Pan-Cancer Proteomics Analysis to Identify Tumor-Enriched and Highly Expressed Cell Surface Antigens as Potential Targets for Cancer Therapeutics.

The National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium (CPTAC) provides unique opportunities for cancer target discovery using protein expression. Proteomics data from CPTAC tumor types have been primarily generated using a multiplex tandem mass tag (TMT) approach, which is designed to provide protein quantification relative to reference samples. However, relative protein expression data are suboptimal for prioritization of targets within a tissue type, which requires additional reprocessing of the original proteomics data to derive absolute quantitation estimation. We evaluated the feasibility of using differential protein analysis coupled with intensity-based absolute quantification (iBAQ) to identify tumor-enriched and highly expressed cell surface antigens, employing tandem mass tag (TMT) proteomics data from CPTAC. Absolute quantification derived from TMT proteomics data was highly correlated with that of label-free proteomics data from the CPTAC colon adenocarcinoma cohort, which contains proteomics data measured by both approaches. We validated the TMT-iBAQ approach by comparing the iBAQ value to the receptor density value of HER2 and TROP2 measured by flow cytometry in about 30 selected breast and lung cancer cell lines from the Cancer Cell Line Encyclopedia. Collections of these tumor-enriched and highly expressed cell surface antigens could serve as a valuable resource for the development of cancer therapeutics, including antibody-drug conjugates and immunotherapeutic agents.

Protein kinase A-dependent phosphorylation stimulates the transcriptional activity of hypoxia-inducible factor 1.

Hypoxia-inducible factor 1 (HIF-1) activates the transcription of genes encoding proteins that enable cells to adapt to reduced O2 availability. Proteins encoded by HIF-1 target genes play a central role in mediating physiological processes that are dysregulated in cancer and heart disease. These diseases are also characterized by increased production of cyclic adenosine monophosphate (cAMP), the allosteric activator of cAMP-dependent protein kinase A (PKA). Using glutathione S-transferase pull-down, coimmunoprecipitation, and mass spectrometry analyses, we demonstrated that PKA interacts with HIF-1α in HeLa cervical carcinoma cells and rat cardiomyocytes. PKA phosphorylated Thr(63) and Ser(692) on HIF-1α in vitro and enhanced HIF transcriptional activity and target gene expression in HeLa cells and rat cardiomyocytes. PKA inhibited the proteasomal degradation of HIF-1α in an O2-independent manner that required the phosphorylation of Thr(63) and Ser(692) and was not affected by prolyl hydroxylation. PKA also stimulated the binding of the coactivator p300 to HIF-1α to enhance its transcriptional activity and counteracted the inhibitory effect of asparaginyl hydroxylation on the association of p300 with HIF-1α. Furthermore, increased cAMP concentrations enhanced the expression of HIF target genes encoding CD39 and CD73, which are enzymes that convert extracellular adenosine 5'-triphosphate to adenosine, a molecule that enhances tumor immunosuppression and reduces heart rate and contractility. These data link stimuli that promote cAMP signaling, HIF-1α-dependent changes in gene expression, and increased adenosine, all of which contribute to the pathophysiology of cancer and heart disease.

Hypoxia induces the breast cancer stem cell phenotype by HIF-dependent and ALKBH5-mediated m6A-demethylation of NANOG mRNA.

N(6)-methyladenosine (m(6)A) modification of mRNA plays a role in regulating embryonic stem cell pluripotency. However, the physiological signals that determine the balance between methylation and demethylation have not been described, nor have studies addressed the role of m(6)A in cancer stem cells. We report that exposure of breast cancer cells to hypoxia stimulated hypoxia-inducible factor (HIF)-1α- and HIF-2α-dependent expression of AlkB homolog 5 (ALKBH5), an m(6)A demethylase, which demethylated NANOG mRNA, which encodes a pluripotency factor, at an m(6)A residue in the 3'-UTR. Increased NANOG mRNA and protein expression, and the breast cancer stem cell (BCSC) phenotype, were induced by hypoxia in an HIF- and ALKBH5-dependent manner. Insertion of the NANOG 3'-UTR into a luciferase reporter gene led to regulation of luciferase activity by O2, HIFs, and ALKBH5, which was lost upon mutation of the methylated residue. ALKBH5 overexpression decreased NANOG mRNA methylation, increased NANOG levels, and increased the percentage of BCSCs, phenocopying the effect of hypoxia. Knockdown of ALKBH5 expression in MDA-MB-231 human breast cancer cells significantly reduced their capacity for tumor initiation as a result of reduced numbers of BCSCs. Thus, HIF-dependent ALKBH5 expression mediates enrichment of BCSCs in the hypoxic tumor microenvironment.

