Robust increase in glucagon secretion after oral protein intake, but not after glucose or lipid intake in Japanese people without diabetes.

Few studies in Asian populations have analyzed how glucagon secretion is affected by ingested glucose, proteins or lipids, individually. To investigate the fluctuations of glucagon secretion after the intake of each of these nutrients, 10 healthy volunteers underwent oral loading tests using each of glucose, proteins and lipids, and blood levels of glucose, insulin and glucagon were measured every 30 min for 120 min. Whereas glucagon secretion was suppressed and minimally affected by oral glucose intake and lipid intake, respectively, oral protein intake robustly increased glucagon secretion, as well as insulin secretion. Further studies are needed to elucidate the mechanism by which protein loading increases glucagon secretion.

Plasma and serum prorenin concentrations in diabetes, hypertension, and renal disease.

Although the renin-angiotensin-aldosterone system plays a crucial role in fluid homeostasis and cardiovascular disease pathophysiology, measurements of plasma prorenin levels are still unavailable in clinical practice. We previously found that prorenin molecules in human blood underwent significant posttranslational modifications and were undetectable using immunological assays that utilized antibodies specifically recognizing unmodified recombinant prorenin. Using a sandwich enzyme-linked immunosorbent assay that captures posttranslationally modified prorenins with their prosegment antibodies, we measured plasma and serum prorenin concentrations in 219 patients with diabetes mellitus, hypertension and/or renal disease and compared them with those of 40 healthy controls. The measured values were not significantly different from those of the healthy controls and were 1,000- to 100,000-fold higher than previously reported levels determined using conventional assay kits. Multiple regression analyses showed that body weight, serum albumin levels, and serum creatinine levels negatively correlated with plasma prorenin levels, while the use of loop diuretics was associated with elevated plasma prorenin levels. Blood pressure, HbA1c, and plasma renin activity were not independent variables affecting plasma prorenin levels. In contrast, serum prorenin levels were unaffected by any of the above clinical parameters. The association of the plasma prorenin concentration with indices reflecting body fluid status suggests the need to scrutinize its role as a biomarker, while serum prorenins are less likely to have immediate diagnostic value.

Effects of luseogliflozin on the secretion of islet hormones and incretins in patients with type 2 diabetes.

The insufficient activity of insulin and the hyperactivity of glucagon are responsible for glucose intolerance in patients with type 2 diabetes. Whereas sodium-glucose cotransporter-2 (SGLT2) inhibitors improve blood glucose levels in patients with type 2 diabetes, their effects on the secretion profiles of glucagon and incretins remain unclear. Therefore, to investigate the effects of the SGLT2 inhibitor luseogliflozin on metabolic and endocrine profiles, 19 outpatients with type 2 diabetes were administered luseogliflozin for 12 weeks. It is of note that all subjects were treated only with diet and exercise therapy, and we were able to investigate the effects of luseogliflozin separately from the effects of other antidiabetic agents. Body weight, body fat mass, fat-free mass, and muscle mass were significantly reduced after 12 weeks of luseogliflozin administration. Glycosylated hemoglobin significantly decreased from the baseline of 8.2% ± 0.8% to 7.3% ± 0.7% (p < 0.0001). The meal tolerance test demonstrated that luseogliflozin significantly recovered glucose tolerance, accompanied by improved insulin resistance and ß-cell function, whereas glucagon secretion was unaffected. Furthermore, GLP-1 secretion was significantly increased after luseogliflozin administration. Thus, luseogliflozin improved metabolic and endocrine profiles accompanied by increased GLP-1 secretion in type 2 diabetic patients without any antidiabetic medication, but did not affect glucagon secretion.

GIP_HUMAN[22-51] is a new proatherogenic peptide identified by native plasma peptidomics.

We recently established a new plasma peptidomic technique and comprehensively identified a large number of low-molecular weight and low-abundance native peptides using a single drop of human plasma. To discover a novel polypeptide that potently modulates the cardiovascular system, we performed a bioinformatics analysis of the large-scale identification results, sequentially synthesized the selected peptide sequences, tested their biological activities, and identified a 30-amino-acid proatherogenic peptide, GIP_HUMAN[22-51], as a potent proatherosclerotic peptide hormone. GIP_HUMAN[22-51] has a common precursor with the glucose-dependent insulinotropic polypeptide (GIP) and is located immediately N-terminal to GIP. Chronic infusion of GIP_HUMAN[22-51] into ApoE-/- mice accelerated the development of aortic atherosclerotic lesions, which were inhibited by co-infusions with an anti-GIP_HUMAN[22-51] antibody. GIP_HUMAN[22-51] increased the serum concentrations of many inflammatory and proatherogenic proteins, whereas neutralising antibodies reduced their levels. GIP_HUMAN[22-51] induced IκB-α degradation and nuclear translocation of NF-κB in human vascular endothelial cells and macrophages. Immunoreactive GIP_HUMAN[22-51] was detected in human tissues but there was no colocalization with the GIP. The plasma GIP_HUMAN[22-51] concentration in healthy humans determined using a stable-isotope tagged peptide was approximately 0.6 nM. This study discovered a novel endogenous proatherogenic peptide by using a human plasma native peptidomic resource.

