Nonfat Cells Research Articles

Effect of carbohydrate level in the diets of Kholmogor cows on milk productivity and milk quality

The effect of the diets with different ratio of easily digestible carbohydrates on milk productivity and milk quality of high-yielding Kholmogor cows during the milking period was studied. The research and production experiment on the cows was conducted in the Arkhangelsk region in 2022. Selection of animals in the control and experimental groups was carried out by the method of pair-analogues under the stall barn system. Two groups of cows participated in the research: experimental and control groups of 10 cows each. The experiment lasted for 100 days. Carbohydrate-protein mixture was used as a carbohydrate supplement to the diets of experimental animals in the amount of 1.5 kg per head per day. Selective milk quality control for mass fraction of fat, protein, lactose, dry matter, milk solids non-fat (MSNF), freezing point, urea and somatic cells were determined on a Bentley Combi 150 high-speed infrared milk analyzer at the Selective Milk Quality Control Laboratory. It was found that the sugar-protein ratio in the experimental group was twice higher (0.58) than in the control group (0.29), due to the presence of carbohydrate supplementation in the diet. The increase in milk productivity of the animals by 4.6 and 0.14% of the protein level in milk at inclusion of carbohydrate-protein mixture in the amount of 1.5 kg per one head per day in the diet was established. A 3.2% decrease in the level of urea in milk was recorded, indicating a more efficient use of amino acids by the animals in the process of metabolism at the same level of absorption of ammonium forms while increasing the level of simple carbohydrates in the diet. The positive effect of this additive on the content of dry skim milk residue, which increased in the milk of the cows of the experimental group by 0.128 absolute % (abs.%), was revealed. Application of the carbohydrate-protein mixture in the amount of 1.5 kg per one head per day and control over the quality of produced products will increase the milk productivity of cows, improve the quality of products, increase the productive longevity of dairy cattle.

Synthesis of a metal oxide affinity chromatography magnetic mesoporous nanomaterial and development of a one-step selective phosphopeptide enrichment strategy for analysis of phosphorylated proteins

Protein phosphorylation is one of the most vital protein post-translational modifications, which regulates numerous biological processes including protein structure transformation, molecular interaction, cell proliferation and apoptosis, as well as various malignant diseases. Though mass spectrometry (MS)-based technologies provide a feasible strategy to analyze phosphorylated proteins, label-free quantitative analysis for phosphopeptides in biological samples is still a challenging task due to sample losses from complicated preparation and necessary phosphopeptide enrichment. In this study, magnetic mesoporous TiO2 microspheres, Fe3O4@nSiO2@mSiO2@TiO2, a metal oxide affinity chromatography material, were synthesized with uniform particle size of 500 nm. The synthesized material exhibited good selectivity (molar ratio of β-casein:BSA = 1:2000, great sensitivity (0.5 fmol μL−1β-casein), and good adsorption capacity (120 mg g−1). Moreover, the proper mesopores on the surface of the enrichment material displayed the unique property of size-exclusion, which could ‘capture’ the trypsin and form a temporary immobilized enzyme reactor, reducing the tryptic digestion procedure from at least 16 h to 2 h. Based on the synthesized material, a facile one-step selective phosphopeptide enrichment method was successfully developed and consisted with protein digestion and phosphopeptide enrichment, which was further applied to selectively enrich phosphopeptides from the complex biological samples of nonfat milk and HepG2 cells, indicating its great feasibility and potential of applications in real sample analysis. More importantly, combining with MS, a label-free phosphoproteomic quantitative strategy was established and performed for determination of the mixture samples containing BSA and various concentrations of β-casein. The results showed that the obtained calibration curve was linear with R = 0.9987 by plotting the intensities of unique phosphopeptide of β-casein (m/z 2061.77) versus the concentrations of β-casein. Our study will provide a novel analytical strategy for selective enrichment of phosphopeptides and facile label-free relative quantitative analysis of phosphorylated proteins.

Molecular characterization and functional analysis of apoptosis-inducing factor (AIF) in palmitic acid-induced apoptosis in Ctenopharyngodon idellus kidney (CIK) cells.

