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OBJECTIVES: Elevated fetuin-A (Fet-A) has been shown to be a risk factor for several metabolic diseases and understanding the effects of moderate weight loss on changes in Fet-A and other inflammatory markers could allow for lifestyle intervention strategies. The association of inflammatory markers and Fet-A with incremental body weight loss is unknown. The objective of the study was to evaluate the association of inflammatory markers, including TNF-alpha (TNF-α), adiponectin, and C-reactive protein (CRP), Fet-A, and it's phosphorylated form, pFet-A, with incremental body weight loss. METHODS: Sixteen men achieved a targeted weight loss of 8% to 10% of initial body weight. In this study, we analyzed changes in serum TNF-α, adiponectin, and CRP inflammatory cytokines to changes in serum serum-Fet-A and pFet-A. We also examined the relationship of changes in cytokine profile to alterations in anthropometrics and other metabolic indices. All statistical analyses were performed using GraphPad Prism version 8.0. RESULTS: A moderate body weight loss of 8% to 10%, significantly decreased serum CRP, but did not affect TNF-α or adiponectin concentrations in individuals with obesity. Serum CRP started to decrease with 4% to 6% body weight loss, demonstrating a mean change in serum CRP concentrations of – 0.15 mg/L and – 0.10 mg/L, for 4%-6% and 8%-10% body weight loss, respectively, for each 1 kg of body weight loss. Weight-loss induced change in serum CRP concentrations were not significantly associated with a decrease in serum Fet-A or pFet-A, although a trend was observed for change in serum pFet-A (r = 0.44, P = 0.09). CONCLUSIONS: A moderate weight loss improved serum inflammatory marker C-reactive protein but did not affect TNF-α or adiponectin concentrations in individuals with obesity. These changes were not significantly associated with a decrease in serum pFet-A although a trend was observed. FUNDING SOURCES: This work was supported by the American Diabetes Association (ADA 7–04-JF-36); the Alabama Agricultural Experiment Station (ALA080–052).

Fetuin-A (Fet-A), a hepatokine associated with insulin resistance, obesity, and incident type 2 diabetes, is shown to exist in both phosphorylated and dephosphorylated forms in circulation. However, studies on fetuin-A phosphorylation status in insulin-resistant conditions and its functional significance are limited. We demonstrate that serum phosphofetuin-A (Ser312) levels were significantly elevated in high-fat diet-induced obese mice, insulin-resistant Zucker diabetic fatty rats, and in individuals with obesity who are insulin resistant. Unlike serum total fetuin-A, serum phosphofetuin-A was associated with body weight, insulin, and markers of insulin resistance. To characterize potential mechanisms, fetuin-A was purified from Hep3B human hepatoma cells. Hep3B Fet-A was phosphorylated (Ser312) and inhibited insulin-stimulated glucose uptake and glycogen synthesis in L6GLUT4 myoblasts. Furthermore, single (Ser312Ala) and double (Ser312Ala + Ser120Ala) phosphorylation-defective Fet-A mutants were without effect on glucose uptake and glycogen synthesis in L6GLUT4 myoblasts. Together, our studies demonstrate that phosphorylation status of Fet-A (Ser312) is associated with obesity and insulin resistance and raise the possibility that Fet-A phosphorylation may play a role in regulation of insulin action.