Circulating prorenin: its molecular forms and plasma concentrations.

The renin-angiotensin-aldosterone system plays pivotal roles in the maintenance of fluid homeostasis and in the pathophysiology of major human diseases. However, the molecular forms of plasma renin/prorenin have not been fully elucidated, and measurements of plasma prorenin levels are still unavailable for clinical practice. We attempted to evaluate the molecular forms of human plasma prorenin and to directly measure its concentration without converting it to renin to determine its activity. Polyacrylamide gel electrophoresis and subsequent immunoblotting using antibodies that specifically recognise prosegment sequences were used to analyse its molecular forms in plasma. We also created a sandwich enzyme-linked immunosorbent assay suitable for directly quantifying the plasma concentration. The plasma level in healthy people was 3.0-13.4 µg/mL, which is from 3 to 4 orders of magnitude higher than the levels reported thus far. Plasma immunoreactive prorenin consists of three major distinct components: a posttranslationally modified full-length protein, an albumin-bound form and a smaller protein truncated at the common C-terminal renin/prorenin portion. In contrast to plasma renin activity, plasma prorenin concentrations were not affected by the postural changes of the donor. Hence, plasma prorenin molecules may be posttranslationally modified/processed or bound to albumin and are present in far higher concentrations than previously thought.

Molecular form and concentration of serum α2-macroglobulin in diabetes.

α2-Macroglobulin is a highly abundant serum protein involved in the development of atherosclerosis and cardiac hypertrophy. However, its circulating molecular form and exact concentrations in human health/diseases are not known. Blue native-polyacrylamide gel electrophoresis of human serum was used to confirm the native conformation of α2-macroglobulin. We created an enzyme-linked immunosorbent assay suitable for quantifying its circulating molecular form and undertook a cross-sectional study to measure its serum levels in 248 patients with diabetes mellitus and 59 healthy volunteers. The predominant circulating molecular form of α2-macroglobulin was the tetramer, whereas its dimer was detectable in patients with high serum levels of α2-macroglobulin. The serum α2-macroglobulin concentration was not associated with glycated hemoglobin or any other glycemic variable as evaluated from 48-h continuous glucose monitoring, but showed close correlation with left ventricular posterior wall thickness, carotid artery intima-media thickness, urinary albumin:creatinine ratio (ACR) and brachial-ankle pulse wave velocity (baPWV). Multivariate analysis revealed only the ACR and baPWV to be independent variables influencing serum levels of α2-macroglobulin. Thus, an increased ACR and baPWV are associated with higher serum concentrations of α2-macroglobulin, and the latter may contribute to the mechanism by which albuminuria increases the risk of developing cardiovascular diseases.

Identification of plasma binding proteins for glucose-dependent insulinotropic polypeptide.

Glucose-dependent insulinotropic polypeptide (GIP), secreted from enteroendocrine K cells, has potent insulin-releasing and extrapancreatic glucoregulatory activities. However, exogenous GIP has less potent biological effects compared with another incretin hormone, GLP-1, which limits its use for the treatment of type 2 diabetes. The fate and secretion of administered native GIP remain unclear. The aim of this study was to identify plasma binding proteins for human GIP. Fluorescent-labelled GIP was added to fresh human plasma and subjected to clear native polyacrylamide gel electrophoresis (CN-PAGE). Then fluorescent protein bands were in-gel trypsin-digested and subjected to liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis, revealing the presence of albumin, immunoglobulin G (IgG) and transferrin. In contrast to GIP, the binding of fluorescent GLP-1 and glucagon to plasma protein fractions were minimal. CN-PAGE analysis of synthetic GIP incubated with human serum albumin, purified IgG or transferrin, and subsequent western blot analysis revealed that GIP binds to each of these proteins. Taken together, these results indicate that GIP readily binds to albumin, IgG and transferrin, three plasma proteins highly abundant in the human peripheral circulation. Separation of protein complexes using CN-PAGE and the identification of in-gel digested proteins by LC-MS/MS analysis provide a promising strategy to identify plasma binding proteins for bioactive peptides.

