effect of fat cell-conditioned medium even in the presence of the angiotensin type 1 receptor antagonist, valsartan. None of the recently defined adipocytokines accounted for the effect. Mineralocorticoid-stimulating activity was heat sensitive and could be blunted by heating fat cell-conditioned medium to 99 degrees C. Centrifugal filtration based on molecular mass revealed at least two releasing factors: a heat sensitive fraction (molecular mass >50 kDa) representing 60% of total activity, and an inactive fraction (molecular mass <50 kDa). However, the recovery rate increased to 92% when combining these two fractions, indicating the interaction of at least two factors. In conclusion, human adipocytes secrete potent mineralocorticoid-releasing factors, suggesting a direct link between obesity and hypertension.