Palmitic acid (PA), the most common saturated free fatty acid, may cause apoptosis when overloaded in non-fat cells. Apoptosis-inducing factor (AIF) is known to translocate from the mitochondria into the nucleus to induce apoptosis. However, it remains to be investigated whether AIF involved in palmitic acid-induced lipoapoptosis in fish. In the present study, we cloned a coding sequence of grass carp (Ctenopharyngodon idella) AIF (CiAIF) gene, and determined its function in Ctenopharyngodon idellus kidney (CIK) cells. The open reading frame (ORF) of CiAIF gene is 1863 bp, encoding a precursor protein of 620 amino acids (aa). Sequence analysis indicated that CiAIF contains a mitochondrial localization sequence, a conserved Pyr_redox and a C-terminal domain. Phylogenetic analyses showed that the CiAIF gene tended to cluster with sequences from Danio rerio. CiAIF gene was ubiquitously expressed in all tested tissues, including heart, liver, spleen, muscle, brain, eye, kidney, intestine, and fat. Moreover, we demonstrated that PA treatment induced the expression level of CiAIF and increases in markers of endoplasmic reticulum (ER) stress and apoptosis. Meanwhile, ER stress-inducing agent thapsigargin (TG) induced CiAIF translocated into the nucleus in CIK cells, whereas the suppression of ER stress inhibited PA-induced CiAIF expression and apoptosis. In addition, overexpression of CiAIF caused apoptosis by upregulating capase9, capase8, and capase3b, and affects protein translation via directly interacting with CieIF3g. Taken together, our data indicate that in Ctenopharyngodon idellus, PA is key elements that affect not only ER stress and mitochondrial apoptosis but also different physiological functions, such as protein translation, and CiAIF might play a key role in this progress.

Loss of p53 Sensitizes Cells to Palmitic Acid-Induced Apoptosis by Reactive Oxygen Species Accumulation.

Palmitic acid, the most common saturated free fatty acid, can lead to lipotoxicity and apoptosis when overloaded in non-fat cells. Palmitic acid accumulation can induce pancreatic β-cell dysfunction and cardiac myocyte apoptosis. Under various cellular stresses, the activation of p53 signaling can lead to cell cycle arrest, DNA repair, senescence, or apoptosis, depending on the severity/type of stress. Nonetheless, the precise role of p53 in lipotoxicity induced by palmitic acid is not clear. Here, our results show that palmitic acid induces p53 activation in a dose- and time-dependent manner. Furthermore, loss of p53 makes cells sensitive to palmitic acid-induced apoptosis. These results were demonstrated in human colon carcinoma cells (HCT116) and primary mouse embryo fibroblasts (MEF) through analysis of DNA fragmentation, flow cytometry, colony formation, and Western blots. In the HCT116 p53−/− cell line, palmitic acid induced greater reactive oxygen species formation compared to the p53+/+ cell line. The reactive oxygen species (ROS) scavengers N-acetyl cysteine (NAC) and reduced glutathione (GSH) partially attenuated apoptosis in the HCT116 p53−/− cell line but had no obvious effect on the p53+/+ cell line. Furthermore, p53 induced the expression of its downstream target genes, p21 and Sesn2, in response to ROS induced by palmitic acid. Loss of p21 also leads to more palmitic acid-induced cell apoptosis in the HCT116 cell line compared with HCT116 p53+/+ and HCT116 p53−/−. In a mouse model of obesity, glucose tolerance test assays showed higher glucose levels in p53−/− mice that received a high fat diet compared to wild type mice that received the same diet. There were no obvious differences between p53−/− and p53+/+ mice that received a regular diet. We conclude that p53 may provide some protection against palmitic acid- induced apoptosis in cells by targeting its downstream genes in response to this stress.

Association between low SIRT1 expression in visceral and subcutaneous adipose tissues and metabolic abnormalities in women with obesity and type 2 diabetes

AimsTo assess the importance of adipose tissue sirtuin 1 (SIRT1) in the regulation of whole-body metabolism in humans with obesity and type 2 diabetes. MethodsIn total, 19 non-diabetic obese women, 19 type 2 diabetic women undergoing gastric bypass surgery, and 27 normal-weight women undergoing gynecological surgery (total 65 women) were enrolled. Their anthropometric variables, abdominal fat distribution and metabolic parameters, serum adiponectin concentrations, and SIRT1 mRNA and protein and adiponectin mRNA expressions in visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) were measured. ResultsSIRT1 mRNA levels in VAT and SAT were similar and these levels were suppressed in obese and type 2 diabetic women compared to normal-weight subjects. These decreases in SIRT1 expression were observed in both adipocytes and non-fat cells. There was a strong association between adipose tissue SIRT1 mRNA and protein levels. Adipose SIRT1 expression correlated inversely with HOMA-IR and other insulin resistance-related parameters. Adipose SIRT1 and adiponectin mRNA expression correlated very strongly and positively. SIRT1 mRNA level in VAT correlated inversely with visceral obesity whereas its expression in SAT correlated negatively with body mass index. ConclusionsAdipose tissue SIRT1 may play a key role in the regulation of whole body metabolic homeostasis in humans. Downregulation of SIRT1 in VAT may contribute to the metabolic abnormalities that are associated with visceral obesity.

