Potential role of the apelin-APJ pathway in sex-related differential cardiotoxicity induced by doxorubicin in mice.

Preclinical and clinical findings suggest sexual dimorphism in cardiotoxicity induced by a chemotherapeutic drug, doxorubicin (DOX). However, molecular alterations leading to sex-related differential vulnerability of heart to DOX toxicity are not fully explored. In the present study, RNA sequencing in hearts of B6C3F1 mice indicated more differentially expressed genes in males than females (224 vs. 19; ≥1.5-fold, False Discovery Rate [FDR] < 0.05) at 1 week after receiving 24 mg/kg total cumulative DOX dose that induced cardiac lesions only in males. Pathway analysis further revealed probable inactivation of cardiac apelin fibroblast signaling pathway (p = 0.00004) only in DOX-treated male mice that showed ≥1.25-fold downregulation in the transcript and protein levels of the apelin receptor, APJ. In hearts of DOX-treated females, the transcript levels of apelin (1.24-fold) and APJ (1.47-fold) were significantly (p < 0.05) increased compared to saline-treated controls. Sex-related differential DOX effect was also observed on molecular targets downstream of the apelin-APJ pathway in cardiac fibroblasts and cardiomyocytes. In cardiac fibroblasts, upregulation of Tgf-ß2, Ctgf, Sphk1, Serpine1, and Timp1 (fibrosis; FDR < 0.05) in DOX-treated males and upregulation of only Tgf-ß2 and Timp1 (p < 0.05) in females suggested a greater DOX toxicity in hearts of males than females. Additionally, Ryr2 and Serca2 (calcium handling; FDR < 0.05) were downregulated in conjunction with 1.35-fold upregulation of Casp12 (sarcoplasmic reticulum-mediated apoptosis; FDR < 0.05) in DOX-treated male mice. Drug effect on the transcript level of these genes was less severe in female hearts. Collectively, these data suggest a likely role of the apelin-APJ axis in sex-related differential DOX-induced cardiotoxicity in our mouse model.

MicroRNA-34a-5p as a promising early circulating preclinical biomarker of doxorubicin-induced chronic cardiotoxicity.

Cardiotoxicity is a serious adverse effect of an anticancer drug, doxorubicin (DOX), which can occur within a year or decades after completion of therapy. The present study was designed to address a knowledge gap concerning a lack of circulating biomarkers capable of predicting the risk of cardiotoxicity induced by DOX. Profiling of 2083 microRNAs (miRNAs) in mouse plasma revealed 81 differentially expressed miRNAs 1 week after 6, 9, 12, 18, or 24 mg/kg total cumulative DOX doses (early-onset model) or saline (SAL). Among these, the expression of seven miRNAs was altered prior to the onset of myocardial injury at 12 mg/kg and higher cumulative doses. The expression of only miR-34a-5p was significantly (false discovery rate [FDR] < 0.1) elevated at all total cumulative doses compared with concurrent SAL-treated controls and showed a statistically significant dose-related response. The trend in plasma miR-34a-5p expression levels during DOX exposures also correlated with a significant dose-related increase in cardiac expression of miR-34a-5p in these mice. Administration of a cardioprotective drug, dexrazoxane, to mice before DOX treatment, significantly mitigated miR-34a-5p expression in both plasma and heart in conjunction with attenuation of cardiac pathology. This association between plasma and heart may suggest miR-34a-5p as a potential early circulating marker of early-onset DOX cardiotoxicity. In addition, higher expression of miR-34a-5p (FDR < 0.1) in plasma and heart compared with SAL-treated controls 24 weeks after 24 mg/kg total cumulative DOX dose, when cardiac function was altered in our recently established delayed-onset cardiotoxicity model, indicated its potential as an early biomarker of delayed-onset cardiotoxicity.

Doxorubicin-induced delayed-onset subclinical cardiotoxicity in mice.