Fetuin‐A (Fet‐A), secreted by the liver and adipose tissue, inhibits insulin receptor tyrosine kinase activity and modulates insulin action. Numerous studies have shown association of elevated serum Fet‐A concentrations with obesity, non‐alcoholic fatty liver disease, and type 2 diabetes. Both moderate body weight loss (5%–10%) and significant body weight loss have been shown to decrease serum Fet‐A and improve insulin sensitivity. Currently, there are no studies examining the effects of a single bout of exercise on serum Fet‐A or Ser312‐pFet‐A (pFet‐A) responses. We hypothesized that a single bout of moderate‐intensity exercise will lower serum Fet‐A and that these changes will be associated with an improvement in insulin sensitivity. Thirty‐one individuals with obesity and 11 individuals with normal body weight were recruited. Participants underwent a single bout of treadmill walking, expending 500 kcal at 60%–70% VO2max. Oral glucose tolerance tests (OGTT) were administered before the single bout of exercise (Pre Ex) and 24 h after exercise (24h Post Ex). In individuals with obesity, we observed a transient elevation of serum Fet‐A concentrations, but not pFet‐A, immediately after exercise (Post Ex). Further, a single bout of exercise decreased glucoseAUC, insulinAUC, and insulin resistance index in individuals with obesity. Consistent with this improvement in insulin sensitivity, we observed that Fet‐AAUC, pFet‐AAUC, 2 h pFet‐A, and 2 h pFet‐A/Fet‐A were significantly lower following a single bout of exercise. Further, reductions in serum Fet‐AAUC 24h Post Ex were correlated with a reduction in insulin resistance index. Together, this suggests that alterations in serum Fet‐A following a single bout of moderate‐intensity endurance exercise may play a role in the improvement of insulin sensitivity. Clinical Trial Registration NCT03478046; https://clinicaltrials.gov/ct2/show/NCT03478046., Serum Fet‐A was transiently elevated immediately after a single bout of exercise. and restored to Pre Ex levels 24 h after a single bout of exercise. Furthermore, serum Fet‐AAUC, pFet‐AAUC, glucoseAUC, insulinAUC, and insulin resistance index were significantly decreased 24 h after a single bout of exercise compared with Pre Ex levels in individuals with obesity. Additionally, the reduction in 24 h Post Ex insulin resistance index was correlated with a reduction in serum Fet‐AAUC, suggesting that alterations in serum Fet‐A and its phosphorylated form, Ser‐312 pFet‐A, may potentially contribute to the observed improvement in insulin sensitivity following a single bout of endurance exercise.

Objective Phosphorylated fetuin-A (pFet-A) inhibits insulin action and has been shown to be associated with obesity and insulin resistance. The objective of this cohort study was to assess the effect of incremental body weight loss on alterations in serum pFet-A and indexes of insulin sensitivity. Methods A total of 16 men with obesity attained a targeted weight loss of 8% to 10% of their initial body weight by achieving an energy expenditure/deficit of 2,000 to 2,500 kcal/wk. Anthropometric assessments and blood samples were obtained every 4 weeks. Weight loss was calculated and partitioned as 2% to 4%, 4% to 6%, 6% to 8%, and 8% to 10% compared with initial body weight. Results Targeted body weight loss of 8% to 10% decreased serum pFet-A, pFet-A:Fet-A ratio, fasting insulin, log(homeostasis model assessment of insulin resistance), quantitative insulin sensitivity check index, adipose insulin resistance, and insulin resistance index significantly. Percent changes in serum pFet-A were associated with percent changes in indexes of insulin sensitivity. Unlike insulin sensitivity indexes, which were altered starting with 6% to 8% weight loss, serum pFet-A levels were significantly decreased by 19.6% starting with 2% to 4% weight loss and decreased by 25.6%, 36.8%, and 42.3% with 4% to 6%, 6% to 8%, and 8% to 10% weight loss, respectively. Conclusions This study reports for the first time that the insulin-sensitizing effects of moderate weight loss are associated with a reduction in serum pFet-A levels.

Epidemiological and observational studies indicate a positive correlation between type 2 diabetes (T2DM) and dementia, with an increased risk of dementia and Alzheimer's disease (AD) associated with insulin-treated diabetes patients. The purpose of this review is to reveal the molecular mechanisms that connect physiological and pathological processes commonly observed in T2DM and AD. Conformational modifications in peptide residues, such as amyloid-β peptide in AD and amylin in T2DM have been shown to instigate formation of insoluble protein aggregates that get deposited in extracellular spaces of brain and pancreatic tissue thus disrupting their normal function. Impaired insulin signaling plays a critical role in AD pathogenesis by reducing IRS-associated PI3 kinase activity and increasing GSK-3β activity. GSK-3β has been suggested to be a component of the γ-secretase complex and is involved in amyloid-β protein precursor processing. GSK-3β along with CDK5 is responsible for hyperphosphorylation of tau leading to the formation of neurofibrillary tangles. In summary, there is evidence to believe that a molecular link connects AD and T2DM and has potential for further investigation toward development of an effective therapeutic target.

