ICH3, a selective alpha7 nicotinic acetylcholine receptor agonist, modulates adipocyte inflammation associated with obesity.

PURPOSE: The alpha7 nicotinic acetylcholine receptor (α7nAChR), involved in the modulation of inflammation and insulin sensitivity, is downregulated in white adipose tissue (WAT) of obese patients. This study aims to test the ability of a selective synthetic α7nAChR agonist, the spirocyclic Δ2-isoxazoline derivative (R)-(-)-ICH3 (ICH3), to counteract acute inflammation and obesity-associated modifications in WAT. METHODS: We employed the LPS-septic shock murine model, human primary adipocytes and diet-induced obese (DIO) mice. Inflammatory factor expression was assessed by ELISA and quantitative real-time PCR. Flow cytometry was employed to define WAT inflammatory infiltrate. Insulin signaling was monitored by quantification of AKT phosphorylation. RESULTS: In the septic shock model, ICH3 revealed antipyretic action and reduced the surge of circulating cytokines. In vitro, ICH3 stimulation (10 µM) preserved viability of human adipocytes, decreased IL-6 mRNA (P < 0.05) and blunted LPS-induced peak of TNFα (P < 0.05) and IL-6 (P < 0.01). Chronic administration of ICH3 to DIO mice was associated with lower numbers of CD8+ T cells (P < 0.05) and to changed WAT expression of inflammatory factors (Hp, P < 0.05; CD301/MGL1, P < 0.01; Arg-1, P < 0.05). As compared to untreated, ICH3 DIO mice exhibited improved insulin signaling in the skeletal muscle (P < 0.01) mirrored by an improved response to glucose load (ipGTT: P < 0.05 at 120 min). CONCLUSIONS: We proved that ICH3 is an anti-inflammatory drug, able to reduce inflammatory cytokines in human adipocytes and to blunt the effects of obesity on WAT inflammatory profile, on glucose tolerance and on tissue insulin sensitivity.

Adipose tissue microbiota in humans: an open issue.

BACKGROUND: A specific 'adipose tissue' microbiota has been recently identified in mice and hypothesized in humans. The purpose of this study was to verify the presence of microbiota of human whole adipose tissue and isolated adipocytes by combining culture-dependent and independent methods. METHODS: Standard microbiological cultural techniques and 16S ribosomal RNA (16S rRNA) gene sequencing (Illumina technology) on DNA and RNA were employed to study (a) whole abdominal subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) from 14 obese and five normal-weight subjects and (b) mature adipocytes isolated from SAT and VAT after collagenase digestion or mechanical separation. To optimize the 16S rRNA gene detection, we used different DNA extraction methods (lysis with proteinase K, proteinase K+lysozyme and microbeads) and amplification procedures (semi-quantitative standard PCR and real-time quantitative PCR). RESULTS: Microbiological cultures were negative in all analyzed samples. In enzymatically isolated adipocytes, 90% of the sequenced bacterial DNA belonged to Clostridium histolyticum, the bacterium from which the collagenase enzyme was isolated. Bacterial 16S rRNA gene was not detected from DNA and RNA of whole SAT and VAT, as well as of mechanically isolated mature adipocytes, even after blocking with a specific primer the nonspecific amplification of human mitochondrial 12S rRNA. CONCLUSIONS: Our results do not support the presence of a human adipose tissue microbiota. In addition, they emphasized the technical problems encountered when applying metagenomic studies to human tissues with very low or absent bacterial load.

Permanence of molecular features of obesity in subcutaneous adipose tissue of ex-obese subjects.