Subclinical cardiotoxicity at low total cumulative doxorubicin (DOX) doses can manifest into cardiomyopathy in long-term cancer survivors. However, the underlying mechanisms are poorly understood. In male B6C3F1 mice, assessment of cardiac function by echocardiography was performed at 1, 4, 10, 17, and 24 weeks after exposure to 6, 9, 12, and 24 mg/kg total cumulative DOX doses or saline (SAL) to monitor development of delayed-onset cardiotoxicity. The 6- or 9-mg/kg total cumulative doses resulted in a significant time-dependent decline in systolic function (left ventricular ejection fraction (LVEF) and fractional shortening (FS)) during the 24-week recovery although there was not a significant alteration in % LVEF or % FS at any specific time point during the recovery. A significant decline in systolic function was elicited by the cardiotoxic cumulative DOX dose (24 mg/kg) during the 4- to 24-week period after treatment compared to SAL-treated counterparts. At 24 weeks after DOX treatment, a significant dose-related decrease in the expression of genes and proteins involved in sarcoplasmic reticulum (SR) calcium homeostasis (Ryr2 and Serca2) was associated with a dose-related increase in the transcript level of Casp12 (SR-specific apoptosis) in hearts. These mice also showed enhanced apoptotic activity in hearts indicated by a significant dose-related elevation in the number of apoptotic cardiomyocytes compared to SAL-treated counterparts. These findings collectively suggest that a steady decline in SR calcium handling and apoptosis might be involved in the development of subclinical cardiotoxicity that can evolve into irreversible cardiomyopathy later in life.

NFκB-inducing kinase inhibits NFκB activity specifically in neurons of the CNS.

The control of NFκB in CNS neurons appears to differ from that in other cell types. Studies have reported induction of NFκB in neuronal cultures and immunostaining in vivo, but others have consistently detected little or no transcriptional activation by NFκB in brain neurons. To test if neurons lack some component of the signal transduction system for NFκB activation, we transfected cortical neurons with several members of this signaling system along with a luciferase-based NFκB-reporter plasmid; RelA was cotransfected in some conditions. No component of the NFκB pathway was permissive for endogenous NFκB activity, and none stimulated the activity of exogenous RelA. Surprisingly, however, the latter was inhibited by cotransfection of NFκB-inducing kinase (NIK). Fluorescence imaging of RelA indicated that co-expression of NIK sequestered RelA in the cytoplasm, similar to the effect of IκBα. NIK-knockout mice showed elevated expression of an NFκB-reporter construct in neurons in vivo. Cortical neurons cultured from NIK-knockout mice showed elevated expression of an NFκB-reporter transgene. Consistent with data from other cell types, a C-terminal fragment of NIK suppressed RelA activity in astrocytes as well as neurons. Therefore, the inhibitory ability of the NIK C-terminus was unbiased with regard to cell type. However, inhibition of NFκB by full-length NIK is a novel outcome that appears to be specific to CNS neurons. This has implications for unique aspects of transcription in the CNS, perhaps relevant to aspects of development, neuroplasticity, and neuroinflammation. Full-length NIK was found to inhibit (down arrow) transcriptional activation of NFκB in neurons, while it elevated (up arrow) activity in astrocytes. Deletion constructs corresponding to the N-terminus or C-terminus also inhibited NFκB in neurons, while only the C-terminus did so in astrocytes. One possible explanation is that the inhibition in neurons occurs via two different mechanisms, including the potential for a neuron-specific protein (e.g., one of the 14-3-3 class) to create a novel complex in neurons, whereas the C-terminus may interact directly with NFκB. [Structure of NIK is based on Liu J., Sudom A., Min X., Cao Z., Gao X., Ayres M., Lee F., Cao P., Johnstone S., Plotnikova O., Walker N., Chen G., and Wang Z. (2012) Structure of the nuclear factor κB-inducing kinase (NIK) kinase domain reveals a constitutively active conformation. J Biol Chem. 287, 27326-27334); N-terminal lobe is oriented at top].

Evaluation of a microphysiological human placental barrier model for studying placental drug transfer.

Understanding drug transport across the placental barrier is important for assessing the potential fetal drug toxicity and birth defect risks. Current in vivo and in vitro models have structural and functional limitations in evaluating placental drug transfer and toxicity. Microphysiological systems (MPSs) offer more accurate and relevant physiological models of human tissues and organs on a miniature scale for drug development and toxicology testing. MPSs for the placental barrier have been recently explored to study placental drug transfer. We utilized a multilayered hydrogel membrane-based microphysiological model composed of human placental epithelial and endothelial cells to replicate the key structure and function of the human placental barrier. A macroscale human placental barrier model was created using a transwell to compare the results with the microphysiological model. Placental barrier models were characterized by assessing monolayer formation, intercellular junctions, barrier permeability, and their structural integrity. Three small-molecule drugs (glyburide, rifaximin, and caffeine) that are prescribed or taken during pregnancy were studied for their placental transfer. The results showed that all three drugs crossed the placental barrier, with transfer rates in the following order: glyburide (molecular weight, MW = 494 Da) < rifaximin (MW = 785.9 Da) < caffeine (MW = 194.19 Da). Using non-compartmental analysis, we estimated human pharmacokinetic characteristics based on in vitro data from both MPS and transwell models. While further research is needed, our findings suggest that MPS holds potential as an in vitro tool for studying placental drug transfer and predicting fetal exposure, offering insights into pharmacokinetics.