Role of Peripheral Blood Mononuclear Cells in the Predisposition of Obese Individuals to Inflammation and Infection

Objective: To compare the production of pro- and anti-inflammatory cytokines by peripheral blood mononuclear cells (PBMC) from obese but otherwise healthy individuals to that of normal-weight volunteers. Methods: 25 healthy normal-weight subjects and 41 obese individuals were enrolled. Weight and height were measured twice. PBMC were examined for their capacity to generate pro-inflammatory (TNF-α, IFN-γ, IL-1β, IL-6, IL-2) and anti-inflammatory IL-10 and IL-1ra) cytokines. Results: PBMC from obese individuals, compared to those from subjects with normal weight showed an increased production of the pro-inflammatory cytokines IL-2 (6.7 ± 0.4. vs. 4.9 ± 0.3 ng/ml; p = 0.003), TNF-α (505 ± 45 vs. 277 ± 32 pg/ml; p = 0.001), and IFN-γ (93.8 ± 6.0 vs. 73.9 ± 2.7 ng/ml; p = 0.0016). However, PBMC from obese individuals produced a lower amount of the anti-inflammatory cytokine IL-10 (651 ±72 pg/ml) versus those from subjects with normal weight (951 ± 133 pg/ml; p = 0.039). Conclusions: The findings imply that obese individuals are in a ‘low-grade inflammatory state', presumed to be connected with metabolic and cardiovascular co morbidities. The surplus of pro-inflammatory cytokines produced by circulating mononuclear cells of obese individuals, together with those secreted by adipocytes and non-fat cells in the adipose tissue, may contribute to the predisposition of obese patients to inflammation and infections.

Adipose tissue-derived PAI-1: A molecular link for thrombo-inflammatory disease states?

Adipose tissue (particularly the visceral adipose tissue of the omentum) was long thought to be a passive, fat-storing cell clump; however, many years ago this view became out-dated. Numerous studies made us aware of the actual character of this contour-determining part of our body: Adipose tissue is considered to be a true organ capable of producing multiple types of hormones, proand anti-inflammatory mediators such as adipokines and other components that are relevant for metabolic control and vascular homeostasis. No wonder that fat tissue-derived factors are dynamically involved in obesity-associated diseases, play an important part in insulin resistance or metabolic syndrome, and are relevant control elements of the cardiovascular system (1). Adipokines constitute a heterogenous group of macromolecules and are divided into those produced by fat cells (such as leptin or adiponectin) and those released by non-fat cells, including several interleukins, tumour necrosis factor (TNF)-α as well as plasminogen activator inhibitor-1 (PAI-1) (2, 3). Moreover, the repertoire of adipokines, particularly when released under stimulatory/inflammatory conditions, may differ considerably between omental and subcutaneous fat tissue. Thus, adipose tissuederived factors may act in a paracrine fashion at the interface with other organs or tissues, as was proposed for the fat–bone relationship (4). Against this background, in this issue of Thrombosis and Haemostasis, Ekstrom et al. (5) provide experimental evidence in humans for a potential cross-talk between an acute inflammatory situation (such as open heart surgery in a small group of patients) and fat. This situation lead to a significant stimulation of PAI-1 gene expression in omental adipose tissue, accompanied by increased plasma levels of PAI-1 antigen over several hours during the post-operative period (5). Consequently, the authors propose that such inflammation-provoked elevated PAI-1 levels would diminish the t-PAmediated fibrinolytic potential in these patients, resulting in an increased risk of myocardial infarction. As the origin of chronically elevated PAI-1 levels in situations of spontaneous arterial thrombosis or early vein graft rejection is largely unknown, this first study in human subjects offers a promising approach to define mechanistic relations between adipose tissue and other disturbed organs with regard to the thrombo-inflammatory axis. Yet, one should be careful to over-interpret these findings (as already indicated by the authors), since several aspects may limit the conclusions of this small study. Further experimental work is required to clarify the following issues: (a) Although the elevation in PAI-1 antigen may also imply increased PAI-1 activity and enhanced inhibition of t-PA (with an increase in t-PA-PAI-1 complexes), these parameters were not determined, indicating that an impact on fibrinolysis may be assumed but was not experimentally demonstrated; (b) in addition to omental tissue being a major origin of the protease inhibitor, elevated plasma PAI-1 levels could be derived from activated platelets as well, which would fit with the known inflammatory role of platelets in different vascular diseases (6); (c) although quantitatively less pronounced when compared to omental fat tissue, epicardial adipose tissue may very well be responsive to acute inflammatory situations during open heart surgery, particularly due to its close proximity to the organ under operation. Here, further analysis to clarify mechanisms of a paracrine crosstalk provoked by adipokines may be worth considering. And finally, (d) the authors did not mention any PAI-1 targeting approaches that may add to an anti-inflammatory regimen with antagonists towards interleukins or TNF-α; here, PAI-1 inhibitors may very well be suitable to limit the risk of vascular diseases, including myocardial infarction, in the post-operative period and beyond (7). Together with their first study in human patients the authors provide some clues for a thrombo-inflammatory connection between open heart surgery and increased PAI-1 production in adipose tissue. These initial findings may stimulate further translational investigations as to the potential role of adipokines and their direct or indirect contribution in thrombosis and haemostasis.