Basal glucagon hypersecretion and response to oral glucose load in prediabetes and mild type 2 diabetes.

Dysregulation of glucagon secretion plays an important role in the pathogenesis of type 2 diabetes (T2DM). However it hasn't been elucidated involvement of glucagon dysregulation in pathophysiology of T2DM. Recently a new glucagon sandwich enzyme-linked immunosorbent assay (ELISA) became available that can measure plasma glucagon level with higher accuracy and simpler procedure than the conventional RIA method. We performed OGTT for adult subjects aged 20-69 years to define normal glucose tolerance (NGT, n = 25), borderline glucose intolerance (defined as pre-diabetes mellitus: preDM, n = 15), or diabetes mellitus (DM, n = 13), and we measured glucagon levels with this new ELISA method at fasting and during OGTT. Plasma glucose, insulin, glucagon and active GLP-1 were also measured. This study took place in diabetes outpatient clinic in Kitasato University Hospital and an affiliated outpatient clinic. PreDM and DM exhibited higher fasting plasma glucagon levels than NGT (34.4 ± 4.6 and 44.1 ± 5.0 vs. 20.6 ± 3.6 pg/mL), and statistical significance was observed between NGT and DM (p < 0.05). There was significant correlation between fasting glucagon level and indexes of insulin sensitivity. During OGTT, glucagon levels were less suppressed in DM and preDM than in NGT, whereas no apparent relationship was observed between glucagon and GLP-1 secretion. Significant positive correlation was observed between glucagon levels during OGTT and fasting TG. In conclusion, subjects with mild T2DM exhibited fasting hyperglucagonemia and insufficient suppression to oral glucose load compared to NGT subjects.

Identification and quantification of plasma free salusin-ß, an endogenous parasympathomimetic peptide.

Salusin-ß is an endogenous parasympathomimetic proatherosclerotic peptide. Salusin-ß was initially predicted from bioinformatic analyses and later immunologically detected in human biofluids. However, elucidation of salusin-ß bioactivity has faced enormous challenges because of its unique physicochemical characteristics that cause it to strongly adhere to laboratory apparatus materials. In the strictest sense, the discovery of bioactive peptides is not complete until their exact native sequences have been confirmed in the peripheral circulation. In this study, we determined the plasma molecular form and levels of free salusin-ß to determine its pathophysiological significance. Ultra-high-yield enrichment and preseparation of non-tryptic human plasma was followed by LC-MS/MS, and full-length salusin-ß and seven different endogenous fragment sequences were identified. We established a new ELISA that specifically detects plasma free salusin-ß without cross-reacting with any of its identified endogenous fragments. Free salusin-ß levels exhibited a profound early morning nadir and rapidly decreased in response to parasympathetic nervous augmentation. Our technical advance in plasma native peptide analysis successfully identified a hard-to-detect bioactive peptide, salusin-ß, together with its formerly unrecognized fragments, and further suggests that conventional immunological measurements of target peptides may not be fully representative.

Methionine sulfoxides in serum proteins as potential clinical biomarkers of oxidative stress.

Oxidative stress contributes to the pathophysiology of a variety of diseases, and circulating biomarkers of its severity remains a topic of great interest for researchers. Our peptidomic strategy enables accurate and reproducible analysis of circulating proteins/peptides with or without post-translational modifications. Conventional wisdom holds that hydrophobic methionines exposed to an aqueous environment or experimental handling procedures are vulnerable to oxidation. However, we show that the mass spectra intensity ratio of oxidized to non-oxidized methionine residues in serum tryptic proteins can be accurately quantified using a single drop of human serum and give stable and reproducible results. Our data demonstrate that two methionine residues in serum albumin (Met-111 and Met-147) are highly oxidized to methionine sulfoxide in patients with diabetes and renal failure and in healthy smokers versus non-smoker controls. This label-free mass spectrometry approach to quantify redox changes in methionine residues should facilitate the identification of additional circulating biomarkers suitable for predicting the development or progression of human diseases.

Physiological fluctuations of human plasma total salusin-ß, an endogenous parasympathomimetic/proatherosclerotic peptide.