HIF-1 regulates CD47 expression in breast cancer cells to promote evasion of phagocytosis and maintenance of cancer stem cells.

Increased expression of CD47 has been reported to enable cancer cells to evade phagocytosis by macrophages and to promote the cancer stem cell phenotype, but the molecular mechanisms regulating CD47 expression have not been determined. Here we report that hypoxia-inducible factor 1 (HIF-1) directly activates transcription of the CD47 gene in hypoxic breast cancer cells. Knockdown of HIF activity or CD47 expression increased the phagocytosis of breast cancer cells by bone marrow-derived macrophages. CD47 expression was increased in mammosphere cultures, which are enriched for cancer stem cells, and CD47 deficiency led to cancer stem cell depletion. Analysis of datasets derived from thousands of patients with breast cancer revealed that CD47 expression was correlated with HIF target gene expression and with patient mortality. Thus, CD47 expression contributes to the lethal breast cancer phenotype that is mediated by HIF-1.

Chemotherapy triggers HIF-1-dependent glutathione synthesis and copper chelation that induces the breast cancer stem cell phenotype.

Triple negative breast cancer (TNBC) accounts for 10-15% of all breast cancer but is responsible for a disproportionate share of morbidity and mortality because of its aggressive characteristics and lack of targeted therapies. Chemotherapy induces enrichment of breast cancer stem cells (BCSCs), which are responsible for tumor recurrence and metastasis. Here, we demonstrate that chemotherapy induces the expression of the cystine transporter xCT and the regulatory subunit of glutamate-cysteine ligase (GCLM) in a hypoxia-inducible factor (HIF)-1-dependent manner, leading to increased intracellular glutathione levels, which inhibit mitogen-activated protein kinase kinase (MEK) activity through copper chelation. Loss of MEK-ERK signaling causes FoxO3 nuclear translocation and transcriptional activation of the gene encoding the pluripotency factor Nanog, which is required for enrichment of BCSCs. Inhibition of xCT, GCLM, FoxO3, or Nanog blocks chemotherapy-induced enrichment of BCSCs and impairs tumor initiation. These results suggest that, in combination with chemotherapy, targeting BCSCs by inhibiting HIF-1-regulated glutathione synthesis may improve outcome in TNBC.

HIF-1α and TAZ serve as reciprocal co-activators in human breast cancer cells.

Hypoxia-inducible factor 1α (HIF-1α) expression is a hallmark of intratumoral hypoxia that is associated with breast cancer metastasis and patient mortality. Previously, we demonstrated that HIF-1 stimulates the expression and activity of TAZ, which is a transcriptional effector of the Hippo signaling pathway, by increasing TAZ synthesis and nuclear localization. Here, we report that direct protein-protein interaction between HIF-1α and TAZ has reciprocal effects: HIF-1α stimulates transactivation mediated by TAZ and TAZ stimulates transactivation mediated by HIF-1α. Inhibition of TAZ expression impairs the hypoxic induction of HIF-1 target genes, such as PDK1, LDHA, BNIP3 and P4HA2 in response to hypoxia, whereas inhibition of HIF-1α expression impairs TAZ-mediated transactivation of the CTGF promoter. Taken together, these results complement our previous findings and establish bidirectional crosstalk between HIF-1α and TAZ that increases their transcriptional activities in hypoxic cells.

Cross-talk between two essential nutrient-sensitive enzymes: O-GlcNAc transferase (OGT) and AMP-activated protein kinase (AMPK).