Chondroitin sulfates play a major role in breast cancer metastasis: a role for CSPG4 and CHST11 gene expression in forming surface P-selectin ligands in aggressive breast cancer cells.

INTRODUCTION: We have previously demonstrated that chondroitin sulfate glycosaminoglycans (CS-GAGs) on breast cancer cells function as P-selectin ligands. This study was performed to identify the carrier proteoglycan (PG) and the sulfotransferase gene involved in synthesis of the surface P-selectin-reactive CS-GAGs in human breast cancer cells with high metastatic capacity, as well as to determine a direct role for CS-GAGs in metastatic spread. METHODS: Quantitative real-time PCR (qRT-PCR) and flow cytometry assays were used to detect the expression of genes involved in the sulfation and presentation of chondroitin in several human breast cancer cell lines. Transient transfection of the human breast cancer cell line MDA-MB-231 with the siRNAs for carbohydrate (chondroitin 4) sulfotransferase-11 (CHST11) and chondroitin sulfate proteoglycan 4 (CSPG4 ) was used to investigate the involvement of these genes in expression of surface P-selectin ligands. The expression of CSPG4 and CHST11 in 15 primary invasive breast cancer clinical specimens was assessed by qRT-PCR. The role of CS-GAGs in metastasis was tested using the 4T1 murine mammary cell line (10 mice per group). RESULTS: The CHST11 gene was highly expressed in aggressive breast cancer cells but significantly less so in less aggressive breast cancer cell lines. A positive correlation was observed between the expression levels of CHST11 and P-selectin binding to cells (P < 0.0001). Blocking the expression of CHST11 with siRNA inhibited CS-A expression and P-selectin binding to MDA-MB-231 cells. The carrier proteoglycan CSPG4 was highly expressed on the aggressive breast cancer cell lines and contributed to the P-selectin binding and CS-A expression. In addition, CSPG4 and CHST11 were over-expressed in tumor-containing clinical tissue specimens compared with normal tissues. Enzymatic removal of tumor-cell surface CS-GAGs significantly inhibited lung colonization of the 4T1 murine mammary cell line (P = 0.0002). CONCLUSIONS: Cell surface P-selectin binding depends on CHST11 gene expression. CSPG4 serves as a P-selectin ligand through its CS chain and participates in P-selectin binding to the highly metastatic breast cancer cells. Removal of CS-GAGs greatly reduces metastatic lung colonization by 4T1 cells. The data strongly indicate that CS-GAGs and their biosynthetic pathways are promising targets for the development of anti-metastatic therapies.

Stearoyl-coenzyme A desaturase 1 gene expression increases after pioglitazone treatment and is associated with peroxisomal proliferator-activated receptor-gamma responsiveness.

CONTEXT AND OBJECTIVE: Stearoyl-coenzyme A desaturase (SCD1) is the rate-limiting enzyme that converts palmitoyl- and stearoyl-coenzyme A to palmitoleoyl- and oleoyl-cownzyme A, respectively. SCD-deficient mice are protected from obesity, and the ob/ob mouse has high levels of SCD. This study was designed to better characterize SCD1 gene and protein expression in humans with varying insulin sensitivity. DESIGN, PARTICIPANTS, AND SETTING: In a university hospital clinical research center setting, SCD1 gene expression was measured in sc adipose and vastus lateralis muscle of 86 nondiabetic subjects; 10 wk of pioglitazone (45 mg daily) and metformin (1000 mg twice daily) treatment were assessed in 36 impaired glucose-tolerant subjects. Adipocytes were treated with pioglitazone, and SCD1 expression was attenuated with small interfering RNA (siRNA) to examine other adipocyte genes. RESULTS: There was no significant relationship between adipose or muscle SCD1 mRNA and either body mass index or insulin sensitivity. After pioglitazone (but not metformin) treatment, there was a 2-fold increase in SCD1 mRNA and protein in adipose tissue. Pioglitazone also increased SCD1 in vitro. There were significant positive correlations between SCD1 and peroxisomal proliferator-activated receptor gamma (PPARgamma) as well as other PPARgamma-responsive genes, including lipin-beta, AGPAT2, RBP4, adiponectin receptors, CD68, and MCP1. When SCD1 expression was inhibited with a siRNA, lipin-beta, AGPAT2, and the adiponectin R2 receptor expression were decreased, and adipocyte MCP-1 was increased. CONCLUSIONS: SCD1 is closely linked to PPARgamma expression in humans, and is increased by PPARgamma agonists. The change in expression of some downstream PPARgamma targets after SCD1 knockdown suggests that PPARgamma up-regulation of SCD1 leads to increased lipogenesis and potentiation of adiponectin signaling.

