Hypoxia and the endocrine and signalling role of white adipose tissue

White adipose tissue is a major endocrine and signalling organ. It secretes multiple protein hormones and factors, termed adipokines (such as adiponectin, leptin, IL-6, MCP-1, TNFalpha) which engage in extensive cross-talk within adipose tissue and with other tissues. Many adipokines are linked to inflammation and immunity and these include cytokines, chemokines and acute phase proteins. In obesity, adipose tissue exhibits a major inflammatory response with increased production of inflammation-related adipokines. It has been proposed that hypoxia may underlie the inflammatory response in adipose tissue and evidence that the tissue is hypoxic in obesity has been obtained in animal models. Cell culture studies have demonstrated that the expression and secretion of key adipokines, including leptin, IL-6 and VEGF, are stimulated by hypoxia, while adiponectin (with an anti-inflammatory action) production falls. Hypoxia also stimulates glucose transport by adipocytes and may have a pervasive effect on cell function within adipose tissue.     

Ontogenesis of muscle and adipose tissues and their interactions in ruminants and other species

The lean-to-fat ratio, that is, the relative masses of muscle and adipose tissue, is a criterion for the yield and quality of bovine carcasses and meat. This review describes the interactions between muscle and adipose tissue (AT) that may regulate the dynamic balance between the number and size of muscle v. adipose cells. Muscle and adipose tissue in cattle grow by an increase in the number of cells (hyperplasia), mainly during foetal life. The total number of muscle fibres is set by the end of the second trimester of gestation. By contrast, the number of adipocytes is never set. Number of adipocytes increases mainly before birth until 1 year of age, depending on the anatomical location of the adipose tissue. Hyperplasia concerns brown pre-adipocytes during foetal life and white pre-adipocytes from a few weeks after birth. A decrease in the number of secondary myofibres and an increase in adiposity in lambs born from mothers severely underfed during early pregnancy suggest a balance in the commitment of a common progenitor into the myogenic or adipogenic lineages, or a reciprocal regulation of the commitment of two distinct progenitors. The developmental origin of white adipocytes is a subject of debate. Molecular and histological data suggested a possible transdifferentiation of brown into white adipocytes, but this hypothesis has now been challenged by the characterization of distinct precursor cells for brown and white adipocytes in mice. Increased nutrient storage in fully differentiated muscle fibres and adipocytes, resulting in cell enlargement (hypertrophy), is thought to be the main mechanism, whereby muscle and fat masses increase in growing cattle. Competition or prioritization between adipose and muscle cells for the uptake and metabolism of nutrients is suggested, besides the successive waves of growth of muscle v. adipose tissue, by the inhibited or delayed adipose tissue growth in bovine genotypes exhibiting strong muscular development. This competition or prioritization occurs through cellular signalling pathways and the secretion of proteins by adipose tissue (adipokines) and muscle (myokines), putatively regulating their hypertrophy in a reciprocal manner. Further work on the mechanisms underlying cross-talk between brown or white adipocytes and muscle fibres will help to achieve better understanding as a prerequisite to improving the control of body growth and composition in cattle.     

Pathways leading to muscle insulin resistance--the muscle--fat connection

Type 2 diabetes is a heterogeneous disease characterized by hyperglycemia and insulin resistance in peripheral tissues such as adipose tissue and skeletal muscle. This review focuses on obesity as one of the major environmental factors contributing to the development of diabetes. It has become evident that adipose tissue represents an active secretory organ capable of releasing a variety of cytokines such as TNFalpha, IL-6, adiponectin and other still unknown factors that might constitute the missing link between adipose tissue and insulin resistance. In fact, adipocyte-derived factors are significantly increased in obesity and represent good predictors of the development of type 2 diabetes. The negative crosstalk between adipocytes and skeletal muscle cells leads to disturbances in muscle cell insulin signalling and insulin resistance involving major pathways in inflammation, cellular stress and mitogenesis. Positive regulators of insulin sensitivity include the adipocyte hormone adiponectin and inhibitors of inflammatory pathways such as JNK-, IKK- and ERK-inhibitors. In summary, a better knowledge of intracellular and intercellular mechanisms by which adipose tissue affects skeletal muscle cell physiology may help to develop new strategies for diabetes treatment.     