OBJECTIVE: Bariatric surgery represents a powerful tool for morbid obesity treatment. However, after stabilization of weight loss that follows surgical interventions, ex-obese patients face the problem of residual tissues removal. Actually, it is unknown whether the characteristics of this residual subcutaneous adipose tissue (SAT) are 'restored' with regard to molecular and morphological features. DESIGN: To clarify this issue, we compared the SAT gene expression profile of ex-obese patients (ExOB-SAT, mean body mass index (BMI): 27.2±1.3 kg m(-2)) with that of lean (normal weight, NW-SAT, mean BMI: 22.6±1.1 kg m(-2)), overweight (OW-SAT, BMI: 27.65±0.2 kg m(-2)) and obese patients, according to BMI classes (OB1-SAT: 30 > or = BMI < or = 34.9, OB2-SAT: 35 > or = BMI < or = 39.9, OB3-SAT: BMI > or = 40). SUBJECTS AND METHODS: A total of 58 samples of SAT were collected during surgical interventions. Gene expression levels were assessed by microarrays and significant genes were validated by RT-qPCR. Adipocyte hypertrophy, inflammatory infiltration and fibrosis were assessed by morphological techniques. RESULTS: Global gene expression in ExOB-SAT was closely related to gene expression of OB3-SAT by hierarchical clustering procedures, in spite of different BMI. Metallothioneins (MT1A and MT2A) were the key over-expressed genes in both groups. At morphologic level, adipocyte hypertrophy and inflammatory infiltration improved after weight loss in ExOB-SAT, despite a persistence of fibrosis. CONCLUSIONS: Taken together, these results demonstrate that SAT gene expression is not fully restored, even after an extensive and stable weight loss. The persistence of 'obesity molecular features' in ExOB-SAT suggests that the molecular signature of adipose tissue is not solely dependent on weight loss and may need longer time period to completely disappear.

The nicotinic acetylcholine receptor α7 in subcutaneous mature adipocytes: downregulation in human obesity and modulation by diet-induced weight loss.

BACKGROUND: It is known that cholinergic anti-inflammatory reflex regulates inflammation in peripheral tissues. Nicotinic acetylcholine receptors (nAChRs) are mediators of this anti-inflammatory pathway and also non-neuronal cells express functional nAChrs. A role for α7-subtype acetylcholine cholinergic receptor (α7nAChR) in insulin sensitivity improvement has already been shown in rodents both in vivo and in vitro. However, no data are available on α7nAChR expression in human adipocytes. OBJECTIVE: To investigate the expression and protein content of α7nAChR in human subcutaneous adipose tissue (SAT) and in isolated mature adipocytes. DESIGN: A total of 39 SAT biopsy specimens obtained from obese and normal-weight subjects were used to assess α7nAChR messenger RNA levels and to stimulate α7nAChR with a specific agonist and antagonist in vitro. Additional SATs from eight non-diabetic obese subjects were also studied, before and after a 3-month lifestyle intervention. RESULTS: α7nAChR expression was significantly lower in the SAT of obese subjects compared with that of normal-weight subjects. In mature adipocytes isolated from morbidly obese subjects (body mass index > 40 kg m(-2)), α7nAChR expression was 75% lower compared with adipocytes from normal-weight subjects. In adipocytes of obese subjects, α7nAChR was downregulated also at protein level. In eight non-diabetic obese subjects, a lifestyle intervention (3 months of diet and physical activity) induced a significant weight loss and an increase in α7nAChR SAT expression. In vitro stimulation of adipocytes with the specific α7nAChR agonist PNU282987 induced a significant anti-inflammatory effect. Furthermore, a similar downregulation of the inflammatory profile, associated with a significant increase in α7nAChR protein level, was observed after genistein stimulation. CONCLUSIONS: These results provide evidence that α7nAChR expression levels are significantly decreased in obese subjects, and that this receptor modulates inflammatory gene expression in human adipocytes. The upregulation of α7nAChR by genistein stimulation opens new insights for the management of low-grade inflammation linked to human obesity.

Transcriptomics applied to obesity and caloric restriction.