Human visfatin expression: relationship to insulin sensitivity, intramyocellular lipids, and inflammation.

CONTEXT: Visfatin (VF) is a recently described adipokine preferentially secreted by visceral adipose tissue (VAT) with insulin mimetic properties. OBJECTIVE: The aim of this study was to examine the association of VF with insulin sensitivity, intramyocellular lipids (IMCL), and inflammation in humans. DESIGN AND PATIENTS: VF mRNA was examined in paired samples of VAT and abdominal sc adipose tissue (SAT) obtained from subjects undergoing surgery. Plasma VF and VF mRNA was also examined in SAT and muscle tissue, obtained by biopsy from well-characterized subjects with normal or impaired glucose tolerance, with a wide range in body mass index (BMI) and insulin sensitivity (S(I)). SETTING: The study was conducted at a University Hospital and General Clinical Research Center. INTERVENTION: S(I) was measured, and fat and muscle biopsies were performed. In impaired glucose tolerance subjects, these procedures were performed before and after treatment with pioglitazone or metformin. MAIN OUTCOME MEASURES: We measured the relationship between VF and obesity, S(I), adipose tissue inflammation, IMCL, and response to insulin sensitizers. RESULTS: No significant difference in VF mRNA was seen between SAT and VAT depots. VAT VF mRNA associated positively with BMI, whereas SAT VF mRNA decreased with BMI. SAT VF correlated positively with S(I), and the association of SAT VF mRNA with S(I) was independent of BMI. IMCL and markers of inflammation (adipose CD68 and plasma TNFalpha) were negatively associated with SAT VF. Impaired glucose tolerance subjects treated with pioglitazone showed no change in SAT VF mRNA despite a significant increase in S(I). Plasma VF and muscle VF mRNA did not correlate with BMI or S(I) or IMCL, and there was no change in muscle VF with either pioglitazone or metformin treatments. CONCLUSION: SAT VF is highly expressed in lean, more insulin-sensitive subjects and is attenuated in subjects with high IMCL, low S(I), and high levels of inflammatory markers. VAT VF and SAT VF are regulated oppositely with BMI.

Retinol binding protein 4 expression in humans: relationship to insulin resistance, inflammation, and response to pioglitazone.

CONTEXT: Retinol binding protein 4 (RBP4) was recently found to be expressed and secreted by adipose tissue, and was strongly associated with insulin resistance. OBJECTIVE: The aim was to determine the relationship between RBP4 and obesity, insulin resistance, and other markers of insulin resistance in humans. DESIGN AND PATIENTS: RBP4 mRNA levels in adipose tissue and muscle of nondiabetic human subjects with either normal or impaired glucose tolerance (IGT) were studied, along with plasma RBP4. RBP4 gene expression was also measured in adipose tissue fractions, and from visceral and sc adipose tissue (SAT) from surgical patients. SETTING: The study was conducted at University Hospital and General Clinical Research Center. INTERVENTION: Insulin sensitivity (S(I)) was measured, and fat and muscle biopsies were performed. In IGT subjects, these procedures were performed before and after treatment with metformin or pioglitazone. MAIN OUTCOME MEASURES: The relationship between RBP4 expression and obesity, S(I), adipose tissue inflammation, and intramyocellular lipid level, and response to insulin sensitizers was measured. RESULTS: RBP4 was expressed predominantly from the adipocyte fraction of SAT. Although SAT RBP4 expression and the plasma RBP4 level demonstrated no significant relationship with body mass index or S(I), there was a strong positive correlation between RBP4 mRNA and adipose inflammation (monocyte chemoattractant protein-1 and CD68), and glucose transporter 4 mRNA. Treatment of IGT subjects with pioglitazone resulted in an increase in S(I) and an increase in RBP4 gene expression in both adipose tissue and muscle, but not in plasma RBP4 level, and the in vitro treatment of cultured adipocytes with pioglitazone yielded a similar increase in RBP4 mRNA. CONCLUSIONS: RBP4 gene expression in humans is associated with inflammatory markers, but not with insulin resistance. The increase in RBP4 mRNA after pioglitazone treatment is unusual, suggesting a complex regulation of this novel adipokine.