Adipose Tissue-Derived Soluble Fms-Like Tyrosine Kinase 1 Is an Obesity-Relevant Endogenous Paracrine Adipokine

Adipose tissue growth depends on angiogenesis. We tested the hypothesis that adipose tissue produces factors relevant to angiogenesis. We obtained fat biopsies in 2 different patient cohorts, cultured adipose-derived stem cells and studied mature adipocytes. We performed microarray, RT-PCR, and Western blotting; studied a rat obesity/metabolic syndrome model; and conducted viral gene transfer experiments in leptin-deficient mice. The microarray identified the splice variant of the vascular endothelial growth factor receptor, the soluble fms-like tyrosine kinase 1 (sFlt-1), as an antiangiogenesis candidate. We verified the expression findings and found that sFlt-1 was secreted by isolated mature human adipocytes. Tumor necrosis factor-α decreased sFlt-1 expression in mature adipocytes, whereas hypoxia had no effect. Separating cells from adipose tissue showed that the highest sFlt-1 expression was present in adipose-tissue nonfat cells rather than in the adipocytes themselves. We also found that sFlt-1 expression and sFlt-1 release by adipose-tissue explants were inversely correlated with body mass index of the corresponding patients but was directly correlated with adiponectin expression. In the obesity/metabolic syndrome rat model, we observed that circulating sFlt-1 levels and sFlt-1 expression in adipose tissue were also inversely correlated with body weight. To model our putative antiangiogenic factor further, we next overexpressed sFlt-1 by viral transfer in a mouse genetic model of leptin deficiency and observed that the transfected mice gained less weight than controls. We suggest that sFlt-1 could act as a paracrine factor inhibiting adipose tissue growth. Local sFlt-1 may regulate angiogenic potential and thereby influence adipose tissue mass.

Release of 12 Adipokines by Adipose Tissue, Nonfat Cells, and Fat Cells From Obese Women

The relative release in vitro of endothelin-1, zinc-alpha2-glycoprotein (ZAG), lipocalin-2, CD14, RANTES (regulated on activation, normal T cell expressed and secreted protein), lipoprotein lipase (LPL), osteoprotegerin (OPG), fatty acid-binding protein 4 (FABP-4), visfatin/PBEF/Nampt, glutathione peroxidase-3 (GPX-3), intracellular cell adhesion molecule 1 (ICAM-1), and amyloid A was examined using explants of human adipose tissue as well as the nonfat cell fractions and adipocytes from obese women. Over a 48-h incubation the majority of the release of LPL was by fat cells whereas that of lipocalin-2, RANTES, and ICAM-1 was by the nonfat cells present in human adipose tissue. In contrast appreciable amounts of OPG, amyloid A, ZAG, FABP-4, GPX-3, CD14, and visfatin/PBEF/Nampt were released by both fat cells and nonfat cells. There was an excellent correlation (r = 0.75) between the ratios of adipokine release by fat cells to nonfat cells over 48 h and the ratio of their mRNAs in fat cells to nonfat cells at the start of the incubation. The total release of ZAG, OPG, RANTES, and amyloid A by incubated adipose tissue explants from women with a fat mass of 65 kg was not different from that by women with a fat mass of 29 kg. In contrast that of ICAM-1, FABP-4, GPX-3, visfatin/PBEF/Nampt, CD14, lipocalin-2, LP, and endothelin-1 was significantly greater in tissue from women with a total fat mass of 65 kg.