Caloric restriction still remains the most efficient way to promote weight loss. Deciphering the molecular basis of adaptation to energy restriction is critical for the tailoring of new therapeutic strategies. This review focuses on the recent input of gene profiling on adipose tissue in obesity pathogenesis and on the new insights on adaptations occurring during very low caloric diet (VLCD) in humans. Hypocaloric diets improve a wide range of metabolic parameters including lipolytic efficiency, insulin sensitivity, and inflammatory profile. In the subcutaneous white adipose tissue (scWAT) the VLCD induced a decrease in the mRNA levels for the antilipolytic alpha2-adrenergic receptor associated with changes in catecholamine-induced adipocyte lipolytic capacity. The improvement in insulin sensitivity was not associated with a change in subcutaneous adipose tissue adiponectin gene expression or in its plasma level, suggesting that adiponectin is not involved in the regulation of VLCD-induced improvement of insulin sensitivity and that there is a small contribution of subcutaneous adipose tissue to plasma adiponectin levels. Pangenomic microarray studies in human scWAT revealed that a panel of inflammatory markers and acute phase reactants were over expressed in obese compared to lean subjects. Caloric restriction improved the inflammatory profile of obese subjects through a decrease of pro-inflammatory factors and an increase of anti-inflammatory molecules. These genes were mostly expressed in the stroma vascular fraction of the adipose tissue. Specific cell-type isolation and immunohistochemistry demonstrated that monocyte/macrophage lineage cells were responsible for the expression of both mRNA and protein inflammatory markers. The acute phase proteins serum amyloid A was highly expressed in mature adipocytes from obese subjects. Caloric restriction decreased both serum amyloid mRNA and circulating levels. Obesity now clearly appears as chronic low-grade inflammation state. Modulation of the inflammatory pathways may represent new therapeutic targets for the treatment of obesity-related complications.

Leptin and UCP1 genes are reciprocally regulated in brown adipose tissue.

In a previous work we showed that only unilocular brown adipocytes express leptin. In order to investigate the relationship between leptin gene expression, brown adipocyte activity (UCP1) and morphology, we studied brown adipose tissues of mice (C57BL, female, 7 weeks old) acclimated at different temperatures (19 degrees C and 28 degrees C). Northern blot analysis revealed higher leptin and lower UCP1 mRNA levels in mice exposed to 28 degrees C than in the group acclimated at 19 degrees C. Also protein expression (immunohistochemistry) differed in the two groups: at 28 degrees C brown adipocytes were positive for leptin and only weakly positive for UCP1, while at 19 degrees C they were leptin-negative and UCP1-positive. In the former group the morphology was mainly unilocular. Our data suggest that in brown adipocytes of warm-acclimated mice leptin expression is closely related to their hypoactive functional stage, as evidenced by their low level of UCP1 synthesis and the morphological rearrangement of the lipid content (unilocularity).

Expression and distribution of heme oxygenase-1 and -2 in rat brown adipose tissue: the modulatory role of the noradrenergic system.

To investigate whether brown adipose tissue (BAT) expresses the inducible (HO-1) and the constitutive (HO-2) isoform of heme oxygenase, reverse transcriptase-polymerase chain reaction, Western blotting and immunohistochemistry were performed on interscapular BAT (IBAT) from rats acclimated at environmental temperature or exposed to cold. Both HO isoforms were detected in rat IBAT. They were immunolocalized in the cytoplasm and/or nuclei of brown adipocytes, in parenchymal capillaries, arteries and in some veins and nerves. Whereas cold exposure did not affect HO-2 expression, it significantly increased the expression of HO-1, both at mRNA (about 3-fold) and protein (about 2-fold) levels, reflecting the increased expression of HO-1 in the brown adipocytes and endothelial cells of parenchymal capillaries. Western blotting of cytosolic and nuclear protein extracts from cultured differentiated brown adipocytes showed that HO-1 and HO-2 are indeed localized in the cytosol and nuclei of brown adipocytes, and that noradrenaline stimulation significantly increased their amount in cytosol but not in the nuclear fraction.

Chemokines control fat accumulation and leptin secretion by cultured human adipocytes.

