Nitric oxide promotes differentiation of rat white preadipocytes in culture

The putative role of nitric oxide (NO) in modulating adipogenesis was investigated in cultured preadipocytes derived from rat white adipose tissue. The NO releasing reagent, hydroxylamine (HA), and nitric oxide synthase (NOS) substrate L-arginine (Arg) had no influence on cell replication. However, both HA and Arg exhibited significant induction on differentiation, as evidenced by increased lipoprotein lipase (LPL) and glycerol-3-phosphate dehydrogenase (GPDH) activities, as well as accelerated triacylglycerol (TG) accumulation. These observations suggested a positive role of NO in modulating adipogenesis. Preadipocytes were found to produce NO, and a approximately 50% increase over basal level was observed on the first 2 days of differentiation. Deprivation of endogenous NOS activity by a non-selective NOS inhibitor, N(G)-monomethyl-L-arginine (NMMA), partially abrogated the differentiation process, implicating a role for endogenous NO to stimulate preadipocyte differentiation. Both NOS isoforms, eNOS and iNOS, were detected in differentiating preadipocytes. Specific iNOS inhibitors (1400W and aminoguanidine) had little influence on NO production and differentiation, suggesting that eNOS rather than iNOS may be the major isoform involved in modulating adipogenesis.     

Expression of endothelial NO synthase, inducible NO synthase, and estrogen receptors alpha and beta in placental tissue of normal, preeclamptic, and intrauterine growth-restricted pregnancies.

In the physiology of placental blood circulation, nitric oxide (NO) synthases seem to play important roles, although their expression in pathological placentas and their role is still unclear. In addition, NO synthase activation seems to be related to estrogen receptor expression. Therefore, the aims of this study were to investigate the expression of estrogen receptors alpha (ERalpha), estrogen receptor beta (ER and the endothelial NO synthase (eNOS), and inducible NO synthase (iNOS) in intrauterine growth-restricted (IUGR) placentas, preeclamptic placentas, and in normal healthy control placentas. Slides of paraffin-embedded placental tissue were obtained after delivery from patients diagnosed with IUGR, preeclampsia, and normal term placentas and analyzed for eNOS, iNOS as well as ERalpha and ERbeta expression. Intensity of immunohistochemical reaction was analyzed using a semiquantitative score and statistical analysis was performed. In addition, Western blot experiments were performed for comparison of staining intensities obtained by immunohistochemistry and western blot. Expression of eNOS, iNOS, and ERbeta is significantly reduced in trophoblast cells of placentas with IUGR. However, preeclamptic placentas demonstrated a significant elevated expression intensity of these proteins compared with normal controls. A different expression of eNOS, iNOS, ERalpha, and ERbeta by human trophoblast cells seems to results in lower NO output and impaired trophoblast invasion. Results obtained in our study provide evidence that reduced expression of the investigated proteins is related to IUGR.     

Study of lactoferrin gene expression in human and mouse adipose tissue,human preadipocytes and mouse 3T3-L1 fibroblasts. Association with adipogenic and inflammatory markers

Lactoferrin is considered an epithelial protein present in different gland secretions. Administration of exogenous lactoferrin is also known to modulate adipogenesis and insulin action in human adipocytes. Here, we aimed to investigate lactoferrin gene expression (real-time polymerase chain reaction) and protein (enzyme-linked immunosorbent assay) levels in human (n=143) and mice adipose tissue samples, in adipose tissue fractions and during human preadipocyte and 3T3-L1 cell line differentiation, evaluating the effects of inducers (rosiglitazone) and disruptors (inflammatory factors) of adipocyte differentiation. Lactoferrin (LTF) gene and protein were detectable at relatively high levels in whole adipose tissue and isolated adipocytes in direct association with low-density lipoprotein-related protein 1 (LRP1, its putative receptor). Obese subjects with type 2 diabetes and increased triglycerides had the lowest levels of LTF gene expression in subcutaneous adipose tissue. Specifically, LTF gene expression was significantly increased in adipocytes, mainly from lean subjects, increasing during differentiation in parallel to adipogenic genes and gene markers of lipid droplets. The induction or disruption of adipogenesis led to concomitant changes (increase and decrease, respectively) of lactoferrin levels during adipocyte differentiation also in parallel to gene markers of adipogenesis and lipid droplet development. The administration of lactoferrin led to autopotentiated increased expression of the LTF gene. The decreased lactoferrin mRNA levels in association with obesity and diabetes were replicated in mice adipose tissue. In conclusion, this is the first observation, to our knowledge, of lactoferrin gene expression in whole adipose tissue and isolated adipocytes, increasing during adipogenesis and suggesting a possible contribution in adipose tissue physiology through LRP1.     

