Zinc-alpha2-glycoprotein, a lipid mobilizing factor, is expressed and secreted by human (SGBS) adipocytes

Zinc-alpha2-glycoprotein (ZAG), a lipid mobilizing factor, is expressed in mouse adipose tissue and is markedly upregulated in mice with cancer cachexia. We have explored whether ZAG is expressed and secreted by human adipocytes, using SGBS cells, and examined the regulation of ZAG expression. ZAG mRNA was detected by RT-PCR in mature human adipocytes and in SGBS cells post-, but not pre-, differentiation to adipocytes. Relative ZAG mRNA levels increased rapidly after differentiation of SGBS cells, peaking at day 8 post-induction. ZAG protein was evident in differentiated adipocytes (by day 3) and also detected in the culture medium (by day 6) post-induction. The PPARgamma agonist rosiglitazone induced a 3-fold increase in ZAG mRNA level, while TNF-alpha led to a 4-fold decrease. Human adipocytes express and secrete ZAG, with ZAG expression being regulated particularly through TNF-alpha and the PPARgamma nuclear receptor. ZAG is a novel adipokine, which may be involved in the local regulation of adipose tissue function.     

The tyrosine kinase receptor HER2 (erbB-2): from oncogenesis to adipogenesis

Recent experimental evidences begin to support the notion that the proto-oncogene HER2 (erbB-2) might unexpectedly function to modulate the adipogenic conversion of preadipocytes. Two opposing scenarios have been proposed, however, to explain the influence of HER2 on adipocyte differentiation. In one hand, down-modulation of HER2 expression and pharmacological reduction of HER2 activity have been related to enhanced adipocyte differentiation. On the contrary, an increased abundance in HER2 has been described in differentiated adipocytes compared with preadipocytes. Considering that expression and activity of the lipogenic enzyme Fatty Acid Synthase (FASN) become up-regulated during adipogenic conversion, we recently hypothesized that a "HER2 --> FASN axis" -a "lipogenic benefit" that has been shown to enhance cancer cell proliferation, survival, chemoresistance and metastasis in biologically aggressive subgroups of breast carcinomas-might also naturally work during the differentiation of preadipocytes. To definitely clarify if the discrepancy between the opposing theories for a role of HER2 during adipocyte differentiation related to the experimental approach utilized to compare the abundance of HER2 in undifferentiated and differentiated adipocytes (i.e., cell lysates containing equivalent protein content versus cell lysates generated from similar cell numbers), we here took advantage of a high content microscopy approach. Using an automated confocal imaging platform, we monitored the expression status of the adipogenic marker FASN and its timing relationship with HER2 not only in individual 3T3-L1 cells but further in whole cultures of 3T3-L1 preadipocytes undergoing adipogenic conversion. Our findings not only confirm a non-oncogenic role for HER2 in the process of adipose differentiation but further suggest that HER2 might represent a previously unrecognized target to manage obesity via the lipogenic enzyme FASN.     

Heparin-binding epidermal growth factor-like growth factor inhibits adipocyte differentiation at commitment and early induction stages

Abstract Adipocytokines, bioactive molecules secreted from adipose tissues, play important roles in physiology, development, and disease. Recently, heparin-binding epidermal growth factor-like growth factor (HB-EGF) was identified as an adipocytokine whose expression correlates with obesity. However, the biological role of fat-secreted HB-EGF is still unclear. In this study, we investigated the effects of HB-EGF on the adipocyte differentiation of C3H10T1/2 pluripotent mesenchymal cells. Upon adipogenic conversion of C3H10T1/2 cells, HB-EGF displayed dynamic changes in expression where an initial decrease was followed by increased levels of expression at later stages. HB-EGF treatment during adipogenic induction inhibited lipid accumulation and decreased the expression of adipocyte molecular markers (fatty acid-binding protein, peroxisome proliferator-activated receptor γ, and CAAT enhancer-binding protein α) and lipogenic genes (glucose transporter, fatty acid synthetase, and lipoprotein lipase). Therefore, HB-EGF has an inhibitory effect on adipocyte differentiation. Administration of HB-EGF at various intervals during adipocyte differentiation revealed that HB-EGF acts during the early stages of adipocyte differentiation, but not at the later stages of differentiation. Furthermore, HB-EGF was able to block the commitment of pluripotent mesenchymal cells to the adipocyte lineage triggered by bone morphogenic protein 4 treatment. These data suggest that HB-EGF acts as a negative regulator of adipogenesis by inhibiting the commitment and early differentiation of the adipose lineage. The inhibitory role of HB-EGF on adipocyte differentiation of pluripotent mesenchymal cells sheds light on potential mechanisms that control adipose tissue homeostasis.     