In addition to their role in inflammation, cytokines like TNFalpha have been reported to regulate the adipose tissue function suggesting a role for these soluble mediators in metabolism. However, it is not known whether adipocytes have the capacity to secrete chemokines, a group of low molecular weight inflammatory mediators that control leukocyte migration into tissues. Here we show that primary cultures of human preadipocytes constitutively produce three chemokines, interleukin-8 (IL-8), macrophage inflammatory protein-1alpha (MIP-1alpha) and monocyte chemotactic protein-1 (MCP-1), while their level of expression is low in mature adipocytes. Upon TNFalpha treatment, the expression of all the three chemokines is upregulated in adipocytes differentiated in vitro. In addition, we describe the presence of seven different chemokine receptors, mainly in mature adipocytes, both in vitro and in human fat tissue sections. Prolonged stimulation of cultured human adipocytes with exogenous chemokines leads to a decrease in lipid content in association with the downregulation of PPARgamma mRNA expression. Moreover, chemokines positively control the secretion of leptin, a hormone that regulates appetite, by a post-transcriptional mechanism. These findings reveal a new role for chemokines in the regulation of adipose tissue and suggest a novel therapeutic basis for the treatment of obesity, diabetes and cachexia.

Adiposity signals, genetic and body weight regulation in humans.

Numerous signals convey information about body fat status from the periphery to the brain areas that control energy homeostasis so that, throughout life, body weight remains nearly stable. These signals mainly originate, either from the adipose tissue, like leptin and to a lesser extent interleukin 6, or from the pancreas, like insulin and amylin. These factors circulate in proportion to body fat mass and they are referred to as "adiposity signals". It is well established, at least for leptin and insulin, that they enter the brain from the plasma where they induce/repress a network of important neuropeptide regulators of energy intake and expenditure. Beside these endocrine signals, a growing amount of literature show data relative to adipocyte-derived molecules, most of them belonging to the cytokine family, like IL6, TNFalpha, IL8, IL10 whose secretion also correlates with body fat mass and that may locally regulate fat mass expansion. Others, like adiponectin, are negatively correlated with body fat mass. These "adiposity molecules" have already been involved in insulin resistance associated with obesity and inflammatory process. They may participate to a complex inter organ dialogue. In this review, we will synthesize data relative to the role played by insulin, leptin and amylin, either alone or through a cross talk, in "energy level sensing" at the brain level. Furthermore, we will develop how "adiposity molecules" through their paracrin and/or autocrin action may contribute to maintain fat mass expansion, therefore representing new adiposity molecules per se. Lastly, since any distortion in the metabolic circuitry of energy homeostasis is susceptible to lead to a pathological status like obesity, the impact of known genetic polymorphisms in genes encoding the adiposity signals will be discussed.

Is obesity an inflammatory illness? Role of low-grade inflammation and macrophage infiltration in human white adipose tissue.

There are at least two scientific evidences of human obesity as a chronic inflammatory illness: first, the well-described moderate increase of inflammatory factors in the circulation in obese subjects, and second, the recent identification of macrophage cells infiltrating the white adipose tissue (WAT). These observations led to a revision of the physiopathology of obesity and its co-morbidities. It has been suggested that the 'low-grade' inflammatory state associates with metabolic and cardiovascular complications of obesity. Weight loss is able to improve this inflammatory state by both significantly decreasing circulating inflammatory molecules and macrophage cell infiltration in WAT depots. However, the mechanisms of WAT macrophage recruitment into the adipose tissue and their role in obesity complications have not been defined. This review aims to point out the knowledge on inflammatory cytokines associated with obesity and focuses on macrophage infiltration in human WAT, discussing their recruitment and role. The interactions of macrophages with adipocytes will certainly be the subject of intense investigations in the future.

Microarray profiling of human white adipose tissue after exogenous leptin injection.

BACKGROUND: Leptin is a secreted adipocyte hormone that plays a key role in the regulation of body weight homeostasis. The leptin effect on human white adipose tissue (WAT) is still debated. OBJECTIVE: The aim of this study was to assess whether the administration of polyethylene glycol-leptin (PEG-OB) in a single supraphysiological dose has transcriptional effects on genes of WAT and to identify its target genes and functional pathways in WAT. MATERIALS AND METHODS: Blood samples and WAT biopsies were obtained from 10 healthy nonobese men before treatment and 72 h after the PEG-OB injection, leading to an approximate 809-fold increase in circulating leptin. The WAT gene expression profile before and after the PEG-OB injection was compared using pangenomic microarrays. Functional gene annotations based on the gene ontology of the PEG-OB regulated genes were performed using both an 'in house' automated procedure and GenMAPP (Gene Microarray Pathway Profiler), designed for viewing and analyzing gene expression data in the context of biological pathways. RESULTS: Statistical analysis of microarray data revealed that PEG-OB had a major down-regulated effect on WAT gene expression, as we obtained 1,822 and 100 down- and up-regulated genes, respectively. Microarray data were validated using reverse transcription quantitative PCR. Functional gene annotations of PEG-OB regulated genes revealed that the functional class related to immunity and inflammation was among the most mobilized PEG-OB pathway in WAT. These genes are mainly expressed in the cell of the stroma vascular fraction in comparison with adipocytes. CONCLUSION: Our observations support the hypothesis that leptin could act on WAT, particularly on genes related to inflammation and immunity, which may suggest a novel leptin target pathway in human WAT.

