Visfatin is released from 3T3-L1 adipocytes via a non-classical pathway

Visfatin is a secretory protein which exerts insulin mimetic and proinflammatory effects, also functioning as an intracellular enzyme to produce NAD. Plasma visfatin levels and visfatin mRNA expression in adipose tissues are increased in obese subjects. Visfatin does not have a decent cleavable signal sequence, and the mechanism, that mediates release of visfatin from adipocytes, remains poorly understood. In this study, we demonstrate that visfatin is released abundantly into culture medium from 3T3-L1 adipocytes. Subcellular fractionation analysis showed that visfatin was localized in the cytosol, but not in nucleus, membrane, vesicles, or mitochondria fractions. Visfatin release was not reduced by Brefeldin A and Monensin, inhibitors of endoplasmic reticulum (ER)-Golgi-dependent secretion. In addition, visfatin was not released on microvesicles. These results suggest that visfatin should be released from 3T3-L1 adipocytes via an ER-Golgi or microvesicles independent pathway.     

Visfatin: gene expression in isolated adipocytes and sequence analysis in obese WOKW rats compared with lean control rats

Recently, Fukuhara et al. have shown that the novel visfatin is predominantly released from visceral adipocytes and shares metabolic functions with insulin. The authors suggested that there is a relationship between visfatin and the metabolic syndrome in humans. These findings prompted us to clarify the visfatin gene expression from visceral and subcutaneous adipocytes in WOKW rats, as an animal model for polygenically inherited metabolic syndrome, compared to lean Dark Agouti (DA) rats. Moreover, visfatin sequence analysis was performed in WOKW, DA rats, and disease-resistant Fisher 344, Lewis, Brown Norway, and Karlsburg wild rats. The relative gene expression of visfatin displays no significant changes in adipocytes from WOKW rats compared with DA and visfatin sequence analysis of the coding region was identical to the GenBank. But, we found length differences of two repeats, GT and GA, in intron 2 between the strains. In summary, the relative visfatin gene expression is not associated with the metabolic syndrome in WOKW rats.     

Immunophilin-ligands FK506 and CsA inhibit HIF1alpha expression by a VHL- and ubiquitin-independent mechanism

Hypoxia inducible factor-1alpha (HIF1alpha) plays a key role in the regulation of genes controlling oxygen supply, glucose metabolism and angiogenesis. Its expression in tumors appears to confer an adaptive advantage to their hypoxic microenvironment. We have evaluated the effect of the immunophilin ligands FK506 and cyclosporin A on HIF1alpha levels in different tumor cell lines. Our results indicate that both drugs are potent suppressors of HIF1alpha expression by accelerating the proteasomal degradation of the protein. Unexpectedly, the suppressive effect of these compounds was found to be independent of the presence of von Hippel Lindau factor and the degree of hydroxylation of the HIF1alpha protein. Moreover, HIF1alpha degradation induced by these compounds did not required ubiquitination, as it was also induced in E1 ligase-incompetent cells.     

Hypoxic induction of human visfatin gene is directly mediated by hypoxia-inducible factor-1

Visfatin has been originally identified as a growth factor for early stage B cells and recently known as an adipokine. Here, we report that hypoxia induces the visfatin mRNA and protein levels in MCF7 breast cancer cells. We also demonstrate that induction of visfatin gene is regulated by hypoxia-inducible factor-1alpha (HIF-1alpha). Moreover, 5'-flanking promoter region of human visfatin gene contains two functional HIF responsive elements (HREs), activating the expression of visfatin. Mutation of these HREs in the visfatin promoter abrogates activation of a luciferase reporter gene driven by visfatin promoter under hypoxia. Taken together, our results demonstrate that visfatin is a new hypoxia-inducible gene of which expression is stimulated through the interaction of HIF-1 with HRE sites in its promoter region.     

Hypoxia increases expression of selective facilitative glucose transporters (GLUT) and 2-deoxy-D-glucose uptake in human adipocytes

Hypoxia modulates the production of key inflammation-related adipokines and may underlie adipose tissue dysfunction in obesity. Here we have examined the effects of hypoxia on glucose transport by human adipocytes. Exposure of adipocytes to hypoxia (1% O₂) for up to 24 h resulted in increases in GLUT-1 (9.2-fold), GLUT-3 (9.6-fold peak at 8 h), and GLUT-5 (8.9-fold) mRNA level compared to adipocytes in normoxia (21% O₂). In contrast, there was no change in GLUT-4, GLUT-10 or GLUT-12 expression. The rise in GLUT-1 mRNA was accompanied by a substantial increase in GLUT-1 protein (10-fold), but there was no change in GLUT-5; GLUT-3 protein was not detected. Functional studies with [³H]2-deoxy-d-glucose showed that hypoxia led to a stimulation of glucose transport (4.4-fold) which was blocked by cytochalasin B. These results indicate that hypoxia increases monosaccharide uptake capacity in human adipocytes; this may contribute to adipose tissue dysregulation in obesity.     