The inflammatory response seen when human omental adipose tissue explants are incubated in primary culture is not dependent upon albumin and is primarily in the nonfat cells

BackgroundThe present studies were designed to investigate the changes in gene expression during in vitro incubation of human visceral omental adipose tissue explants as well as fat cells and nonfat cells derived from omental fat.MethodsAdipose tissue was obtained from extremely obese women undergoing bariatric surgery. Explants of the tissue as well as fat cells and the nonfat cells derived by digestion with collagenase were incubated for 20 minutes to 48 h. The expression of interleukin 1β [IL-1β], tumor necrosis factor α [TNFα], interleukin 8 [IL-8], NFκB1p50 subunit, hypoxia-inducible factor 1α [HIF1α], omentin/intelectin, and 11β-hydroxysteroid dehydrogenase 1 [11β-HSD1] mRNA were measured by qPCR as well as the release of IL-8 and TNFα.ResultsThere was an inflammatory response at 2 h in explants of omental adipose tissue that was reduced but not abolished in the absence of albumin from the incubation buffer for IL-8, IL-1β and TNFα. There was also an inflammatory response with regard to upregulation of HIF1α and NFκB1 gene expression that was unaffected whether albumin was present or absent from the medium. In the nonfat cells derived by a 2 h collagenase digestion of omental fat there was an inflammatory response comparable but not greater than that seen in tissue. The exception was HIF1α where the marked increase in gene expression was primarily seen in intact tissue. The inflammatory response was not seen with respect to omentin/intelectin. Over a subsequent 48 h incubation there was a marked increase in IL-8 mRNA expression and IL-8 release in adipose tissue explants that was also seen to the same extent in the nonfat cells incubated in the absence of fat cells.ConclusionThe marked inflammatory response seen when human omental adipose tissue is incubated in vitro is reduced but not abolished in the presence of albumin with respect to IL-1β, TNFα, IL-8, and is primarily in the nonfat cells of adipose tissue.

Release of Inflammatory Mediators by Human Adipose Tissue Is Enhanced in Obesity and Primarily by the Nonfat Cells: A Review

This paper considers the role of putative adipokines that might be involved in the enhanced inflammatory response of human adipose tissue seen in obesity. Inflammatory adipokines [IL-6, IL-10, ACE, TGFβ1, TNFα, IL-1β, PAI-1, and IL-8] plus one anti-inflammatory [IL-10] adipokine were identified whose circulating levels as well as in vitro release by fat are enhanced in obesity and are primarily released by the nonfat cells of human adipose tissue. In contrast, the circulating levels of leptin and FABP-4 are also enhanced in obesity and they are primarily released by fat cells of human adipose tissue. The relative expression of adipokines and other proteins in human omental as compared to subcutaneous adipose tissue as well as their expression in the nonfat as compared to the fat cells of human omental adipose tissue is also reviewed. The conclusion is that the release of many inflammatory adipokines by adipose tissue is enhanced in obese humans.

Intricacies of Fat

One of the most exciting cell biology fields of study concerns the physiology and pathology of fat. The basic assumptions once held concerning the function of adipose tissue have been shown to be oversimplified or sometimes completely wrong. Fat does more than store excess energy; it is actually the largest endocrine organ in the body, and it may be one of the most active. Adipocytes release hormones and other molecules that act on nearby tissues and travel through the vasculature to distant sites, such as the brain, skeletal muscle, and liver. Under conditions of normal weight, those signals help the body to suppress hunger, utilize glucose, and decrease the risk of cardiovascular disease. However, under conditions of obesity, the hormones (or the proteins that bind the hormones) become abnormal and can result in states of chronic inflammation leading to diabetes and heart disease. In addition, excessive fat can lead to the accumulation of lipid droplets in nonfat cells, including skeletal and cardiac muscle. Although some lipid droplets are used as an immediate source of energy for cells, large numbers of stored droplets can cause cellular damage and cell death. The purposes of this article are to review the normal and deviant signals released by fat cells, to draw a link between those signals and chronic diseases such as diabetes, and to discuss the role of exercise in reversing some of the deviant signaling perpetrated by excess fat.