Troglitazone inhibits the expression of inducible nitric oxide synthase in adipocytes in vitro and in vivo study in 3T3-L1 cells and Otsuka Long-Evans Tokushima Fatty rats

The aim of this study was to determine the mechanism of troglitazone action on nitric oxide (NO) production via inducible NO synthase (iNOS) in adipocytes in vitro and in vivo. The treatment of 3T3-L1 adipocytes with the combination of lipopolysaccharide (LPS), tumor necrosis factor-alpha and interferon-gamma synergistically induced de novo iNOS expression leading to enhanced NO production. The NO production was inhibited by co-treatment with aminoguanidine or N-nitro-L-arginine methylester hydrochloride. Troglitazone inhibited the NO production in a dose dependent manner by the suppression of iNOS expression. In the 24 week-old Otsuka Long-Evans Tokushima Fatty (OLETF) rats, the mean weight and the blood glucose were 21% and 30%, respectively, higher than in their lean counterparts. The serum nitrite concentration was increased after injection of LPS (4 mg/kg, i.p.), more markedly in OLETF rats than in the lean rats. The epididymal fats from LPS-injected groups, but not the ones from the non-injected groups, expressed mRNA and protein of iNOS. Troglitazone pre-treatment blocked the LPS-induced expression of iNOS in adipose tissue and the increase in serum nitrite concentration. These results suggest that troglitazone inhibits the cytokine-induced NO production in adipocytes by blocking iNOS expression both in vitro and in vivo.     

Adipogenesis-related increase of semicarbazide-sensitive amine oxidase and monoamine oxidase in human adipocytes

A strong induction of semicarbazide-sensitive amine oxidase (SSAO) has previously been reported during murine preadipocyte lineage differentiation but it remains unknown whether this emergence also occurs during adipogenesis in man. Our aim was to compare SSAO and monoamine oxidase (MAO) expression during in vitro differentiation of human preadipocytes and in adipose and stroma-vascular fractions of human fat depots. A human preadipocyte cell strain from a patient with Simpson-Golabi-Behmel syndrome was first used to follow amine oxidase expression during in vitro differentiation. Then, human preadipocytes isolated from subcutaneous adipose tissues were cultured under conditions promoting ex vivo adipose differentiation and tested for MAO and SSAO expression. Lastly, human adipose tissue was separated into mature adipocyte and stroma-vascular fractions for analyses of MAO and SSAO at mRNA, protein and activity levels. Both SSAO and MAO were increased from undifferentiated preadipocytes to lipid-laden cells in all the models: 3T3-F442A and 3T3-L1 murine lineages, human SGBS cell strain or human preadipocytes in primary culture. In human subcutaneous adipose tissue, the adipocyte-enriched fraction exhibited seven-fold higher amine oxidase activity and contained three- to seven-fold higher levels of mRNAs encoded by MAO-A, MAO-B, AOC3 and AOC2 genes than the stroma-vascular fraction. MAO-A and AOC3 genes accounted for the majority of their respective MAO and SSAO activities in human adipose tissue. Most of the SSAO and MAO found in adipose tissue originated from mature adipocytes. Although the mechanism and role of adipogenesis-related increase in amine oxidase expression remain to be established, the resulting elevated levels of amine oxidase activities found in human adipocytes may be of potential interest for therapeutic intervention in obesity.     

Resistance to endotoxic shock in endothelial nitric-oxide synthase (eNOS) knock-out mice: a pro-inflammatory role for eNOS-derived no in vivo

The expression of inducible nitric-oxide synthase (iNOS) and subsequent "high-output" nitric oxide (NO) production underlies the systemic hypotension, inadequate tissue perfusion, and organ failure associated with septic shock. Therefore, modulators of iNOS expression and activity, both endogenous and exogenous, are important in determining the magnitude and time course of this condition. We have shown previously that NO from the constitutive endothelial NOS (eNOS) is necessary to obtain maximal iNOS expression and activity following exposure of murine macrophages to lipopolysaccharide (LPS). Thus, eNOS represents an important regulator of iNOS expression in vitro. Herein, we validate this hypothesis in vivo using a murine model of sepsis. A temporal reduction in iNOS expression and activity was observed in LPS-treated eNOS knock-out (KO) mice as compared with wild-type animals; this was reflected in a more stable hemodynamic profile in eNOS KO mice during endotoxaemia. Furthermore, in human umbilical vein endothelial cells, LPS leads to the activation of eNOS through phosphoinositide 3-kinase- and Akt/protein kinase B-dependent enzyme phosphorylation. These data indicate that the pathogenesis of sepsis is characterized by an initial eNOS activation, with the resultant NO acting as a co-stimulus for the expression of iNOS, and therefore highlight a novel pro-inflammatory role for eNOS.     