Nutrient-sensitive pathways regulate both O-GlcNAc transferase (OGT) and AMP-activated protein kinase (AMPK), cooperatively connecting metabolic homeostasis to regulation of numerous intracellular processes essential for life. Similar to phosphorylation, catalyzed by kinases such as AMPK, O-GlcNAcylation is a highly dynamic Ser/Thr-specific post-translational modification of nuclear, cytoplasmic, and mitochondrial proteins catalyzed exclusively by OGT. OGT and AMPK target a multitude of intracellular proteins, with the net effect to protect cells from the damaging effects of metabolic stress. Despite hundreds of studies demonstrating significant overlap in upstream and downstream signaling processes, no study has investigated if OGT and AMPK can directly regulate each other. We show acute activation of AMPK alters the substrate selectivity of OGT in several cell lines and nuclear localization of OGT in C2C12 skeletal muscle myotubes. Nuclear localization of OGT affects O-GlcNAcylation of numerous nuclear proteins and acetylation of Lys-9 on histone 3 in myotubes. AMPK phosphorylates Thr-444 on OGT in vitro; phosphorylation of Thr-444 is tightly associated with AMPK activity and nuclear localization of OGT in myotubes, and phospho-mimetic T444E-OGT exhibits altered substrate selectivity. Conversely, the α- and γ-subunits of AMPK are O-GlcNAcylated, O-GlcNAcylation of the γ1-subunit increases with AMPK activity, and acute inhibition of O-GlcNAc cycling disrupts activation of AMPK. We have demonstrated significant cross-talk between the O-GlcNAc and AMPK systems, suggesting OGT and AMPK may cooperatively regulate nutrient-sensitive intracellular processes that mediate cellular metabolism, growth, proliferation, and/or tissue function.

Hypoxia-inducible factor 1 mediates TAZ expression and nuclear localization to induce the breast cancer stem cell phenotype.

Intratumoral hypoxia, which is associated with breast cancer metastasis and patient mortality, increases the percentage of breast cancer stem cells (BCSCs) but the underlying molecular mechanisms have not been delineated. Here we report that hypoxia-inducible factor 1 (HIF-1) triggers the expression and activity of TAZ, a transcriptional co-activator that is required for BCSC maintenance, through two discrete mechanisms. First, HIF-1 binds directly to the WWTR1 gene and activates transcription of TAZ mRNA. Second, HIF-1 activates transcription of the SIAH1 gene, which encodes a ubiquitin protein ligase that is required for the hypoxia-induced ubiquitination and proteasome-dependent degradation of LATS2, a kinase that inhibits the nuclear localization of TAZ. Inhibition of HIF-1α, TAZ, or SIAH1 expression by short hairpin RNA blocked the enrichment of BCSCs in response to hypoxia. Human breast cancer database analysis revealed that increased expression (greater than the median) of both TAZ and HIF-1 target genes, but neither one alone, is associated with significantly increased patient mortality. Taken together, these results establish a molecular mechanism for induction of the BCSC phenotype in response to hypoxia.

Myelin-associated glycoprotein (MAG) protects neurons from acute toxicity using a ganglioside-dependent mechanism.

Myelin-associated glycoprotein (MAG), a protein expressed on the innermost wrap of myelin, contributes to long-term axon stability as evidenced by progressive axon degeneration in Mag-null mice. Recently, MAG was also found to protect axons from acute toxic insults. In the current study, rat dorsal root ganglion neurons were cultured on control substrata and substrata adsorbed with myelin proteins. Neurons on myelin-adsorbed surfaces were resistant to acute degeneration of neurites induced by vincristine, a cancer chemotherapeutic agent with neuropathic side effects. Myelin-mediated protection was reversed by anti-MAG antibody and was absent when cells were cultured on extracts from Mag-null mouse myelin, confirming the protective role of MAG. Gangliosides (sialylated glycosphingolipids) are one functional class of axonal receptors for MAG. In the current studies, a direct role for gangliosides in mediating the acute protective effects of MAG was established. Treatment of neurons with sialidase, an enzyme that cleaves the terminal sialic acids required for MAG binding, reversed MAG's protective effect, as did treatment with (1R,2R)-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol, an inhibitor of glycosphingolipid biosynthesis. In contrast, treatment with phosphatidylinositol-specific phospholipase C, an enzyme that cleaves Nogo receptors (NgR, another class of MAG receptor), or with a peptide inhibitor of an NgR-associated signaling molecule p75(NTR), failed to diminish MAG-mediated protection. Inhibiting the Rho-associated protein kinase ROCK reversed protection. We conclude that MAG protects neurites from acute toxic insult via a ganglioside-mediated signaling pathway that involves activation of RhoA. Understanding MAG-mediated protection may provide opportunities to reduce axonal damage and loss.

Cross-talk between GlcNAcylation and phosphorylation: roles in insulin resistance and glucose toxicity.