Lipin expression is attenuated in adipose tissue of insulin-resistant human subjects and increases with peroxisome proliferator-activated receptor gamma activation.

Lipin-alpha and -beta are the alternatively spliced gene products of the Lpin1 gene, whose product lipin is required for adipocyte differentiation. Lipin deficiency causes lipodystrophy, fatty liver, and insulin resistance in mice, whereas adipose tissue lipin overexpression results in increased adiposity but improved insulin sensitivity. To assess lipin expression and its relation to insulin resistance in humans, we examined lipin-alpha and -beta mRNA levels in subjects with normal or impaired glucose tolerance. We found higher expression levels of both lipin isoforms in lean, insulin-sensitive subjects. When compared with normal glucose-tolerant subjects, individuals with impaired glucose tolerance were more insulin resistant, demonstrated higher levels of intramyocellular lipids (IMCLs), and expressed approximately 50% lower levels of lipin-alpha and -beta. In addition, there was a strong inverse correlation between adipose tissue lipin expression and muscle IMCLs but no evidence for an increase in muscle lipid oxidation. After treatment of the impaired glucose-tolerant subjects with insulin sensitizers for 10 weeks, pioglitazone (but not metformin) resulted in a 60% increase in the insulin sensitivity index (Si) and a 32% decrease in IMCLs (both P < 0.01), along with an increase in lipin-beta (but not lipin-alpha) expression by 200% (P < 0.005). Lipin expression in skeletal muscle, however, was not related to obesity or insulin resistance. Hence, high adipose tissue lipin expression is found in insulin-sensitive subjects, and lipin-beta expression increases following treatment with pioglitazone. These results suggest that increased adipogenesis and/or lipogenesis in subcutaneous fat, mediated by the LPIN1 gene, may prevent lipotoxicity in muscle, leading to improved insulin sensitivity.

An HLA-E single chain trimer inhibits human NK cell reactivity towards porcine cells.

HLA-E, when expressed by pig cells, could alleviate human natural killer (NK) cell-mediated rejection of porcine xenografts by providing a potent inhibitory ligand for human NK cells expressing CD94/NKG2A. Yet cell-surface expression of HLA-E on porcine epithelial (LLC-PK1) cells was not observed after transfection with an expression vector harboring HLA-E alone or in combination with an expression vector containing human beta2m. A single chain trimer (SCT) of HLA-E consisting of, in the following order (from N- to C-terminus), the leader peptide of human beta2m, VMAPRTLIL (an HLA-E-binding peptide), a 15 amino acid linker, mature human beta2m, a 20 amino acid linker, and mature HLA-E heavy chain was engineered. Cell-surface expression and correct folding of HLA-E SCT was shown by FACS analyses of stably transfected LLC-PK1 cells. Untransfected LLC-PK1 cells were readily lysed by the NK cell lines NKL and NK-92, while LLC-PK1 cells expressing HLA-E SCT were almost completely protected. In addition, the HLA-E SCT recapitulates the peptide dependent properties of normal HLA-E trimeric complexes in that an HLA-E SCT with an hsp60 derived peptide, though expressed at the cell-surface, did not inhibit NK cell-mediated lysis. The HLA-E SCT, which conferred protection against NK cell-mediated killing, also inhibited NK cell IFN-gamma secretion elicited by co-culture of NKL cells with LLC-PK1 cells. Thus, HLA-E SCT, in which all three components of a normal HLA-E protein complex are in one polypeptide chain, is immunologically functional as it is able to modulate NK cell cytotoxicity and cytokine secretion.

Molecular cloning and characterization of a porcine UL16 binding protein (ULBP)-like cDNA.

UL16 binding proteins (ULBPs) are ligands for the NK cell activating receptor NKG2D. A cDNA encoding a porcine ULBP-like protein (PULBP) was cloned and the predicted amino acid sequence exhibited 35-52% identity to human ULBPs. Southern blot analysis suggested that there is only one ULBP-like gene in the pig genome. Transcripts of PULBP and another potential NKG2D ligand, MIC2, were detected by RT-PCR in a wide range of tissues. Recombinant PULBP-Fc and human ULBP2-Fc fusion proteins were made and used to examine PULBP binding to porcine PBMCs and a human NK cell line (NKL cells). PULBP-Fc bound to a subpopulation of porcine PBMCs but not NKL cells. Conversely, human ULBP2-Fc did not bind to porcine PBMCs but did stably interact with NKL cells.