Inducible disruption of autophagy in the lung causes airway hyper-responsiveness

Objective

Central adiposity and inflammation play key roles in the development of insulin resistance through the effects of pro-inflammatory adipokines such as IL-6, but the effect of infiltrating adipocytes in skeletal muscle tissues is not known. Communications between muscle cells and fat cells may contribute to the inflammatory response associated with insulin resistance.

Methods

In this study we used a co-culture system of skeletal muscle (L6) and adipocyte (3T3-L1) cell lines to study expression of the inflammatory cytokine IL-6 and changes in insulin signaling. This model could mimic the adipocytes infiltrating myocytes that is commonly seen in obese patients.

Results

When plated alone the L6 cells express IL-6 mRNA and secrete IL-6 protein, both of which are increased when the cells are challenged with the bacterial lipopolysaccharide (LPS). In contrast, the 3T3-L1 cells had very little expression of IL-6 mRNA or protein. Co-culture of 3T3-L1 pre-adipocytes with L6 cells, at a density ratio of 1:10, respectively, increased IL-6 expression significantly and decreased insulin-stimulated Akt phosphorylation. To examine the role of IL-6 in insulin sensitivity we incubated the L6 cells with IL-6. A brief challenge of L6 cells with IL-6 enhanced insulin-stimulated Akt phosphorylation. In contrast, incubation of the L6 cells with IL-6 for 96 h markedly decreased insulin-stimulated Akt phosphorylation.

Conclusion

The enhanced IL-6 mRNA expression and IL-6 release in L6 myocytes co-cultured with 3T3-L1 cells indicate an important interaction between adipocytes and myocytes. This observation may shed some light on the long-standing enigma of obesity-induced insulin resistance where infiltration of the skeletal muscle by preadipocytes/adipocytes is evident.     

Identification and Validation of Novel Contraction-Regulated Myokines Released from Primary Human Skeletal Muscle Cells

Proteins secreted by skeletal muscle, so called myokines, have been shown to affect muscle physiology and additionally exert systemic effects on other tissues and organs. Although recent profiling studies have identified numerous myokines, the amount of overlap from these studies indicates that the secretome of skeletal muscle is still incompletely characterized. One limitation of the models used is the lack of contraction, a central characteristic of muscle cells. Here we aimed to characterize the secretome of primary human myotubes by cytokine antibody arrays and to identify myokines regulated by contraction, which was induced by electrical pulse stimulation (EPS). In this study, we validated the regulation and release of two selected myokines, namely pigment epithelium derived factor (PEDF) and dipeptidyl peptidase 4 (DPP4), which were recently described as adipokines. This study reveals that both factors, DPP4 and PEDF, are secreted by primary human myotubes. PEDF is a contraction-regulated myokine, although PEDF serum levels from healthy young men decrease after 60 min cycling at VO₂max of 70%. Most interestingly, we identified 52 novel myokines which have not been described before to be secreted by skeletal muscle cells. For 48 myokines we show that their release is regulated by contractile activity. This profiling study of the human skeletal muscle secretome expands the number of myokines, identifies novel contraction-regulated myokines and underlines the overlap between proteins which are adipokines as well as myokines.     

Characterization of the human visceral adipose tissue secretome

Adipose tissue is an endocrine organ involved in storage and release of energy but also in regulation of energy metabolism in other organs via secretion of peptide and protein hormones (adipokines). Especially visceral adipose tissue has been implicated in the development of metabolic syndrome and type 2 diabetes. Factors secreted by the stromal-vascular fraction contribute to the secretome and modulate adipokine secretion by adipocytes. Therefore, we aimed at the characterization of the adipose tissue secretome rather than the adipocyte cell secretome. The presence of serum proteins and intracellular proteins from damaged cells, released during culture, may dramatically influence the dynamic range of the sample and thereby identification of secreted proteins. Part of the study was therefore dedicated to the influence of the culture setup on the quality of the final sample. Visceral adipose tissue was cultured in five experimental setups, and the quality of resulting samples was evaluated in terms of protein concentration and protein composition. The best setup involved one wash after the 1st h in culture followed by two or three additional washes within an 8-h period, starting after overnight culture. Thereafter tissue was maintained in culture for an additional 48-114 h to obtain the final sample. For the secretome experiment, explants were cultured in media containing L-[(13)C(6),(15)N(2)]lysine to validate the origin of the identified proteins (adipose tissue- or serum-derived). In total, 259 proteins were identified with > or =99% confidence. 108 proteins contained a secretion signal peptide of which 70 incorporated the label and were considered secreted by adipose tissue. These proteins were classified into five categories according to function. This is the first study on the (human) adipose tissue secretome. The results of this study contribute to a better understanding of the role of adipose tissue in whole body energy metabolism and related diseases.     