Comparison of messenger RNA distribution for 60 proteins in fat cells vs the nonfat cells of human omental adipose tissue

The messenger RNA (mRNA) distribution of 60 proteins was examined in the 3 fractions obtained by collagenase digestion (fat cells and the nonfat cells comprising the tissue remaining after collagenase digestion [matrix] and the stromovascular cells) of omental adipose tissue obtained from morbidly obese women undergoing bariatric surgery. Fat cells were enriched by at least 3-fold as compared with nonfat cells in the mRNAs for retinol binding protein 4, angiotensinogen, adipsin, glutathione peroxidase 3, uncoupling protein 2, peroxisome proliferator–activated receptor γ, cell death–inducing DFFA-like effector A, fat-specific protein 27, 11 β-hydroxysteroid dehydrogenase 1, glycerol channel aquaporin 7, NADPH:quinone oxidoreductase 1, cyclic adenosine monophosphate phosphodiesterase 3B, glyceraldehyde-3-phosphate dehydrogenase, insulin receptor, and amyloid A1. Fat cells were also enriched by at least 26-fold in the mRNAs for proteins involved in lipolysis such as hormone-sensitive lipase, lipoprotein lipase, adipose tissue triglyceride lipase, and FAT/CD36. The relative distribution of mRNAs in cultured preadipocytes was also compared with that of in vitro differentiated adipocytes derived from human omental adipose tissue. Cultured preadipocytes had far lower levels of the mRNAs for inflammatory proteins than the nonfat cells of omental adipose tissue. The nonfat cells were enriched by at least 5-fold in the mRNAs for proteins involved in the inflammatory response such as tumor necrosis factor α, interleukin l β, cyclooxygenase 2, interleukin 24, interleukin 6, and monocyte chemoattractant protein 1 plus the mRNAs for osteopontin, vaspin, endothelin, angiotensin II receptor 1, butyrylcholinesterase, lipocalin 2, and plasminogen activator inhibitor 1. The cells in the adipose tissue matrix were enriched at least 3-fold as compared with the isolated stromovascular cells in the mRNAs for proteins related to the inflammatory response, as well as osteopontin and endothelial nitric oxide synthase. We conclude that the mRNAs for inflammatory proteins are primarily present in the nonfat cells of human omental adipose tissue.

Identification of omentin mRNA in human epicardial adipose tissue: comparison to omentin in subcutaneous, internal mammary artery periadventitial and visceral abdominal depots

The purpose of this study was to determine the relative distribution of omentin and visfatin mRNA in human epicardial, peri-internal mammary, upper thoracic, upper abdominal and leg vein subcutaneous adipose tissue as well as the distribution of omentin in the nonfat cells and adipocytes of human omental adipose tissue. Omentin is found in human omentum but not subcutaneous fat. Omentin and visfatin are considered markers of visceral abdominal fat. The mRNA content of omentin and visfatin was measured by qRT-PCR analysis of fat samples removed from humans undergoing cardiac or bariatric surgery. Omentin mRNA in internal mammary fat was 3.5%, that in the upper thoracic subcutaneous fat was 4.7% while that in the other subcutaneous fat depots was less than 1% of omentin in epicardial fat. The distribution of visfatin mRNA did not vary between the five depots. Omentin mRNA was preferentially expressed in the nonfat cells of omental adipose tissue since the omentin mRNA content of isolated adipocytes was 9% of that in nonfat cells, and similar results were seen for visfatin. The amount of omentin mRNA in differentiated adipocytes was 0.3% and that of visfatin 4% of that in nonfat cells. The amount of omentin mRNA in preadipocytes was virtually undetectable while that of visfatin was 3% of that in freshly isolated nonfat cells from omental adipose tissue. Omentin mRNA is predominantly found in epicardial and omental human fat whereas visfatin mRNA is found to the same extent in epicardial, subcutaneous and omental fat.

Release in vitro of adipsin, vascular cell adhesion molecule 1, angiotensin 1–converting enzyme, and soluble tumor necrosis factor receptor 2 by human omental adipose tissue as well as by the nonfat cells and adipocytes

The relative release in vitro of adipsin, vascular cell adhesion molecule (VCAM) 1, angiotensin 1–converting enzyme (ACE), and soluble tumor necrosis factor α receptor 2 (sTNFR2) by explants of human omental and subcutaneous adipose tissue as well as the nonfat cell fractions and adipocytes from morbidly obese gastric bypass women was compared with that by tissue from obese abdominoplasty patients. Release of VCAM-1 and ACE by omental adipose tissue explants was 220% and 80% greater, respectively, over 48 hours of incubation than that by subcutaneous adipose tissue explants. However, this difference was not seen when release by adipocytes derived from omental fat was compared with that by adipocytes from subcutaneous fat. The release of adipsin and sTNFR2 by omental adipose tissue explants did not differ from that by subcutaneous tissue adipose tissue. The release of adipsin by tissue explants over 48 hours was 100-fold greater than that of VCAM-1, ACE, or sTNFR2. Most of the release of all 4 adipokines was due to the nonfat cells because adipsin release by adipocytes was only 13% of that by the nonfat cells derived from the same amount of adipose tissue, whereas ACE release by adipocytes was 7% and release of VCAM-1 as well as sTNFR2 by adipocytes was 4% or less of that by nonfat cells. The total release in vitro of ACE and sTNFR2, but not that of adipsin or VCAM-1, was enhanced in adipose tissue explants from morbidly obese women as compared with those by explants derived from obese women. We conclude that although human adipose tissue explants release appreciable amounts of adipsin and far smaller amounts of VCAM-1, ACE, and sTNFR2 in vitro, more than 87% of the release is due to the nonfat cells present in adipose tissue. Furthermore, the enhanced release of VCAM-1 and ACE seen in omental adipose tissue explants as compared with explants of subcutaneous adipose tissue is due to release by nonfat cells.