O-linked beta-N-acetylglucosamine (O-GlcNAc) is a dynamic posttranslational modification that, analogous to phosphorylation, cycles on and off serine and/or threonine hydroxyl groups. Cycling of O-GlcNAc is regulated by the concerted actions of O-GlcNAc transferase and O-GlcNAcase. GlcNAcylation is a nutrient/stress-sensitive modification that regulates proteins involved in a wide array of biological processes, including transcription, signaling, and metabolism. GlcNAcylation is involved in the etiology of glucose toxicity and chronic hyperglycemia-induced insulin resistance, a major hallmark of type 2 diabetes. Several reports demonstrate a strong positive correlation between GlcNAcylation and the development of insulin resistance. However, recent studies suggest that inhibiting GlcNAcylation does not prevent hyperglycemia-induced insulin resistance, suggesting that other mechanisms must also be involved. To date, proteomic analyses have identified more than 600 GlcNAcylated proteins in diverse functional classes. However, O-GlcNAc sites have been mapped on only a small percentage (<15%) of these proteins, most of which were isolated from brain or spinal cord tissue and not from other metabolically relevant tissues. Mapping the sites of GlcNAcylation is not only necessary to elucidate the complex cross-talk between GlcNAcylation and phosphorylation but is also key to the design of site-specific mutational studies and necessary for the generation of site-specific antibodies, both of which will help further decipher O-GlcNAc's functional roles. Recent technical advances in O-GlcNAc site-mapping methods should now finally allow for a much-needed increase in site-specific analyses to address the functional significance of O-GlcNAc in insulin resistance and glucose toxicity as well as other major biological processes.

Linking hepatic transcriptional changes to high-fat diet induced physiology for diabetes-prone and obese-resistant mice.

Insulin resistance is characterized by high insulin levels and decreased responsiveness of tissues to the clearance of glucose from the bloodstream. This study maintained the diabetes-prone C57BL/6J and obese-resistant A/J mice strains on a high-fat diet for twelve weeks to transcriptionally profile the liver for changes caused by high fat diet. In the eighth week of the experiment, the C57BL/6J mice began exhibiting signs of insulin resistance, while the A/J mice did not show any such indications during the course of the experiment. A regression model of partial least squares between serum insulin measurements and the liver gene expression profile for the C57BL/6J mice on a high-fat diet was constructed in an effort to quantitatively link the physiological measurement with the gene expressions. A series of discriminating genes between high fat and chow fed mice was generated for both the C57BL/6J and A/J strains. These discriminatory genes contain information about the mechanisms responsible for the development of insulin resistance, and the compensation for a high fat diet, respectively. The results identified several genes involved in the development of insulin resistance and serve as a framework for other studies involving other organs affected by this systemic disease.

Total and high-molecular-weight adiponectin in breast cancer: in vitro and in vivo studies.

BACKGROUND: Obesity is a major risk factor for breast cancer. We hypothesized that obesity-induced decreases in total and/or high-molecular-weight (HMW) adiponectin levels may underlie this association. METHODS: We measured serum total and HMW adiponectin in a hospital-based case-control study of 74 female breast cancer patients and 76 controls. In parallel, expression of adiponectin and its receptors AdipoR1/R2 were measured in tissue samples using RT-PCR, and protein expression of AdipoR1/R2 was localized and quantified using immunohistochemistry. Finally, we documented AdipoR1/R2 expression in several breast cancer cell lines and studied adiponectin signaling and the effect of adiponectin on proliferation in the T47D breast cancer cell line in vitro. RESULTS: Women with the highest adiponectin levels had a 65% reduced risk of breast cancer (P = 0.04). This association became stronger after adjustment for age, body mass index, and hormonal and reproductive factors (P = 0.02). Modeling HMW instead of total adiponectin produced similar results and did not offer any additional predictive value. Breast cancer cells expressed AdipoR1/R2 but not adiponectin. Expression of AdipoR1, but not AdipoR2, was higher in tumor tissue than both adjacent and control tissues. Exposure of T47D cells to adiponectin significantly inhibited the percentage of viable cells to 86% and proliferation to 66% but had no effect on apoptosis. These effects were associated with activation of ERK1/2 but not AMP-activated protein kinase or p38MAPK. CONCLUSION: These studies suggest that adiponectin may act as a biomarker of carcinogenesis and may constitute a molecular link between obesity and breast cancer.

Regulation of adiponectin and its receptors in response to development of diet-induced obesity in mice.