Adipokines oversecreted by omental adipose tissue in human obesity

Central-omental obesity plays a causative role in the pathogenesis of the metabolic syndrome. Adipokines are involved in the pathogenesis of this syndrome. However, adipokines secreted by omental adipose tissue (OAT) are still poorly characterized in human obesity. Therefore, we searched for novel adipokines abnormally secreted by OAT in obesity and examined their relationships with some features of metabolic syndrome and the respective contribution of adipocytes vs. stromal-vascular cells. OAT from obese and nonobese men was fractionated into adipocytes and SV cells, which were then cultured. Medium was screened by medium-scale protein arrays and ELISAs. Adipokine mRNA levels were measured by real-time RT-qPCR. We detected 16 cytokines secreted by each cellular fraction of lean and obese subjects. Of the 16 cytokines, six adipokines were newly identified as secretory products of OAT, which were dysregulated in obesity: three chemokines (growth-related oncogen factor, RANTES, macrophage inflammatory protein-1beta), one interleukin (IL-7), one tissue inhibitor of metalloproteinases (TIMP-1), and one growth factor (thrombopoietin). Their secretion and expression were enhanced in obesity, with a relatively similar contribution of the two fractions. The higher proportion of macrophages and endothelial cells in obesity may contribute to this enhanced production as well as changes in intrinsic properties of hypertrophied adipocytes. Accordingly, mRNA concentrations of most of these adipokines increased during adipocyte differentiation. Eventually, expression of the investigated adipokines did correlate with several features of the metabolic syndrome. In conclusion, six adipokines were newly identified as oversecreted by OAT in obesity. These adipokines may link obesity to its cardiovascular or metabolic comorbidities.     

The secretory function of adipocytes in the physiology of white adipose tissue

White adipose tissue, previously regarded as a passive lipid storage site, is now viewed as a dynamic tissue. It has the capacity to actively communicate by sending and receiving different types of signals. An overview of these signals, the external modulators that affect adipose tissue and the secreted signaling molecules, the adipokines, is presented. The secretory function is highlighted in relation to energy metabolism, inflammation and the extracellular matrix and placed in the context of adipose tissue biology. We observe that the endocrine function of adipocytes receives much attention, while its paracrine and autocrine functions are underestimated. Also, we provide examples that species specificity should not be neglected. We conclude that adipose tissue primarily is an energy storage organ, well supported by its secretory function.     

The adipose organ at a glance

The main parenchymal cells of the adipose organ are adipocytes. White adipocytes store energy, whereas brown adipocytes dissipate energy for thermogenesis. These two cell types with opposing functions can both originate from endothelial cells, and co-exist in the multiple fat depots of the adipose organ – a feature that I propose is crucial for this organ’s plasticity. This poster review provides an overview of the adipose organ, describing its anatomy, cytology, physiological function and histopathology in obesity. It also highlights the remarkable plasticity of the adipose organ, explaining theories of adipocyte transdifferentiation during chronic cold exposure, physical exercise or lactation, as well as in obesity. White-to-brown adipocyte transdifferentiation is of particular medical relevance, because animal data indicate that higher amounts of brown adipose tissue are positively associated with resistance to obesity and its co-morbidities, and that ‘browning’ of the adipose organ curbs these disorders.     