Stanniocalcin-1 is induced by hypoxia inducible factor in rat alveolar epithelial cells

Alveolar type II (ATII) cells remain differentiated and express surfactant proteins when cultured at an air–liquid (A/L) interface. When cultured under submerged conditions, ATII cells dedifferentiate and change their gene expression profile. We have previously shown that gene expression under submerged conditions is regulated by hypoxia inducible factor (HIF) signaling due to focal hypoxia resulting from ATII cell metabolism. Herein, we sought to further define gene expression changes in ATII cells cultured under submerged conditions. We performed a genome wide microarray on RNA extracted from rat ATII cells cultured under submerged conditions for 24–48 h after switching from an A/L interface. We found significant alterations in gene expression, including upregulation of the HIF target genes stanniocalcin-1 (STC1), tyrosine hydroxylase (Th), enolase (Eno) 2, and matrix metalloproteinase (MMP) 13, and we verified upregulation of these genes by RT-PCR. Because STC1, a highly evolutionarily conserved glycoprotein with anti-inflammatory, anti-apoptotic, anti-oxidant, and wound healing properties, is widely expressed in the lung, we further explored the potential functions of STC1 in the alveolar epithelium. We found that STC1 was induced by hypoxia and HIF in rat ATII cells, and this induction occurred rapidly and reversibly. We also showed that recombinant human STC1 (rhSTC1) enhanced cell motility with extended lamellipodia formation in alveolar epithelial cell (AEC) monolayers but did not inhibit the oxidative damage induced by LPS. We also confirmed that STC1 was upregulated by hypoxia and HIF in human lung epithelial cells. In this study, we have found that several HIF target genes including STC1 are upregulated in AECs by a submerged condition, that STC1 is regulated by hypoxia and HIF, that this regulation is rapidly and reversibly, and that STC1 enhances wound healing moderately in AEC monolayers. However, STC1 did not inhibit oxidative damage in rat AECs stimulated by LPS in vitro. Therefore, alterations in gene expression by ATII cells under submerged conditions including STC1 were largely induced by hypoxia and HIF, which may be relevant to our understanding of the pathogenesis of various lung diseases in which the alveolar epithelium is exposed to relative hypoxia.     

Mitochondrial nitric oxide synthase is constitutively active and is functionally upregulated in hypoxia

Nitric oxide is a potent modulator of mitochondrial respiration, ATP synthesis, and K_ATP channel activity. Recent studies show the presence of a potentionally new isoform of the nitric oxide synthase (NOS) enzyme in mitochondria, although doubts have emerged regarding the physiological relevance of mitochondrial NOS (mtNOS). The aim of the present study were to: (i) examine the existence and distribution of mtNOS in mouse tissues using three independent methods, (ii) characterize the cross-reaction of mtNOS with antibodies against the known isoforms of NOS, and (iii) investigate the effect of hypoxia on mtNOS activity. Nitric oxide synthase activity was measured in isolated brain and liver mitochondria using the arginine to citrulline conversion assay. Mitochondrial NOS activity in the brain was significantly higher than in the liver. The calmodulin inhibitor calmidazolium completely inhibited mtNOS activity. In animals previously subjected to hypoxia, mtNOS activity was significantly higher than in the normoxic controls. Antibodies against the endothelial (eNOS), but not the neuronal or inducible isoform of NOS, showed positive immunoblotting. Immunogold labeling of eNOS located the enzyme in the matrix and the inner membrane using electron microscopy. We conclude that mtNOS is a constitutively active eNOS-like isoform and is involved in altered mitochondrial regulation during hypoxia.     

Pathologic HIF1α signaling drives adipose progenitor dysfunction in obesity

1. Download : Download high-res image (245KB)
2. Download : Download full-size image

     

Suppression of hypoxia inducible factor-1alpha (HIF-1alpha) by YC-1 is dependent on murine double minute 2 (Mdm2)

Inhibition of HIF-1alpha activity provides an important strategy for the treatment of cancer. Recently, 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1) has been identified as an anti-HIF-1alpha drug in cancer therapy with unclear molecular mechanism. In the present study, we aimed to investigate the effect and mechanism of YC-1 on HIF-1alpha in a hepatocellular carcinoma cell line under hypoxic condition, which was generated by incubating cells with 0.1% O(2). The phenotypic and molecular changes of cells were determined by cell proliferation assay, apoptosis assay, luciferase promoter assay, and Western blot analysis. YC-1 arrested tumor cell growth in a dose-dependent manner, whereas it did not induce cell apoptosis. Hypoxia-induced upregulation of HIF-1alpha was suppressed by YC-1 administration. YC-1 inhibited HIF-1alpha protein synthesis under normoxia and affected protein stability under hypoxia. YC-1 suppressed the expression of total and phosphorylated forms of murine double minute 2 (Mdm2), whereas this inhibitory effect was blocked by overexpression of Mdm2. In conclusion, YC-1 suppressed both protein synthesis and stability of HIF-1alpha in HCC cells, and its inhibitory effects on HIF-1alpha were dependent on Mdm2.