Adiponectin and its receptors play an important role in energy homeostasis and insulin resistance, but their regulation remains to be fully elucidated. We hypothesized that high-fat diet would decrease adiponectin but increase adiponectin receptor (AdipoR1 and AdipoR2) expression in diet-induced obesity (DIO)-prone C57BL/6J and DIO-resistant A/J mice. We found that circulating adiponectin and adiponectin expression in white adipose tissue are higher at baseline in C57BL/6J mice compared with A/J mice. Circulating adiponectin increases at 10 wk but decreases at 18 wk in response to advancing age and high-fat feeding. However, adiponectin levels corrected for visceral fat mass and adiponectin mRNA expression in WAT are affected by high-fat feeding only, with both being decreased after 10 wk in C57BL/6J mice. Muscle AdipoR1 expression in both C57BL/6J and A/J mice and liver adipoR1 expression in C57BL/6J mice increase at 18 wk of age. High-fat feeding increases both AdipoR1 and AdipoR2 expression in liver in both strains of mice and increases muscle AdipoR1 expression in C57BL/6J mice after 18 wk. Thus advanced age and high-fat feeding, both of which are factors that predispose humans to obesity and insulin resistance, are associated with decreasing adiponectin and increasing AdipoR1 and/or AdipoR2 levels.

Circulating adiponectin and expression of adiponectin receptors in human skeletal muscle: associations with metabolic parameters and insulin resistance and regulation by physical training.

CONTEXT: Adiponectin, an adipocyte-secreted hormone, is associated with insulin resistance and the metabolic syndrome. OBJECTIVE: The physiological regulation of circulating adiponectin levels and mRNA expression of its receptors (AdipoR1 and AdipoR2) in skeletal muscle remains to be fully elucidated. DESIGN/PATIENTS: We assessed circulating adiponectin and AdipoR1/R2 mRNA expression in human skeletal muscle in a cross-sectional study of 140 subjects with normal or impaired glucose tolerance or type 2 diabetes. In the context of an interventional study, the same measurements were performed in 60 of these subjects (20/glucose tolerance group) before and after 4 wk of physical training. Finally, we measured these same variables in addition to protein levels of AMP kinase (AMPK), acetyl phosphorylated AMPK, coenzyme A carboxylase, phosphorylated coenzyme A carboxylase, and phosphatidylinositol 3-kinase in muscle before and after 3 h of intensive exercise in a subgroup of five subjects. SETTING: This study was performed at an academic clinical research center. RESULTS: Circulating adiponectin was negatively associated, whereas AdipoR1/R2 mRNA levels were positively associated with obesity, glucose and lipid levels, and insulin resistance. Physical training for 4 wk resulted in increased circulating adiponectin levels and AdipoR1/R2 mRNA expression in muscle. Exercise for 3 h increased AdipoR1/R2 mRNA expression as well as phosphorylation of AMPK and acetyl coenzyme A carboxylase in muscle, but had no effect on circulating adiponectin. CONCLUSIONS: Adiponectin, AdipoR1, and AdipoR2 are all associated with body composition, insulin sensitivity, and metabolic parameters. Physical training increases circulating adiponectin and mRNA expression of its receptors in muscle, which may mediate the improvement of insulin resistance and the metabolic syndrome in response to exercise.

Natural resistance, iron and infection: a challenge for clinical medicine.

Natural resistance to infection, which does not depend on antibiotics, is a powerful protective mechanism common to all mankind that has been responsible for the survival of our species during countless millennia in the past. The normal functioning of this complex system of phagocytic cells and tissue fluids is entirely dependent on an extremely low level of free ionic iron (10(-18) M) in tissue fluids. This low-iron environment is maintained by the unsaturated iron-binding proteins transferrin and lactoferrin, which depend on well-oxygenated tissues, where a relatively high oxidation-reduction potential (Eh) and pH are essential for the binding of ferric iron. Freely available iron is derived from iron overload, free haem compounds, or hypoxia in injured tissue leading to a fall in Eh and pH. This can severely damage or abolish normal bactericidal mechanisms in tissue fluids leading to overwhelming growth of bacteria or fungi. The challenge for clinical medicine is to reduce or eliminate the presence of freely available iron in clinical disease. In injured or hypoxic tissue, treatment with hyperbaric oxygen might prove very useful by increasing tissue oxygenation and restoring normal bactericidal mechanisms in tissue fluids, which would be of huge benefit to the patient.