Unexpected blockade of adipocyte differentiation by K-7174: implication for endoplasmic reticulum stress

Preadipocytes constitutively express GATA-2 and GATA-3 that are required to halt the cells at the undifferentiated stage. However, we unexpectedly found that K-7174, a GATA-specific inhibitor, did not induce but rather inhibited differentiation of 3T3-L1 preadipocytes. It was associated with lack of lipid accumulation, blunted expression of adipocyte markers including adiponectin and peroxisome proliferator-activated receptor gamma (PPARgamma), and sustained expression of a preadipocyte marker monocyte chemoattractant protein 1 (MCP-1). Subsequent experiments revealed that K-7174 had the potential to induce endoplasmic reticulum (ER) stress evidenced by induction of GRP78 and CHOP. Other inducers of ER stress completely reproduced the effects of K-7174 including suppression of lipid accumulation, blockade of induction of adiponection and PPARgamma and maintenance of MCP-1 expression. These results indicated a possibility that ER stress suppresses adipocyte differentiation and that GATA inhibitor K-7174 has the potential for interfering with adipogenesis through induction of ER stress.     

The retinoblastoma-histone deacetylase 3 complex inhibits PPARgamma and adipocyte differentiation

The retinoblastoma protein (RB) has previously been shown to facilitate adipocyte differentiation by inducing cell cycle arrest and enhancing the transactivation by the adipogenic CCAAT/enhancer binding proteins (C/EBP). We show here that the peroxisome proliferator-activated receptor gamma (PPARgamma), a nuclear receptor pivotal for adipogenesis, promotes adipocyte differentiation more efficiently in the absence of RB. PPARgamma and RB were shown to coimmunoprecipitate, and this PPARgamma-RB complex also contains the histone deacetylase HDAC3, thereby attenuating PPARgamma's capacity to drive gene expression and adipocyte differentiation. Dissociation of the PPARgamma-RB-HDAC3 complex by RB phosphorylation or by inhibition of HDAC activity stimulates adipocyte differentiation. These observations underscore an important function of both RB and HDAC3 in fine-tuning PPARgamma activity and adipocyte differentiation.     

Adipogenic differentiating agents regulate expression of fatty acid binding protein and CD36 in the J744 macrophage cell line

Adipocyte fatty acid binding protein (aP2) is a key mediator of intracellular transport and metabolism of fatty acids. Its expression during adipocyte differentiation is regulated through the actions of peroxisome proliferator-activated receptor gamma (PPARgamma) and CCAAT/enhancer binding protein alpha (C/EBPalpha). Macrophages also express aP2, and the lack of macrophage aP2 significantly reduces atherosclerotic lesion size in hypercholesterolemic mice. We investigated the regulation of expression of macrophage aP2 and CD36, a fatty acid membrane binding protein and scavenger receptor, in response to the adipogenic agents isobutylmethylxanthine (IBMX), insulin, and dexamethasone, a combination of agents shown to induce fibroblast-to-adipocyte differentiation. Treatment of J774 macrophages with adipogenic agents significantly induced aP2 mRNA expression, while CD36 expression was inhibited. Dexamethasone was essential and sufficient to induce aP2 expression, and insulin had a synergistic effect. However, IBMX antagonized induced-aP2 expression. aP2 protein expression and [14C]oleic acid uptake by macrophages were also increased by dexamethasone. Unlike what occurs in adipocytes, adipogenic agents had mixed effects on the expression of PPARgamma and C/EBPalpha in macrophages. Our data demonstrate differences in the regulation of aP2 in adipocytes and macrophages and show that macrophage aP2 expression by adipogenic agents is independent of the PPARgamma and/or C/EBPalpha signaling pathway.     

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.     

Adipocytic differentiation and liver x receptor pathways regulate the accumulation of triacylglycerols in human vascular smooth muscle cells

Lipid accumulation by vascular smooth muscle cells (VSMC) is a feature of atherosclerotic plaques. In this study we describe two mechanisms whereby human VSMC foam cell formation is driven by de novo synthesis of fatty acids leading to triacylglycerol accumulation in intracellular vacuoles, a process distinct from serum lipoprotein uptake. VSMC cultured in adipogenic differentiation medium accumulated lipids and were induced to express the adipocyte marker genes adipsin, adipocyte fatty acid-binding protein, C/EBPalpha, PPARgamma, and leptin. However, complete adipocyte differentiation was not observed as numerous genes present in mature adipocytes were not detected, and the phenotype was reversible. The rate of lipid accumulation was not affected by PPARgamma agonists, but screening for the effects of other nuclear receptor agonists showed that activation of the liver X receptors (LXR) dramatically promoted lipid accumulation in VSMC. Both LXRalpha and LXRbeta were present in VSMC, and their activation with TO901317 resulted in induction of the lipogenic genes fatty acid synthetase, sterol regulatory element binding protein (SREBP1c), and stearoyl-CoA desaturase. 27-Hydroxycholesterol, an abundant oxysterol synthesized by VSMC acted as an LXR antagonist and, therefore, may have a protective role in preventing foam cell formation. Immunohistochemistry showed that VSMC within atherosclerotic plaques express adipogenic and lipogenic markers, suggesting these pathways are present in vivo. Moreover, the development of an adipogenic phenotype in VSMC is consistent with their known phenotypic plasticity and may contribute to their dysfunction in atherosclerotic plaques and, thus, impinge on plaque growth and stability.