dRetinol-binding protein 4 (RBP4), the sole retinol transporter in blood, is secreted from adipocytes and liver. Serum RBP4 levels correlate highly with insulin resistance, other metabolic syndrome factors, and cardiovascular disease. Elevated serum RBP4 causes insulin resistance, but the molecular mechanisms are unknown. Here we show that RBP4 induces expression of proinflammatory cytokines in mouse and human macrophages and thereby indirectly inhibits insulin signaling in cocultured adipocytes. This occurs through activation of c-Jun N-terminal protein kinase (JNK) and Toll-like receptor 4 (TLR4) pathways independent of the RBP4 receptor, STRA6. RBP4 effects are markedly attenuated in JNK1 ؊/؊ JNK2 ؊/؊ macrophages and TLR4؊/؊ macrophages. Because RBP4 is a retinol-binding protein, we investigated whether these effects are retinol dependent. Unexpectedly, retinol-free RBP4 (apo-RBP4) is as potent as retinol-bound RBP4 (holo-RBP4) in inducing proinflammatory cytokines in macrophages. Apo-RBP4 is likely to be physiologically significant since RBP4/retinol ratios are increased in serum of lean and obese insulin-resistant humans compared to ratios in insulin-sensitive humans, indicating that higher apo-RBP4 is associated with insulin resistance independent of obesity. Thus, RBP4 may cause insulin resistance by contributing to the development of an inflammatory state in adipose tissue through activation of proinflammatory cytokines in macrophages. This process reveals a novel JNK-and TLR4-dependent and retinol-and STRA6-independent mechanism of action for RBP4.O besity is a major risk factor for insulin resistance, which is a critical pathogenic factor in type 2 diabetes (50). Determination of the physiologic and cellular mechanisms linking obesity to type 2 diabetes could lead to development of new prevention and treatment approaches. Multiple mechanisms may contribute, including abnormal production of adipocyte-secreted proteins (adipokines) (1,15,29), infiltration of white adipose tissue (WAT) with proinflammatory macrophages (42), and aberrant lipid deposition in tissues such as muscle and liver (51). These mechanisms are not mutually exclusive. For example, adipokines can affect inflammation and lipid deposition in tissues (15).Serum retinol-binding protein 4 (RBP4) is an adipokine and is also secreted by liver. RBP4 levels are increased in obese and insulin-resistant humans and mouse models, and genetic or pharmacologic elevation of serum RBP4 causes insulin resistance in normal mice (19,31,65). Although many studies show strong correlations of serum RBP4 levels with obesity and the severity of insulin resistance (9,16,27,35), others do not (8,17,32,46), as reviewed in reference 32. This may result from the use of different populations of human subjects or from methodological issues with RBP4 assays (18,32,64). Many studies also show that serum RBP4 levels correlate with other components of the metabolic syndrome in humans, including hypertension (47, 54, 64), dyslipidemia (41, 47, 64, 67), waist/hip ratio (31...

Plasma proteins represent an important part of the human proteome. Although recent proteomics research efforts focus largely on determining the overall number of proteins circulating in plasma, it is equally important to delineate protein variations among individuals, because they can signal the onset of diseases and be used as biological markers in diagnostics. To date, there has been no systematic proteomics effort to characterize the breadth of structural modifications in individual proteins in the general population. In this work, we have undertaken a population proteomics study to define gene-and protein-level diversity that is encountered in the general population. Twenty-five plasma proteins from a cohort of 96 healthy individuals were investigated through affinity-based mass spectrometric assays. A total of 76 structural forms͞variants were observed for the 25 proteins within the samples cohort. Posttranslational modifications were detected in 18 proteins, and point mutations were observed in 4 proteins. The frequency of occurrence of these variations was wide-ranged, with some modifications being observed in only one sample, and others detected in all 96 samples. Even though a relatively small cohort of individuals was investigated, the results from this study illustrate the extent of protein diversity in the human population and can be of immediate aid in clinical proteomics͞biomarker studies by laying a basal-level statistical foundation from which protein diversity relating to disease can be evaluated.mass spectrometry ͉ population proteomics ͉ protein modifications

Background-The myocardium secretes B-type natriuretic peptide (BNP) in response to stimuli associated with heart failure (HF). However, high immunoreactive-BNP levels in patients with HF are associated with a paradoxical lack of natriuretic response. We hypothesized that commercially available assays for immunoreactive BNP do not reflect the bioactivity of the natriuretic peptide system, because they measure both unprocessed inactive pro-BNP and mature BNP 1-32. We describe an assay for the detection of bioactive BNP 1-32 and confirm very low concentrations in plasma from HF patients. Methods and Results-We developed a quantitative mass spectrometry immunoassay to capture endogenous BNP peptides using high affinity antibodies. Bound BNP and its truncated fragments were detected by matrix assisted laser desorption ionization-time of flight mass spectrometry based on their predicted masses. Mass spectrometry immunoassay revealed rapid in vitro degradation of BNP 1-32 in plasma, which requires plasma collection in the presence of high protease inhibitor concentrations. In 11 of 12 HF patients BNP 1-32 was detectable, ranging from 25 to 43 pg/mL. Several degraded forms of BNP were also detected at similarly low levels. In contrast, parallel measurements of immunoreactive BNP using the Biosite assay ranged from 900 to 5000 pg/mL. Conclusions-Detection of endogenous BNP 1-32 requires special preservation of plasma samples. Mass spectrometry immunoassay technology demonstrates that HF patients have low levels of BNP 1-32. Commercially available immunoreactive-BNP assays overrepresent biological activity of the natriuretic peptide system because they cannot distinguish between active and inactive forms. This observation may, in part, explain the "natriuretic paradox." (Circ Heart Fail. 2008;1:258-264.)

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