Regulation of mouse hepatic genes in response to diet induced obesity, insulin resistance and fasting induced weight reduction.

BACKGROUND: Obesity is associated with insulin resistance that can often be improved by caloric restriction and weight reduction. Although many physiological changes accompanying insulin resistance and its treatment have been characterized, the genetic mechanisms linking obesity to insulin resistance are largely unknown. We used DNA microarrays and RT-PCR to investigate significant changes in hepatic gene transcription in insulin resistant, diet-induced obese (DIO)-C57/BL/6J mice and DIO-C57/BL/6J mice fasted for 48 hours, whose weights returned to baseline levels during these conditions. RESULTS: Transcriptional profiling of hepatic mRNA revealed over 1900 genes that were significantly perturbed between control, DIO, and fasting/weight reduced DIO mice. From this set, our bioinformatics analysis identified 41 genes that rigorously discriminate these groups of mice. These genes are associated with molecular pathways involved in signal transduction, and protein metabolism and secretion. Of particular interest are genes that participate in pathways responsible for modulating insulin sensitivity. DIO altered expression of genes in directions that would be anticipated to antagonize insulin sensitivity, while fasting/weight reduction partially or completely normalized their levels. Among these discriminatory genes, Sh3kbp1 and RGS3, may have special significance. Sh3kbp1, an endogenous inhibitor of PI-3-kinase, was upregulated by high-fat feeding, but normalized to control levels by fasting/weight reduction. Because insulin signaling occurs partially through PI-3-kinase, increased expression of Sh3kbp1 by DIO mice may contribute to hepatic insulin resistance via inhibition of PI-3-kinase. RGS3, a suppressor of G-protein coupled receptor generation of cAMP, was repressed by high-fat feeding, but partially normalized by fasting/weight reduction. Decreased expression of RGS3 may augment levels of cAMP and thereby contribute to increased, cAMP-induced, hepatic glucose output via phosphoenolpyruvate carboxykinase (PCK1), whose mRNA levels were also elevated. CONCLUSION: These findings demonstrate that hepatocytes respond to DIO and weight reduction by controlling gene transcription in a variety of important molecular pathways. Future studies that characterize the physiological significance of the identified genes in modulating energy homeostasis could provide a better understanding of the mechanisms linking DIO with insulin resistance.

Iron and infection: the heart of the matter.

Bacterial resistance to antibiotics is a major threat to clinical medicine. However, natural resistance to bacterial infection, which does not depend on antibiotics, is a powerful protective mechanism common to all mankind. The availability of iron is the heart of the matter and the successful functioning of these antibacterial systems depends entirely upon an extremely low level of free ionic iron (10(-18) M) in normal tissue fluids. This in turn depends on well-oxygenated tissues where the oxidation-reduction potential (Eh) and pH control the binding of iron by unsaturated transferrin and lactoferrin. Bacterial virulence is greatly enhanced by freely available iron, such as that in fully-saturated transferrin or free haemoglobin. Following trauma a fall in tissue Eh and pH due to ischaemia, plus the reducing powers of bacteria, can make iron in transferrin freely available and abolish the bactericidal properties of tissue fluids with disastrous results for the host. Hyperbaric oxygen is a possible therapeutic measure that could restore normal bactericidal systems in infected tissues by raising the Eh and pH.

Recombinant methionyl human leptin administration to achieve high physiologic or pharmacologic leptin levels does not alter circulating inflammatory marker levels in humans with leptin sufficiency or excess.

A role for high leptin levels in the proinflammatory state associated with obesity has been proposed on the basis of observational studies, but a recent interventional study employing administration of long-acting pegylated leptin resulting in very high pharmacologic levels in obese subjects did not support this idea. These interventional studies have not yet been independently confirmed, however, and varying levels and duration of hyperleptinemia as well as the presence of comorbidities such as diabetes have not yet been investigated as potential effect modifiers. We performed three interventional studies involving administration of recombinant methionyl human leptin (r-metHuLeptin) to lean, otherwise healthy obese, and obese diabetic subjects to investigate whether increasing circulating leptin levels over a wide spectrum of values (from low physiologic to high pharmacologic) would alter serum levels of inflammatory markers and other cytokines important in the T helper cell response. Increasing leptin levels from low physiologic to high physiologic in lean men and from higher physiologic to low pharmacologic in obese men over 3 d did not alter serum interferon-gamma, IL-10, TNF-alpha, monocyte chemoattractant protein-1, or soluble intercellular adhesion molecule-1. In obese subjects with type 2 diabetes mellitus, the administration of r-metHuLeptin for 4 or 16 wk, resulting in high pharmacologic leptin levels, did not activate the TNF-alpha system or increase cytokines or inflammatory markers, including IL-10, IL-6, C-reactive protein, monocyte chemoattractant protein-1, and soluble intercellular adhesion molecule-1. These findings do not support an etiopathogenic role for leptin in proinflammatory states associated with leptin excess such as obesity and have direct relevance for the potential future therapeutic use of r-metHuLeptin in humans.