CHST11 gene expression and DNA methylation in breast cancer.

Our previously published data link P-selectin-reactive chondroitin sulfate structures on the surface of breast cancer cells to metastatic behavior of cells. We have shown that a particular sulfation pattern mediated by the expression of carbohydrate (chondroitin 4) sulfotransferase-11 (CHST11) correlates with P-selectin binding and aggressiveness of human breast cancer cell lines. The present study was performed to evaluate the prognostic value of CHST11 expression and determine whether aberrant DNA methylation controls CHST11 expression in breast cancer. Publicly available datasets were used to examine the association of CHST11 expression to aggressiveness and progression of breast cancer. Methylation status was analyzed using bisulfite genomic sequencing. 5-aza-2'-deoxycytidine (5AzadC) was used for DNA demethylation. Reduced representation bisulfite sequencing was performed in the CpG island of CHST11 with a minimum coverage of 10. Quantitative real-time RT-PCR was employed to confirm the expression profile of CHST11 in breast cancer cell lines. Flow cytometry was also used to confirm the expression of the CHST11 product, chondroitin sulfate A (CS-A). The expression of CHST11 was significantly higher in basal-like and Her2-amplified cell lines compared to luminal cell lines. CHST11 was also highly expressed in cancer tissues compared to normal tissues and the expression levels were significantly associated with tumor progression. We observed very low levels of DNA methylation in a CpG island of CHST11 in basal-like cells but very high levels in the same region in luminal cells. Treatment of MCF7 cells, a luminal cell line with very low expression of CHST11, with 5AzadC increased the expression of CHST11 and its immediate product, CS-A, in a dose-dependent manner. These results suggest that CHST11 may play a direct role in progression of breast cancer and that its expression is controlled by DNA methylation. Therefore, in addition to CHST11 mRNA levels, the methylation status of this gene also has potential as a prognostic biomarker.

Metabotropic glutamate receptors inhibit microglial glutamate release.

Pro-inflammatory stimuli evoke an export of glutamate from microglia that is sufficient to contribute to excitotoxicity in neighbouring neurons. Since microglia also express various glutamate receptors themselves, we were interested in the potential feedback of glutamate on this system. Several agonists of mGluRs (metabotropic glutamate receptors) were applied to primary rat microglia, and the export of glutamate into their culture medium was evoked by LPS (lipopolysaccharide). Agonists of group-II and -III mGluR ACPD [(1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid] and L-AP4 [L-(+)-2-amino-4-phosphonobutyric acid] were both capable of completely blocking the glutamate export without interfering with the production of NO (nitric oxide); the group-I agonist tADA (trans-azetidine-2,4-dicarboxylic acid) was ineffective. Consistent with the possibility of feedback, inhibition of mGluR by MSPG [(R,S)-α-2-methyl-4sulfonophenylglycine] potentiated glutamate export. As the group-II and -III mGluR are coupled to Gαi-containing G-proteins and the inhibition of adenylate cyclase, we explored the role of cAMP in this effect. Inhibition of cAMP-dependent protein kinase [also known as protein kinase A (PKA)] by H89 mimicked the effect of ACPD, and the mGluR agonist had its actions reversed by artificially sustaining cAMP through the PDE (phosphodiesterase) inhibitor IBMX (isobutylmethylxanthine) or the cAMP mimetic dbcAMP (dibutyryl cAMP). These data indicate that mGluR activation attenuates a potentially neurotoxic export of glutamate from activated microglia and implicate cAMP as a contributor to this aspect of microglial action.

Adipose triglyceride lipase expression in human adipose tissue and muscle. Role in insulin resistance and response to training and pioglitazone.