Salusin-ß is an endogenous bioactive peptide that systemically exerts acute parasympathomimetic hemodynamic actions and locally induces atherogenesis. Due to its unique physicochemical characteristics to immediately adhere to all types of plastic and glassware, its plasma concentrations have only been successfully determined very recently. Using a total of 50 healthy adults (median age 28 years, range 24-57 years), we evaluated whether circulating salusin-ß levels are affected by the autonomic nervous functions. Plasma total salusin-ß levels obtained during daytime ambulatory monitoring of heart rate variability showed strong negative correlations with variables reflecting parasympathetic nervous activity, high frequency amplitude (HF; r=-0.27, p=0.0018) and the square root of the mean squared differences of successive normal-to-normal intervals (RMSSD; r=-0.19, p=0.0292), but did not with low frequency amplitude (LF) or LF/HF, variables influenced by sympathetic nervous activity. Because early morning nadir in the diurnal variation of plasma total salusin-ß levels appeared to follow the nighttime parasympathetic nervous activity peak as quantified by HF and RMSSD, we determined whether parasympathetic stimulation reduces plasma salusin-ß levels. Both Valsalva maneuver (p<0.05) and urination (p<0.05) significantly reduced plasma total salusin-ß levels. Despite the fact that salusin-ß is the sole endogenous parasympathomimetic peptide identified to date, the current results argue against the contention that physiological parasympathetic augmentation is the consequences of upregulated circulating salusin-ß. Rather, circulating salusin-ß levels are suppressed following physiological parasympathetic stimulation and appear to constitute a negative feedback relationship with the parasympathetic nervous system.

Acquired von Willebrand syndrome associated with Hashimoto's thyroiditis and subcutaneous mucosa-associated lymphoid tissue lymphoma.

Acquired von Willebrand syndrome (AVWS) is a rare bleeding disorder with laboratory findings similar to those of congenital von Willebrand disease. We herein report a case of AVWS associated with Hashimoto's thyroiditis and subcutaneous mucosa-associated lymphoid tissue lymphoma. An IgG autoantibody against von Willebrand factor (VWF) was detected. The antibody bound to VWF but did not inhibit VWF activity. Rapid clearance of VWF seemed to be the cause of AVWS in the present case. VWF-containing concentrates stopped the bleeding. Even if such a complication is rare, for AVWS patients, prompt recognition of the underlying mechanism can save lives.

Circulating levels of human salusin-ß, a potent hemodynamic and atherogenesis regulator.

Using bioinformatics analysis, we previously identified salusin-ß, an endogenous bioactive peptide with diverse physiological activities. Salusin-ß is abundantly expressed in the neuroendocrine system and in systemic endocrine cells/macrophages. Salusin-ß acutely regulates hemodynamics and chronically induces atherosclerosis, but its unique physicochemical characteristics to tightly adhere to all types of plastic and glassware have prevented elucidation of its precise pathophysiological role. To quantitate plasma total salusin-ß concentrations, we produced rabbit and chicken polyclonal antibodies against the C- and N-terminal end sequences, circumvented its sticky nature, and successfully established a sandwich enzyme-linked immunosorbent assay (ELISA). Salusin-ß was abundantly present in the plasma of healthy volunteers, ranging from 1.9 to 6.6 nmol/L. Reverse phase-high performance liquid chromatography analysis showed that a single immunoreactive salusin-ß peak coincided with synthetic authentic salusin-ß. Plasma salusin-ß concentrations were unaffected by postural changes and by potent vasopressin release stimuli, such as hypertonic saline infusion or smoking. However, salusin-ß concentrations showed significant circadian variation; concentrations were high during the daytime and reached the lowest concentrations in the early morning. Plasma salusin-ß levels in subjects with diabetes mellitus, coronary artery disease, and cerebrovascular disease showed distinctly higher levels than healthy controls. Patients with panhypopituitarism combined with complete central diabetes insipidus also showed significantly higher plasma salusin-ß levels. Therefore, the ELISA system developed in this study will be useful for evaluating circulating total salusin-ß levels and for confirming the presence of authentic salusin-ß in human plasma. The obtained results suggest a limited contribution of the neuroendocrine system to peripheral total salusin-ß concentrations and a role for plasma total salusin-ß concentrations as an indicator of systemic vascular diseases.

Emerging roles for vasoactive peptides in diagnostic and therapeutic strategies against atherosclerotic cardiovascular diseases.