Recombinant methionyl human leptin administration activates signal transducer and activator of transcription 3 signaling in peripheral blood mononuclear cells in vivo and regulates soluble tumor necrosis factor-alpha receptor levels in humans with relative leptin deficiency.

Studies of congenital complete leptin deficiency in animals and humans support a role for leptin in regulating immune function. Whether acquired relative leptin deficiency affects immunological parameters in healthy humans remains unknown. We thus used experimental models of relative leptin deficiency and recombinant methionyl human leptin (r-metHuLeptin) administration in humans to investigate whether r-metHuLeptin would activate signaling pathways in peripheral blood mononuclear cells (PBMCs) and whether acquired relative leptin deficiency and/or increasing circulating leptin levels into the physiologic range would change PBMC subpopulations and cytokines important in the T-helper cell and systemic immune responses. We found that r-metHuLeptin administration to healthy humans activates signal transducer and activator of transcription-3 signaling in PBMCs in vivo. Neither short-term leptin deficiency, induced by 3-d complete fasting, nor physiologic r-metHuLeptin replacement for the same period of time had a major effect on PBMC subpopulations or serum cytokines in healthy men. In contrast, normalizing serum leptin levels over 8 wk in lean women with relative leptin deficiency for 5.1 +/- 1.4 yr (mean +/- se) due to chronic energy deficit increased soluble TNFalpha receptor levels, indicating activation of the TNFalpha system. These findings suggest that relative leptin deficiency due to more long-term energy deprivation is associated with defects in immunological parameters that may be corrected with exogenous r-metHuLeptin administration. Further studies are warranted to assess the implications of acquired relative hypoleptinemia and/or r-metHuLeptin administration on the immunosuppression associated with energy- and leptin-deficient states in humans.

Circulating resistin in lean, obese, and insulin-resistant mouse models: lack of association with insulinemia and glycemia.

Resistin is an adipocyte-secreted hormone proposed to link obesity with insulin resistance and diabetes, but no previous study has performed a joint quantitative evaluation of white adipose tissue (WAT) resistin mRNA expression and serum levels in relation to insulinemia and glycemia in mice. We have thus comparatively assessed WAT resistin mRNA expression and serum resistin levels in lean C57BL/6J mice and various mouse models of obesity, including diet-induced obese (DIO) C57BL/6J mice, high fat-fed TNF-alpha-/- mice, and brown adipose tissue (BAT)-deficient uncoupling protein-diphtheria toxin A chain (UCP1-DTA) mice. We also studied whether treatment with the weight-reducing and insulin-sensitizing compounds, MTII, an alpha-melanocyte-stimulating hormone analog, or CNTF(Ax15), a ciliary neurotrophic factor analog, alters resistin mRNA expression and/or circulating levels in lean and DIO C57BL/6J mice. We find that resistin mRNA expression is similar in DIO and lean C57BL/6J mice, as well as in TNF-alpha-/- and wild-type (WT) mice. Circulating resistin levels, however, are higher in DIO C57BL/6J, high fat-fed TNF-alpha-/-, and UCP1-DTA mice compared with lean controls. Moreover, although resistin mRNA expression is upregulated by MTII treatment for 24 h and downregulated by CNTF(Ax15) treatment for 3 or 7 days, circulating resistin levels are not altered by MTII or CNTF(Ax15) treatment. In addition, serum resistin levels, but not resistin mRNA expression levels, are correlated with body weight, and neither resistin mRNA expression nor serum resistin levels are correlated with serum insulin or glucose levels. We conclude that transcriptional regulation of resistin in WAT does not correlate with circulating resistin levels and that circulating resistin is unlikely to play a major endocrine role in insulin resistance or glycemia in mice.