Adipose triglyceride lipase (ATGL) catalyzes the first step in adipocyte and muscle triglyceride hydrolysis, and comparative gene identification-58 (CGI-58) is an essential cofactor. We studied the expression of ATGL and CGI-58 in human adipose and muscle and examined correlations with markers of muscle fatty acid oxidation. Nondiabetic volunteers were studied. Subjects with impaired glucose tolerance were treated with pioglitazone or metformin for 10 weeks. Subjects with normal glucose tolerance underwent a 12-week training program. We examined changes in ATGL and CGI-58 with obesity and insulin resistance, and effects of exercise and pioglitazone. Adipose triglyceride lipase messenger RNA (mRNA) expression showed no correlation with either body mass index or insulin sensitivity index in either adipose or muscle. However, adipose ATGL protein levels were inversely correlated with body mass index (r = -0.64, P < .02) and positively correlated with insulin sensitivity index (r = 0.67, P < .02). In muscle, ATGL mRNA demonstrated a strong positive relationship with carnitine palmitoyltransferase I mRNA (r = 0.82, P < .0001) and the adiponectin receptors AdipoR1 mRNA (r = 0.71, P < .0001) and AdipoR2 mRNA (r = 0.74, P < .0001). Muscle CGI-58 mRNA was inversely correlated with intramyocellular triglyceride in both type 1 (r = -0.35, P < .05) and type 2 (r = -0.40, P < .05) fibers. Exercise training resulted in increased muscle ATGL, and pioglitazone increased adipose ATGL by 31% (P < .05). Pioglitazone also increased ATGL in adipocytes. Adipose ATGL protein is decreased with insulin resistance and obesity; and muscle ATGL mRNA is associated with markers of fatty acid oxidation in muscle, as is CGI-58. The regulation of ATGL and CGI-58 has important implications for the control of lipotoxicity.

Muscle inflammatory response and insulin resistance: synergistic interaction between macrophages and fatty acids leads to impaired insulin action.

Obesity is characterized by adipose tissue expansion as well as macrophage infiltration of adipose tissue. This results in an increase in circulating inflammatory cytokines and nonesterified fatty acids, factors that cause skeletal muscle insulin resistance. Whether obesity also results in skeletal muscle inflammation is not known. In this study, we quantified macrophages immunohistochemically in vastus lateralis biopsies from eight obese and eight lean subjects. Our study demonstrates that macrophages infiltrate skeletal muscle in obesity, and we developed an in vitro system to study this mechanistically. Myoblasts were isolated from vastus lateralis biopsies and differentiated in culture. Coculture of differentiated human myotubes with macrophages in the presence of palmitic acid, to mimic an obese environment, revealed that macrophages in the presence of palmitic acid synergistically augment cytokine and chemokine expression in myotubes, decrease IkappaB-alpha protein expression, increase phosphorylated JNK, decrease phosphorylated Akt, and increase markers of muscle atrophy. These results suggest that macrophages alter the inflammatory state of muscle cells in an obese milieu, inhibiting insulin signaling. Thus in obesity both adipose tissue and skeletal muscle inflammation may contribute to insulin resistance.

Thrombospondin-1 is an adipokine associated with obesity, adipose inflammation, and insulin resistance.

OBJECTIVE: We examined the relationship between the expression of thrombospondin (TSP)1, an antiangiogenic factor and regulator of transforming growth factor-beta activity, obesity, adipose inflammation, and insulin resistance. RESEARCH DESIGN AND METHODS: TSP1 gene expression was quantified in subcutaneous adipose tissue (SAT) of 86 nondiabetic subjects covering a wide range of BMI and insulin sensitivity, from visceral adipose (VAT) and SAT from 14 surgical patients and from 38 subjects with impaired glucose tolerance randomized to receive either pioglitazone or metformin for 10 weeks. An adipocyte culture system was also used to assess the effects of pioglitazone and coculture with macrophages on TSP1 gene expression. RESULTS: TSP1 mRNA was significantly associated with obesity (BMI) and insulin resistance (low insulin sensitivity index). Relatively strong positive associations were seen with markers of inflammation, including CD68, macrophage chemoattractant protein-1, and plasminogen activator inhibitor (PAI)-1 mRNA (r >/= 0.46, P = 0.001 for each), that remained significant after controlling for BMI and S(i). However, TSP1 mRNA was preferentially expressed in adipocyte fraction, whereas inflammatory markers predominated in stromal vascular fraction. Coculture of adipocytes and macrophages augmented TSP1 gene expression and secretion from both cell types. Pioglitazone (not metformin) treatment resulted in a 54% decrease (P < 0.04) in adipose TSP gene expression, as did in vitro pioglitazone treatment of adipocytes. CONCLUSIONS: TSP1 is a true adipokine that is highly expressed in obese, insulin-resistant subjects; is highly correlated with adipose inflammation; and is decreased by pioglitazone. TSP1 is an important link between adipocytes and macrophage-driven adipose tissue inflammation and may mediate the elevation of PAI-1 that promotes a prothrombotic state.

Characterization of porcine TAP genes: alternative splicing of TAP1.