Effects of a dammarane-type saponin,ginsenoside Rd,in nicotine-induced vascular endothelial injury

BackgroundPanax notoginseng (Burk.) F.H. Chen is a traditional medicinal plant widely used to prevent and treat cardiovascular diseases. Ginsenoside Rd (GRd) is a major bioactive component of P. notoginseng, but specific effects on cardiovascular disease-related pathogenic processes are rarely studied, especially vascular endothelial injury.PurposeThis study investigated the potential protective efficacy of GRd against nicotine-induced vascular endothelial cell injury, disruption of vascular nitric oxide (NO) signaling, aberrant endothelium–monocyte adhesion, platelet aggregation, and vasoconstriction.Study design/MethodsVascular endothelial injury and functional disruption were investigated in cultured human umbilical vein endothelial cells (HUVECs) by biochemical assays for nitric oxide (NO) and angiotensin II (Ang II), immunofluorescence (IF) and western blotting for expression analyses of apoptosis- related proteins, endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), Ang II type receptor 1 (AGTR1), toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), and nuclear factor-kappa B (NF-κB). In addition, vascular protection by GRd was examined in nicotine-administered Sprague-Dawley (SD) rats by serum NO and Ang II assays, and by hematoxylin-eosin (HE) and immunostaining of aorta. We also examined effects of GRd on monocyte (THP-1 cells) adhesion assays, adenosine diphosphate (ADP)-induced platelet aggregation, and phenylephrine (PE)-induced vasoconstriction of isolated rat aortic rings.ResultsIn HUVECs, nicotine significantly suppressed NO production, enhanced Ang II production, downregulated eNOS expression, and upregulated expression levels of AGTR1, TLR4, MyD88, NF-κB, iNOS, Bax/Bcl-2 ratio, cleaved caspase-3, and cytochrome c (cyt c). All of these changes were significantly reversed by GRd. In rats, oral GRd reversed the reduction NO and enhanced Ang II production in serum induced by nicotine administration, and HE staining revealed protection of aortic endothelial cells. In addition, GRd reversed nicotine-mediated enhancement of HUVECs-monocyte adhesion, inhibited ADP-induced platelet aggregation and PE-induced vasoconstriction.ConclusionGRd may prevent nicotine-induced cardiovascular diseases by preserving normal vascular endothelial NO signaling, suppressing platelet aggregation and vasoconstriction, and by preventing endothelial cell–monocyte adhesion.     

Effect of calcitonin gene-related peptide on nitric oxide production in osteoblasts: an experimental study

The aim of this study was to investigate the in vitro effects and regulatory mechanism of CGRP (calcitonin gene-related peptide) on NO (nitric oxide) production in osteoblasts. MOB (primary human mandibular osteoblasts) and osteoblast-like cells (MG-63) were either cultured with CGRP or co-incubated with inhibitors targeting eNOS (endothelial nitric oxide synthase), iNOS (inducible nitric oxide synthase), nNOS (neuronal nitric oxide synthase) and [Ca2+]i (intracellular Ca2+). The NO concentration in cell culture supernatants was measured during the first 24 h using the Griess test; cellular NO was marked with the fluorescent marker DAF-FM, DA (3-amino, 4-aminomethyl-2',7'-difluorescein; diacetate) and measured by fluorescence microscopy from 1 to 4 h after treatment. eNOS and iNOS mRNA expression levels were measured by quantitative RT-PCR during the first 24 h after treatment. CGRP-induced NO production in the supernatants was high between 1 to 12 h, while cellular NO was highest between 1 to 2 h after treatment and returned to basal levels by 3 h. Both in MG-63 cells and MOBs, the most effective CGRP concentration was 10 nM with a peak time of 1 h. CGRP-induced NO production decreased when eNOS activity was inhibited or when voltage-dependent L-type Ca2+ channels were blocked at 4 h. CGRP was not able to induce changes in iNOS or eNOS mRNA levels and had no effect on the cytokine-induced increase of iNOS expression. Our results suggest that CGRP transiently induces NO production in osteoblasts by elevating intracellular Ca2+ to stimulate the activity of eNOS in vitro.     