Coronary artery disease (CAD) arising from atherosclerosis remains the most common cause of death and morbidity worldwide, although its risk factors, such as hypertension, dyslipidemia, and diabetes, have been individually treated with increasingly improved outcomes. Therefore, it is important to develop diagnostic and therapeutic windows for CAD. Many classical vasoactive hormones, inflammatory cytokines, and oxidative products have been implicated as potential biomarkers. Our recent studies have shown that high levels of the pro-atherogenic vasoactive agents, serotonin and urotensin II, which are potent vasoconstrictors, can be used as biomarkers for CAD. In subsequent trials, we unraveled anti- and pro-atherogenic roles for more recently identified peptides. Anti-atherogenic peptides include the adipocytokine adiponectin, the neuronal growth factor heregulin-ß1 (neuregulin-1 type I), the incretin hormone, glucagon-like peptide-1 (GLP-1), and a peptide recently identified by an in silico approach, salusin-α. Atherogenic roles have been demonstrated by cellular, animal, and clinical experiments, which indicate that human adiponectin, heregulin-ß1, GLP-1, and salusin-α attenuate the development of atherosclerotic lesions by suppressing macrophage foam cell formation via down-regulation of acyl-CoA:cholesterol acyltransferase-1. Circulating levels of these peptides in the blood are markedly decreased in CAD patients compared with those in non-CAD patients. Receiver operating characteristic curve analyses have shown that salusin-α is the most useful biomarker for detecting CAD among the four peptides examined. Therefore, salusin-α, alone or in various combinations with heregulin-ß1, adiponectin, and/or GLP-1, is a candidate biomarker for predicting CAD. Further, anti-atherogenic peptides could potentially serve as useful therapeutic targets for atherosclerotic cardiovascular diseases.

Activation of coagulation by lenalidomide-based regimens for the treatment of multiple myeloma.

We investigated the procoagulant effects of lenalidomide (Len)-based regimens in vitro focusing on tissue factor (TF) and phosphatidylserine (PS). We examined the effects of a pharmacological concentration of Len with or without the corticosteroid dexamethasone (Dex) and the proteasome inhibitor bortezomib (Bor) using the human vascular endothelial cell line EAhy926 and the monocytic cell lines THP-1 and U937. Cell-surface procoagulant activity (PCA) was induced by Dex-containing regimens in all lines. Expression of TF antigen on the cell surface and of TF mRNA was markedly increased by Dex-containing regimens. PS exposure was increased modestly by a Len-based regimen. PS exposure was increased modestly in EAhy926 cells, and markedly increased in THP-1 and U937 cells by Bor-containing treatment. An anti-TF monoclonal antibody almost completely blocked the induced PCA. When Len is given in combination with Dex, PCA may be induced on endothelial cells and monocytes through TF expression and PS exposure.

Release of salusin-beta from human monocytes/macrophages.

Salusin-alpha and salusin-beta are related bioactive peptides biosynthesized from the same precursor, prosalusin. Despite the potent hemodynamic and proatherosclerotic activities of salusin-beta, its exact distribution and biological functions remain largely undetermined because of technical difficulties associated with its unique physicochemical characteristics, such as marked adhesiveness to polypropylene and polystyrene. By circumventing these problems, we recently established a specific radioimmunoassay for detecting immunoreactive human salusin-beta. In the current study, we demonstrated the release of salusin-beta from the human monoblastic leukemia cell lines, THP-1 and U937. Dilution curves of extracted conditioned media from both cells were parallel with those of standard human salusin-beta by radioimmunoassay. Reverse-phase high performance liquid chromatography coupled with radioimmunoassay detection of the culture supernatants revealed a major immunoreactive component that co-eluted with authentic salusin-beta. Both cell lines secreted salusin-beta-like immunoreactivity (LI) into serum-free media as a function of time (1234.3 + or - 122.7 and 186.7 + or - 9.1 fmol/10(5) cells per 24h). When THP-1 and U937 cells differentiated into macrophages after incubation with 2-O-tetradecanoylphorbol-13-acetate (TPA), they secreted far greater amounts of salusin-beta-LI into the culture supernatant (3351.9 + or - 899.3 and 1545.8 + or - 183.3 fmol/10(5) cells per 24h). TPA treatment accelerated the processing of prosalusin into its cleaved fragments, suggesting that the increased secretion of salusin-beta-LI in THP-1-derived macrophages was caused by the enhanced intracellular processing of prosalusin. Stimulation with the inflammatory cytokines, tumor necrosis factor alpha (TNF-alpha) and lipopolysaccharide (LPS), resulted in increased secretion of salusin-beta without inducing expression of the gene for preprosalusin, suggesting that TNF-alpha and LPS stimulated the release of salusin-beta. These data demonstrate that salusin-beta, which induces macrophage foam cell formation, is secreted in its authentic form from human monocytes/macrophages.