The transporter associated with antigen processing (TAP) is a heterodimer composed of TAP1 and TAP2 subunits that belong to the ATP-binding cassette family of transporters. TAP translocates small peptides (usually 8- to 12-amino-acid-long) from the cytosol to the endoplasmic reticulum for subsequent loading onto the major histocompatibility complex (MHC) class I molecules. The translocated peptides are required for the stable cell surface expression of MHC class I molecules. Virus-encoded proteins, which inhibit TAP activity, include ICP47 from herpes simplex virus and US6 from human cytomegalovirus. We have previously shown that ICP47 downregulated porcine MHC class I [swine leukocyte Ag class I (SLA I)] cell-surface expression in the pig epithelial cell line PK(15). Here we show that SLA I cell-surface expression in the pig epithelial cell line LLC-PK1 is relatively unaffected by expression of ICP47. Anticipating that this might be due to differences in the primary structure of TAP1 or TAP2 expressed by these two cell lines, cDNAs from PK(15) and LLC-PK1 encoding the complete open reading frames of porcine TAP1 and TAP2 were cloned and sequenced. Porcine TAP1 and TAP2 exhibited 80% amino acid identity with their human orthologs. Two splice variants of TAP1 were found. In LLC-PK1 cells, an alternatively spliced TAP1 transcript was detected, which was predicted to encode a protein with nine fewer amino acids. While the deleted amino acids may be in close proximity to the putative peptide/ICP47-binding site, we were unable to demonstrate that this imparted an apparent resistance to the effects of ICP47 on SLA I surface expression.

The lipogenic enzymes DGAT1, FAS, and LPL in adipose tissue: effects of obesity, insulin resistance, and TZD treatment.

Acyl-coenzyme A:diacylglycerol transferase (DGAT), fatty acid synthetase (FAS), and LPL are three enzymes important in adipose tissue triglyceride accumulation. To study the relationship of DGAT1, FAS, and LPL with insulin, we examined adipose mRNA expression of these genes in subjects with a wide range of insulin sensitivity (SI). DGAT1 and FAS (but not LPL) expression were strongly correlated with SI. In addition, the expression of DGAT1 and FAS (but not LPL) were higher in normal glucose-tolerant subjects compared with subjects with impaired glucose tolerance (IGT) (P < 0.005). To study the effects of insulin sensitizers, subjects with IGT were treated with pioglitazone or metformin for 10 weeks, and lipogenic enzymes were measured in adipose tissue. After pioglitazone treatment, DGAT1 expression was increased by 33 +/- 10% (P < 0.05) and FAS expression increased by 63 +/- 8% (P < 0.05); however, LPL expression was not altered. DGAT1, FAS, and LPL mRNA expression were not significantly changed after metformin treatment. The treatment of mice with rosiglitazone also resulted in an increase in adipose expression of DGAT1 by 2- to 3-fold, as did the treatment of 3T3 F442A adipocytes in vitro with thiazolidinediones. These data support a more global concept suggesting that adipose lipid storage functions to prevent peripheral lipotoxicity.

Pioglitazone induces apoptosis of macrophages in human adipose tissue.

Metabolic syndrome and type 2 diabetes mellitus are associated with an increased number of macrophage cells that infiltrate white adipose tissue (WAT). Previously, we demonstrated that the treatment of subjects with impaired glucose tolerance (IGT) with the peroxisome proliferator-activated receptor gamma (PPARgamma) agonist pioglitazone resulted in a decrease in macrophage number in adipose tissue. Here, adipose tissue samples from IGT subjects treated with pioglitazone were examined for apoptosis with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining. TUNEL-positive cells were identified, and there was a significant 42% increase in TUNEL-positive cells following pioglitazone treatment. Overlay experiments with anti-CD68 antibody demonstrated that most of the TUNEL-positive cells were macrophages. To determine whether macrophage apoptosis was a direct or indirect effect of pioglitazone treatment, human THP1 cells were treated with pioglitazone in vitro, demonstrating increased TUNEL staining in a dose- and time-dependent manner. Furthermore, the appearance of the active proteolytic subunits of caspase-3 and caspase-9 were detected in cell lysate from THP1 cells and also increased in a dose- and time-dependent manner following pioglitazone treatment. Pretreatment with a PPARgamma inhibitor, GW9662, prevented pioglitazone induction of the apoptotic pathway in THP1 cells. Differentiated human adipocytes did not show any significant increase in apoptosis after treatment in vitro with piolgitazone. These findings indicate that PPARgamma has distinct functions in different cell types in WAT, such that pioglitazone reduces macrophage infiltration by inducing apoptotic cell death specifically in macrophages through PPARgamma activation.