Chemokine Expression in Inflamed Adipose Tissue Is Mainly Mediated by NF-κB

Immune cell infiltration of expanding adipose tissue during obesity and its role in insulin resistance has been described and involves chemokines. However, studies so far have focused on a single chemokine or its receptor (especially CCL2 and CCL5) whereas redundant functions of chemokines have been described. The objective of this work was to explore the expression of chemokines in inflamed adipose tissue in obesity. Human and mouse adipocytes were analyzed for expression of chemokines in response to inflammatory signal (TNF-α) using microarrays and gene set enrichment analysis. Gene expression was verified by qRT-PCR. Chemokine protein was determined in culture medium with ELISA. Chemokine expression was investigated in human subcutaneous adipose tissue biopsies and mechanism of chemokine expression was investigated using chemical inhibitors and cellular and animal transgenic models. Chemokine encoding genes were the most responsive genes in TNF-α treated human and mouse adipocytes. mRNA and protein of 34 chemokine genes were induced in a dose-dependent manner in the culture system. Furthermore, expression of those chemokines was elevated in human obese adipose tissue. Finally, chemokine expression was reduced by NF-κB inactivation and elevated by NF-κB activation. Our data indicate that besides CCL2 and CCL5, numerous other chemokines such as CCL19 are expressed by adipocytes under obesity-associated chronic inflammation. Their expression is regulated predominantly by NF-κB. Those chemokines could be involved in the initiation of infiltration of leukocytes into obese adipose tissue.     

Expression of the adrenomedullin gene in adipose tissue

Adrenomedullin (AM) is a potent vasodilating peptide originally isolated from human pheochromocytoma cells. This report concerns the expression and secretion of AM from adipose tissue. Northern blot analysis demonstrated marked expression of AM mRNA in mouse adipose tissue. Expression levels in adipose tissues were 2.5-3.2 times higher than in the kidney. AM mRNA level in mature adipocytes was 7.3 times higher than in the stroma-vascular fraction of adipose tissue. In mature adipocyte culture, time-dependent increase of AM peptide concentration in the culture medium was detected. AM expression was also detected in human subcutaneous adipose tissue. Adipose AM expression significantly increased in obesity mouse model, high-fat diet fed mice and ob/ob mice. These results suggest that adipose tissue, especially mature adipocytes, is major source of AM in the body, and that adipocyte-derived AM plays a pathophysiological role in obesity.     

Adipokine gene expression in dog adipose tissues and dog white adipocytes differentiated in primary culture.

Obesity and its associated disorders are increasing in companion animals, particularly in dogs. We have investigated whether genes encoding key adipokines, some of which are implicated in the pathologies linked to obesity, are expressed in canine adipose tissues. Using RT-PCR, mRNAs encoding the following adipokines were detected in dog white adipose tissue: adiponectin, leptin, angiotensinogen, plasminogen activator inhibitor-1, IL-6, haptoglobin, metallothionein-1 and 2, and nerve growth factor. The adipokine mRNAs were present in all fat depots examined. Fractionation of adipose tissue by collagenase digestion showed that each gene was expressed in mature adipocytes. The mRNA for TNFalpha was not evident in adipose tissue, but was detected in isolated adipocytes. Fibroblastic preadipocytes from gonadal white fat were differentiated into adipocytes in primary culture and adipokine expression examined before and after differentiation (days 0 and 11, respectively). Each adipokine gene expressed in dog white adipocytes was also expressed in the differentiated cells. These results demonstrate that dog white adipose tissue expresses major adipokine genes, expression being in the adipocytes. Investigation of adipokine production and function will provide insight into the mechanisms involved in obesity-related pathologies in dogs and serve as a model for the related human diseases.     

Identification of new genes involved in human adipogenesis and fat storage

Since the worldwide increase in obesity represents a growing challenge for health care systems, new approaches are needed to effectively treat obesity and its associated diseases. One prerequisite for advances in this field is the identification of genes involved in adipogenesis and/or lipid storage. To provide a systematic analysis of genes that regulate adipose tissue biology and to establish a target-oriented compound screening, we performed a high throughput siRNA screen with primary (pre)adipocytes, using a druggable siRNA library targeting 7,784 human genes. The primary screen showed that 459 genes affected adipogenesis and/or lipid accumulation after knock-down. Out of these hits, 333 could be validated in a secondary screen using independent siRNAs and 110 genes were further regulated on the gene expression level during adipogenesis. Assuming that these genes are involved in neutral lipid storage and/or adipocyte differentiation, we performed InCell-Western analysis for the most striking hits to distinguish between the two phenotypes. Beside well known regulators of adipogenesis and neutral lipid storage (i.e. PPARγ, RXR, Perilipin A) the screening revealed a large number of genes which have not been previously described in the context of fatty tissue biology such as axonemal dyneins. Five out of ten axonemal dyneins were identified in our screen and quantitative RT-PCR-analysis revealed that these genes are expressed in preadipocytes and/or maturing adipocytes. Finally, to show that the genes identified in our screen are per se druggable we performed a proof of principle experiment using an antagonist for HTR2B. The results showed a very similar phenotype compared to knock-down experiments proofing the "druggability". Thus, we identified new adipogenesis-associated genes and those involved in neutral lipid storage. Moreover, by using a druggable siRNA library the screen data provides a very attractive starting point to identify anti-obesity compounds targeting the adipose tissue.     