Profiling of adipokines secreted from human subcutaneous adipose tissue in response to PPAR agonists

The role of PPARs in the regulation of human adipose tissue secretome has received little attention despite its potential importance in the therapeutic actions of PPAR agonists. Here, we have investigated the effect of selective PPARgamma, PPARalpha, and PPARbeta/delta agonists on the production of adipokines by human subcutaneous adipose tissue. Antibody arrays were used to measure secreted factors in media from cultured adipose tissue explants. Sixteen proteins were produced in significant amounts. Activation of PPARs regulated the production of five proteins. Treatments with the three PPAR agonists decreased the secretion of leptin and interleukin-6. PPARalpha and beta/delta agonists markedly enhanced hepatocyte growth factor secretion whereas PPARbeta/delta down-regulated angiogenin and up-regulated TIMP-1 release. Hepatocyte growth factor, interleukin-6, and TIMP-1 are chiefly expressed in cells from the stromal vascular fraction whereas angiogenin is expressed in both adipocytes and cells from the stromal vascular fraction. Our data show that PPAR agonists modulate secretion of bioactive molecules from the different cell types composing human adipose tissue.     

Plasma cells and Fc receptors in human adipose tissue--lipogenic and anti-inflammatory effects of immunoglobulins on adipocytes

We have previously reported high immunoglobulin expression in human omental adipose tissue. The aim of this work was to investigate plasma cell density and Fc receptor (FcR) expression in human adipose tissue depots and in vitro effects of immunoglobulins on adipocyte function. Plasma cell density was higher in the visceral compared to the subcutaneous depot (10.0+/-1.56% and 5.2+/-0.98%, respectively, n=20, p<0.05). Microarray analysis revealed expression of four FcR genes in adipose tissue; FCGR2A, FCGR2B, FCER1G, and FCGRT. FCGR2A was highly expressed in adipocytes in both depots and this was verified by immunohistochemistry. Expression of IL-1beta and IL-6 was markedly reduced in adipocytes after incubation with the Fc moiety of immunoglobulin G (Fc) (p<0.01). Furthermore, Fc stimulated adipocyte lipogenesis as potently as insulin (p<0.05), but did not influence lipolysis. In conclusion, immunoglobulins produced by plasma cells in human adipose tissue could influence adipocyte metabolism and cytokine production.     

Thiazolidinedione induces the adipose differentiation of fibroblast-like cells resident within bovine skeletal muscle.

To investigate the role of peroxisome proliferator-activated receptor gamma (PPARgamma) in adipocyte formation within the skeletal muscle of beef cattle, fibroblast-like cells were isolated from the longissimus muscle of cattle and cultured with activators of murine PPARgammaA thiazolidinedione T-174, which is a specific ligand for PPARgamma, stimulated adipose differentiation (evaluated by counting differentiated adipocytes under microscopic observation) in a dose-dependent fashion. A peroxisome proliferator Wy14,643 which strongly activates the alpha isoform of murine PPAR also stimulated differentiation but its potency was weaker than that of T-174. Unexpectedly, 15-deoxy-Delta12,14-prostaglandin J2, which is believed to be an endogenous ligand for PPARgamma, could not induce adipose differentiation in doses which have been found to be effective on rodent cells. Immunoblotting analysis confirmed the significant expression of PPARgamma protein in fibroblast-like cell cultures prepared from bovine skeletal muscle. In conclusion, bovine skeletal muscle contains adipose precursor cells expressing functionally active PPARgamma.     

Exosomal proteins constitute an essential part of the human adipose tissue secretome

Adipose tissue is an endocrine organ, secreting various adipokines, either directly or via extracellular vesicles, including exosomes. Exosomes are vesicles of 40–150 nm size that represent a novel concept of biomolecule release. We purified exosomes from isolated primary human preadipocytes differentiated to mature adipocytes. The analyses of these exosomal preparations by LC-MS identified 884 proteins, so called exoadipokines. The comparison of exoadipokines with previously identified human exosome-associated proteins in ExoCarta database show an overlap of 817 proteins, but also revealed 67 proteins not assigned to human exosomes, yet. We further compared all exoadipokines to our previously reported reference secretome of human adipose tissue (http://diabesityprot.org/), finding 212 common proteins, whereas 672 proteins were specific for the exosomal fraction. Bioinformatic analyses revealed that the 212 common proteins can be assigned to all major functions of adipose tissue secreted proteins e.g. molecules involved in fibrotic processes or inflammation. In contrast, the exosome-specific proteins were rather assigned to signaling pathways and membrane-mediated processes. In conclusion, the isolation of exosomes allows to further specify the functionality of adipokines and exoadipokines as part of the adipocyte secretome in signaling and interorgan crosstalk.     