Serum amyloid A: production by human white adipocyte and regulation by obesity and nutrition.

AIMS/HYPOTHESIS: The acute-phase proteins, serum amyloid As (SAA), are precursors of amyloid A, involved in the pathogenesis of AA amyloidosis. This work started with the characterisation of systemic AA amyloidosis concurrent with SAA overexpression in the subcutaneous white adipose tissue (sWAT) of an obese patient with a leptin receptor deficiency. In the present study a series of histopathological, cellular and gene expression studies was performed to assess the importance of SAA in common obesity and its possible production by mature adipocytes. MATERIALS AND METHODS: Gene expression profiling was performed in the sWAT of two extremely obese patients with a leptin receptor deficiency. Levels of the mRNAs of the different SAA isoforms were quantified in sWAT cellular fractions from lean subjects and from obese subjects before and after a very-low-calorie diet. These values were subsequently compared with serum levels of SAA in these individuals. In addition, histopathological analyses of sWAT were performed in lean and obese subjects. RESULTS: In sWAT, the expression of SAA is more than 20-fold higher in mature adipocytes than in the cells of the stroma vascular fraction (p<0.01). Levels of SAA mRNA expression and circulating levels of the protein are sixfold (p<0.001) and 3.5-fold (p<0.01) higher in obese subjects than in lean subjects, respectively. In lean subjects, 5% of adipocytes are immunoreactive for SAA, whereas the corresponding value is greater than 20% in obese subjects. Caloric restriction results in decreases of 45-75% in levels of the transcripts for the SAA isoforms and in circulating levels of the protein. CONCLUSIONS/INTERPRETATION: The results of the present study indicate that SAA is expressed by sWAT, and its production at this site is regulated by nutritional status. If amyloidosis is seen in the context of obesity, it is possible that production of SAA by adipocytes could be a contributory factor.

Leptin modulates extracellular matrix molecules and metalloproteinases: possible implications for trophoblast invasion.

Leptin is a circulating hormone which plays an important role in the regulation of energy balance, haemopoiesis and reproduction. Leptin and its receptor (leptin-R) are localized in human placental tissue but their function is not known. In this study we have investigated the expression of leptin and leptin-R in the human placenta with particular attention to extravillous cytotrophoblastic cell islands and cell columns which play a pivotal role in trophoblast invasion and placental growth. We demonstrate that leptin-R immunoreactivity shows a strong expression in the distal extravillous cytotrophoblastic cells of cell columns invading the basal plate, whereas leptin expression is homogeneously expressed in all the cellular components of cell columns. Since the invasive ability of the distally located extravillous cytotrophoblast of cell columns is known to be regulated by a variety of proteases and some extracellular matrix molecules, we tested the influence of leptin on the in-vitro production of matrix metalloproteinase (MMP)-2, MMP-9 and fetal fibronectin (fFN) by cytotrophoblastic cells. We demonstrate that leptin increases, in a dose-dependent manner, the secretion of immunoreactive MMP-2 and fFN and enhances the activity of MMP-9 in cultured cytotrophoblastic cells. Our results suggest that leptin and leptin-R could have a role in the invasive processes of the extravillous cytotrophoblastic cells by modulating the expression of MMPs. In addition, these results provide a foundation for studying pathological conditions characterized by insufficient or excessive trophoblast invasion.