Monocyte chemoattractant protein-1 and nitric oxide promote adipogenesis in a model that mimics obesity

Objective: An increasing body of evidence is emerging linking adipogenesis and inflammation. Obesity, alone or as a part of the metabolic syndrome, is characterized by a state of chronic low‐level inflammation as revealed by raised plasma levels of inflammatory cytokines and acute‐phase proteins. If inflammation can, in turn, increase adipose tissue growth, this may be the basis for a positive feedback loop in obesity. We have developed a tissue engineering model for growing adipose tissue in the mouse that allows quantification of increases in adipogenesis. In this study, we evaluated the adipogenic potential of the inflammogens monocyte chemoattractant protein (MCP)‐1 and zymosan‐A (Zy) in a murine tissue engineering model. Research Methods and Procedures: MCP‐1 and Zy were added to chambers filled with Matrigel and fibroblast growth factor 2. To analyze the role of inducible nitric oxide synthase (iNOS), the iNOS inhibitor aminoguanidine was added to the chamber. Results: Our results show that MCP‐1 generated proportionally large quantities of new adipose tissue. This neoadipogenesis was accompanied by an ingrowth of macrophages and could be mimicked by Zy. Aminoguanidine significantly inhibited the formation of adipose tissue. Discussion: Our findings demonstrate that low‐grade inflammation and iNOS expression are important factors in adipogenesis. Because fat neoformation in obesity and the metabolic syndrome is believed to be mediated by macrophage‐derived proinflammatory cytokines, this adipose tissue engineering system provides a model that could potentially be used to further unravel the pathogenesis of these two metabolic disorders.     

The pro-inflammatory cytokine IL-18 is expressed in human adipose tissue and strongly upregulated by TNFalpha in human adipocytes

White adipocytes have been examined as a potential source of interleukin-18 (IL-18), the circulating levels of which are increased in obesity. IL-18 gene expression was evident in human subcutaneous and visceral adipose tissue, and expression occurred in mature adipocytes and the stromal-vascular fraction. Expression of the IL-18 receptor complex (IL-18Ralpha and IL-18Rbeta) and the IL-18 binding protein (IL-18BP) genes was also observed, mirroring that of IL-18. IL-18 mRNA level increased rapidly (within 2h) and dramatically (>900-fold) in response to TNFalpha in human adipocytes differentiated in culture. IL-18 protein was detected in lysates of cultured adipocytes, though not in the medium. There was a small increase in IL-18 in lysates of adipocytes treated with TNFalpha, but the protein was again undetectable in the medium. IL-18 may be part of the inflammatory cascade within adipose tissue; however, human adipocytes do not appear to secrete significant amounts of IL-18.     

CLA differently regulates adipogenesis in stromal vascular cells from porcine subcutaneous adipose and skeletal muscle

Conjugated linoleic acid (CLA), a mixture of isomers of linoleic acid, has previously been shown to be able to decrease porcine subcutaneous (SC) adipose tissue levels while increasing the count of intramuscular (IM) adipose tissue in vivo. However, the underlying mechanisms through which it acts are poorly understood. The objective of this study was to investigate the different effects of CLA on adipogenesis in cultured SC adipose tissue and IM stromal vascular cells obtained from neonatal pigs. As shown here, trans-10, cis-12 CLA decreased the expression of adipocyte-specific genes as well as adipose precursor cell numbers and the accumulation of lipid in cultured SC adipose tissue stromal vascular cells. However, the cis-9, trans-11 CLA did not alter adipogenesis in SC cultures. On the other hand, both CLA isomers increased the expression of adipocyte-specific genes in IM cultures, together with the increasing accumulation of lipid and Oil Red O-stained cells. Collectively, these data show that CLA decreases SC adipose tissue but increases IM adipose tissue by different regulation of adipocyte-specific gene expression. These results suggest that adipogenesis in IM adipocytes differs from that in SC adipocytes.