Pref-1 and ADSF/resistin: two secreted factors inhibiting adipose tissue development.

Adipose tissue is the source of a wide array of factors of great biological significance that are involved in many aspects of organism physiology, including appetite control and peripheral metabolism. Here, we describe two secreted factors from adipose tissue that inhibit adipogenesis. Pref-1 is a preadipocyte secreted factor synthesized as a transmembrane protein that undergoes proteolitic cleavage to generate two distinct soluble forms. In vitro assays have demonstrated that only the large soluble form of Pref-1 is biologically active and inhibits adipocyte differentiation. In vivo, mice lacking Pref-1 expression show accelerated fat deposition, perinatal mortality and growth retardation as well as distinct skeletal malformations, highlighting the importance of Pref-1 during mouse development in addition to its role in adipose tissue development. ADSF/resistin is secreted by adipocytes and inhibits adipose cells differentiation in vitro. Its function is still unclear, but its expression and high circulating levels have been associated with an impairment of insulin action. The findings show that Pref-1 and possibly ADSF/resistin secretion control fat cell differentiation and adipose tissue development.     

Functional studies of mesenchymal stem cells derived from adult human adipose tissue

Recent evidence suggests that cells with the properties of human mesenchymal stem cells (hMSCs) can be derived from adult peripheral tissues, including adipose tissue, muscle and dermis. We isolated hMSCs from the stromal-vascular portion of subcutaneous adipose tissue from seven adult subjects. These cells could be readily differentiated into cells of the chondrocyte, osteocyte and adipocyte lineage demonstrating their multipotency. We studied the functional properties of hMSCs-derived adipocytes and compared them with adipocytes differentiated from hMSCs obtained from bone marrow (BM-hMSC). The two cell types displayed similar lipolytic capacity upon stimulation with catecholamines, including a pronounced antilipolytic effect mediated through alpha2A-adrenoceptors, a typical trait in human but not rodent fat cells. Furthermore, both cell types secreted the fat cell-specific factors leptin and adiponectin in comparable amounts per time unit. The fat tissue-derived hMSCs retained their differentiation capacity up to at least fifteen passages. We conclude that hMSCs derived from adult human adipose tissue can be differentiated into fully functional adipocytes with a similar, if not identical, phenotype as that observed in cells derived from BM-hMSCs. Human adipose-tissue-derived MSCs could therefore constitute an efficient and easily obtainable renewable cellular source for studies of adipocyte biology.     

Palmitate- and lipopolysaccharide-activated macrophages evoke contrasting insulin responses in muscle cells

Factors secreted by macrophages contribute to whole body insulin resistance, acting in part on adipose tissue. Muscle is the major tissue for glucose disposal, but how macrophage-derived factors impact skeletal muscle glucose uptake is unknown, or whether the macrophage environment influences this response. We hypothesized that conditioned medium from macrophages pretreated with palmitate or LPS would directly affect insulin action and glucose uptake in muscle cells. L6-GLUT4myc myoblasts were exposed to conditioned medium from RAW 264.7 macrophages pretreated with palmitate or LPS. Conditioned medium from palmitate-treated RAW 264.7 macrophages inhibited myoblast insulin-stimulated glucose uptake, GLUT4 translocation, and Akt phosphorylation while activating JNK p38 MAPK, decreasing IkappaBalpha, and elevating inflammation markers. Surprisingly, and opposite to its effects on adipose cells, conditioned medium from LPS-treated macrophages stimulated myoblast insulin-stimulated glucose uptake, GLUT4 translocation, and Akt phosphorylation without affecting stress kinases or inflammation indexes. This medium had markedly elevated IL-10 levels, and IL-10, alone, potentiated insulin action in myoblasts and partly reversed the insulin resistance imparted by medium from palmitate-treated macrophages. IL-10 neutralizing antibodies blunted the positive influence of LPS macrophage-conditioned medium. We conclude that myoblasts and adipocytes respond differently to cytokines. Furthermore, depending on their environment, macrophages negatively or positively influence muscle cells. Macrophages exposed to palmitate produce a mixture of proinflammatory cytokines that reduce insulin action in muscle cells; conversely, LPS-activated macrophages increase insulin action, likely via IL-10. Macrophages may be an integral element in glucose homeostasis in vivo, relaying effects of circulating factors to skeletal muscle.     