Regulation of adiponectin release and demonstration of adiponectin mRNA as well as release by the non-fat cells of human omental adipose tissue

Adiponectin is an adipokine produced by adipose tissue. The present studies examined the in vitro release of adiponectin by human omental adipose tissue explants as well as the mRNA content of freshly isolated non-fat cells and adipocytes plus cultured preadipocytes and adipocytes derived from omental fat. The release of adiponectin was reduced while that of interleukin-8 (IL-8) was enhanced in tissue explants from morbidly obese women. The release of adiponectin was also reduced by one-third in explants from morbidly obese diabetic women while that of IL-8 was unaffected by diabetes. The release of adiponectin was enhanced by insulin and by inhibition of endogenous tumor necrosis factor (TNFalpha) using etancercept. Adiponectin was released in appreciable amounts by the undigested matrix obtained by collagenase digestion of adipose tissue. The release of adiponectin by non-fat cells (matrix+SV cells) was comparable to that by the adipocytes derived from the same amount of tissue while the adiponectin mRNA content of the pooled matrix and SV cell fractions was 40% of that in intact tissue. The adiponectin mRNA content was 48-fold greater in isolated adipocytes than in non-fat cells derived from adipose tissue. In contrast, the amount of adiponectin mRNA in vitro differentiated omental adipocytes was 1 x l0(6)-fold greater than that in cultured preadipocytes while that of leptin was 3 x 10(4)-fold greater. Adiponectin mRNA is present in the non-fat cells of freshly isolated adipose tissue and release by the non-fat cells derived from a gram of adipose tissue is comparable to that by adipocytes isolated from the same amount of tissue. While leptin is only released by mature adipocytes, adiponectin is released by both the non-fat cells and the fat cells derived from human omental adipose tissue.

Transforming Growth Factor β1 release by human adipose tissue is enhanced in obesity

The present studies examined the effect of obesity in humans on the release of transforming growth factor β1 (TGF- β1) by human adipose tissue. The regulation of TGF- β1 release by adipose tissue as well as the question of whether its release is due to the adipocytes or the nonfat cells in adipose tissue was also examined. There was a statistically significant ( r = 0.50) correlation between the body mass index of the fat donors and the subsequent release of TGF- β1 release by subcutaneous adipose tissue. There was also a positive correlation between total TGF- β1 release by adipose tissue explants and body fat content ( r = 0.69). The question of whether tumor necrosis factor α (TNF- α) and/or interleukin 1 β (IL-1 β) regulate the release of TGF- β1 was investigated by incubation of adipose tissue explants with a soluble human TNF- α receptor (etanercept) and a neutralizing antihuman IL-1 β antibody. The release of TGF- β1 over 48 hours by adipose tissue explants was significantly enhanced in the presence of both the inhibitor of TNF- α and of IL-1 β. It is of interest, in view of the elevated circulating insulin in blood of morbidly obese women, that the release of TGF- β1 by adipose tissue was enhanced in the presence of insulin. The question of whether the release of TGF- β1 by human adipose tissue explants was primarily due to adipocytes, as is the case for leptin, or the nonfat cells present in human adipose tissue, as is the case for IL-8 and prostaglandin E 2, was examined. The release of TGF- β1 was primarily by the nonfat cells of human adipose tissue because release by adipocytes was less than 10% of that by the nonfat cells of adipose tissue.