Insulin stimulates interleukin-6 and tumor necrosis factor-alpha gene expression in human subcutaneous adipose tissue

High circulating levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) are found in patients with hyperinsulinemia. Insulin stimulates release of IL-6 from adipocyte cultures, and it stimulates IL-6 gene expression in insulin-resistant, but not control, rat skeletal muscle. In addition, TNF-alpha may be involved in the pathogenesis of insulin resistance. Therefore, we studied the effect of insulin on IL-6 and TNF-alpha gene expression in human skeletal muscle and adipose tissue. Nine healthy young volunteers participated in the study. They underwent a 6-h hyperinsulinemic euglycemic clamp at a fixed insulin infusion rate, with blood glucose clamped at fasting level. Blood samples drawn at 0, 1, 2, 3, 4, 5, and 6 h were analyzed for IL-6 and TNF-alpha. Muscle and fat biopsies, obtained at 0, 2, 4, and 6 h, were analyzed for IL-6 and TNF-alpha mRNA with real-time PCR. IL-6 mRNA increased 11-, 3-, and 5-fold at 2, 4, and 6 h, respectively, in adipose tissue (ANOVA P = 0.027), whereas there was no significant effect of insulin on skeletal muscles. Plasma IL-6 increased during insulin stimulation. TNF-alpha mRNA increased 2.4-, 1.4-, and 2.2-fold in adipose tissue (ANOVA P = 0.001) and decreased 0.74-, 0.64-, and 0.68-fold in muscle tissue (ANOVA P = 0.04). Plasma levels of TNF-alpha were constant. In conclusion, the finding that insulin stimulates IL-6 and TNF-alpha gene expression in adipose tissue only and inhibits the TNF-alpha production in skeletal muscles suggests a differential regulation of muscle- and adipose tissue-derived IL-6 and TNF-alpha.     

甘油诱导的骨骼肌损伤修复过程肌间脂形态学及肌分泌因子表达变化

研究发现在甘油诱导的小鼠肌肉损伤修复过程中可能存在肌间脂的沉积,而肌肉分泌因子（myokines）作为特殊的蛋白参与了肌肉与脂肪的多种生理过程.为研究肌肉内注射甘油后对肌间脂生成的影响,以及注射后肌肉分泌因子在肌肉损伤后修复及肌间脂沉积过程中的表达趋势,本文选用三月龄C57BL/6品系小鼠,右腿胫骨前肌注射50% HBSS(V/V）甘油,左腿胫骨前肌注射等量的HBSS缓冲液作为对照.取注射后不同时期小鼠的胫骨前肌,冰冻切片技术检测肌肉再生及肌间脂沉积状况,实时定量PCR检测各分泌因子（IL-6、IL-15、MSTN、FNDC5、FGF21、myonectin和Insl6）的mRNA表达变化,酶联免疫分析（ELISA）检测分泌因子的蛋白表达变化.结果表明,在甘油诱导的肌肉损伤再生修复过程中存在肌间脂的生成,同时IL-6、Insl6、FGF21和IL-15的mRNA相对表达量在肌肉损伤修复过程中的前、中期变化明显,而MSTN和myonectin的mRNA相对表达量则在中、后期变化明显. IL-6、Insl6的蛋白表达量在前、中期明显升高.综上所述,甘油注射可引起肌肉损伤修复,并在这一过程中伴随着肌间脂的沉积,而肌肉分泌因子作为肌肉与脂肪之间的信息交换因子可能参与了肌肉损伤后的再生修复以及肌间脂的形成.