Regulation of monocyte chemoattractant protein 1 (MCP-1) release by explants of human visceral adipose tissue

Monocyte chemoattractant protein-1 (MCP-1) is a chemokine involved in monocyte recruitment during inflammation whose plasma level is elevated in obesity. The present studies were designed to examine the release of MCP-1 in primary culture by explants of visceral adipose tissue from morbidly obese women. Most of the MCP-1 released by adipose tissue explants was derived from the nonfat cells in adipose tissue. The release of MCP-1 by adipose tissue explants was upregulated almost five-fold between 3 and 48 h of incubation. Approximately half of this upregulation was due to the release of endogenous tumor necrosis factor alpha (TNFalpha) and IL-1beta based on the ability of a combination of a soluble TNFalpha receptor (etanercept) and a blocking antibody against IL-1beta to reduce MCP-1 release. The release of MCP-1 over 48 h was unaffected by insulin or dexamethasone but significantly reduced by the combination of both agents. MCP-1 release was reduced by 60% in the presence of an inhibitor of the nuclear factor kappaB (NF-kappaB) pathway. There were no significant effects of inhibitors of p44/42 mitogen-activated protein kinase (ERK), Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK) pathways on MCP-1 release. However, inhibition of MCP-1 release in the presence of inhibitors of both the p38 MAPK and NF-kappaB pathways was greater than that seen with only the NF-kappaB inhibitor. The present data shows that MCP-1 formation is upregulated over a 48-h incubation of primary explants of visceral adipose tissue. Half of this upregulation is dependent upon endogenous TNFalpha and Il-1beta and involves the p38 MAPK and NF-kappaB pathways.

Involvement of multiple signaling pathways in the post-bariatric induction of IL-6 and IL-8 mRNA and release in human visceral adipose tissue

The present studies were designed to determine the site of and the mechanism for the rapid increase in IL-6 and IL-8 mRNA observed in human visceral adipose tissue after removal during laparoscopic bariatric surgery. Upregulation of IL-6 and IL-8 mRNA as well as their release were seen within 3 h whether one intact piece of tissue or minced pieces of adipose tissue were incubated in vitro. Most of the IL-6 and IL-8 mRNA content of visceral adipose tissue after 3 h of incubation was in the non-fat cells. Actinomcyin D markedly reduced the upregulation of IL-6 and IL-8 mRNA. Incubation of adipose tissue explants with a soluble TNFα receptor (etanercept) plus a blocking antibody against IL-lβ reduced by 55% the increase in IL-6 mRNA and by 42% that of IL-8 mRNA seen between 1 and 5 h of incubation. The upregulation of IL-8 and IL-6 mRNA accumulation as well as their release over a 2 or 4 h incubation was reduced by around 50% in the presence of an inhibitor of the p38 MAPK or an inhibitor of the NFκB pathway and by 85% in the presence of both inhibitors. The data suggest that the relative trauma and/or hypoxia that occurs when adipose tissue is removed results in the release of TNFα and IL-1β. These cytokines, and probably other factors as well, enhance IL-6 and IL-8 mRNA accumulation in human adipose tissue explants through mechanisms involving the p38 MAPK and NFκB pathways

TNFalpha release by the nonfat cells of human adipose tissue.

The primary aim was to investigate the relative importance of the adipocytes vs the nonfat cells present in human adipose tissue with respect to release of immunoreactive tumor necrosis factor-alpha (TNFalpha). The second aim was to examine the correlation between body mass index (BMI) and the subsequent release of adiponectin and TNFalpha by explants of human subcutaneous and visceral adipose tissue incubated in primary culture for 48 h. We found that the maximal release of TNFalpha was seen during the first 4 h of a 48-h incubation by explants of human adipose tissue in primary culture. Over 95% of the TNFalpha released to the medium by human adipose tissue explants over a 4-h incubation came from the nonfat cells present in the adipose tissue. The release of TNFalpha by the nonfat cells released during collagenase digestion was slightly higher than that by the cells present in the adipose tissue matrix after collagenase digestion. TNFalpha release by the combined matrix and isolated nonfat cells was greater than that by explants of tissue indicating some upregulation induced by collagenase digestion. Immunoreactive TNFalpha disappeared from the medium with a half-time of approximately 10 h. There was a positive correlation coefficient of 0.79 between TNFalpha release by tissue explants and the BMI of the fat donors as well as a correlation of 0.52 between BMI and release by adipocytes. TNFalpha release negatively correlated [-0.60] with adiponectin release by adipose tissue. The release of TNFalpha was far less than that of adiponectin or IL-6, and less than that of plasminogen activator inhibitor-1, hepatocyte growth factor, or leptin over a 4-h incubation of human adipose tissue explants. TNFalpha release over 4 h was enhanced by lipopolysaccharide and inhibited by a cyclooxygenase-2 inhibitor. The release of TNFalpha by adipose tissue of obese humans is primarily due to the nonfat cells present in adipose tissue. TNFalpha is a short-lived adipokine whose release by human adipose tissue in primary culture correlates with the BMI of the fat donors.