The nuclear receptor peroxisome proliferator-activated receptor (PPAR)gamma plays a key role in regulating whole body glucose homeostasis and insulin sensitivity. Although it is expressed most highly in adipose, it is also present at lower levels in many tissues, including skeletal muscle. The role muscle PPARgamma plays in metabolic regulation and in mediating the antidiabetic effects of the thiazolidinediones is not understood. The goal of this work was to examine the molecular and physiological effects of PPARgamma activation in muscle cells. We found that pharmacological activation of PPARgamma in primary cultured myocytes, and genetic activation of muscle PPARgamma in muscle tissue of transgenic mice, induced the production of adiponectin directly from muscle cells. This muscle-produced adiponectin was functional and capable of stimulating adiponectin signaling in myocytes. In addition, elevated skeletal muscle PPARgamma activity in transgenic mice provided a significant protection from high-fat diet-induced insulin resistance and associated changes in muscle phenotype, including reduced myocyte lipid content and an increase in the proportion of oxidative muscle fiber types. Our findings demonstrate that PPARgamma activation in skeletal muscle can have a significant protective effect on whole body glucose homeostasis and insulin resistance and that myocytes can produce and secrete functional adiponectin in a PPARgamma-dependent manner. We propose that activation of PPARgamma in myocytes induces a local production of adiponectin that acts on muscle tissue to improve insulin sensitivity.

Background Fetuin-A, a liver-secreted phosphoprotein and physiological inhibitor of insulin receptor tyrosine kinase, is associated with insulin resistance, metabolic syndrome (MetS), and an increased risk for type 2 diabetes. However, studies on the modulation of circulating levels of fetuin-A are limited. The goal of this study was to determine the effect of niacin administration on serum total- and phosphorylated fetuin-A (phosphofetuin-A) concentrations in individuals with MetS and correlate with changes in serum lipids, insulin sensitivity, and markers of inflammation. Methods Fifteen sedentary, obese, male participants, who met the National Cholesterol Education Program Adult Treatment Panel III (NCEP ATP III) criteria for MetS, were treated with extended-release niacin (Niaspan) for 6 weeks. Blood samples were obtained before and after treatment with niacin. Results Serum fetuin-A and phosphofetuin-A concentrations were decreased following niacin administration (p < 0.005). Changes in fetuin-A concentrations were correlated with changes in triglyceride (r = 0.62, p = 0.01) and C-reactive protein (CRP) concentrations (r = 0.58, p < 0.05) after niacin treatment. Changes in high-density lipoproteins (HDL)-cholesterol following niacin intervention were negatively correlated with changes in serum fetuin-A (p < 0.05) and phosphofetuin-A concentrations (p < 0.05). Serum cortisol levels were significantly elevated after niacin administration. Conclusions Niacin treatment lowers serum total- and phosphofetuin-A concentrations in individuals with MetS, and these changes correlate with the beneficial changes in serum lipids. Because niacin is known to induce insulin resistance, these findings suggest that fetuin-A may not be a mediator of niacin-induced insulin resistance but it may blunt the insulin resistance induced by niacin by decreasing its circulating concentrations. Copyright © 2009 John Wiley & Sons, Ltd.

Saliva has been progressively studied as a non-invasive and relatively stress-free diagnostic alternative to blood. Currently, saliva testing is used for clinical assessment of hormonal perturbations, detection of HIV antibodies, DNA analysis, alcohol screening, and drug testing. Recently, there has been increasing interest in evaluating the diagnostic potential of saliva in obesity, inflammation, and insulin-resistance. Current literature has demonstrated elevated levels of inflammatory biomarkers including C-reactive protein, tumor necrosis factor-α, interleukin-6, and interferon-γ in saliva of obese/overweight children and adults. Salivary antioxidant status has also been studied as a measure of oxidative stress in individuals with type 2 diabetes. Further, several studies have demonstrated correlations of salivary markers of stress and insulin resistance including cortisol, insulin, adiponectin, and resistin with serum concentrations. These findings suggest the potential diagnostic value of saliva in health screening and risk stratification studies, particularly in the pediatric population, with implications for inflammatory, metabolic and cardiovascular conditions. However, additional studies are required to standardize saliva collection and storage procedures, validate analytical techniques for biomarker detection, and establish reference ranges for routine clinical use. The purpose of this review is to summarize and evaluate recent advancements in using saliva as a diagnostic tool for inflammation and insulin-resistance.

The elevated level of histidine-rich glycoprotein was considered a risk factor of inherited thrombophilia. However, the mode of action remains largely unclear. In the current study, we employ poly-l-histidine (PLH) mimicking the histidine-rich region and determine whether PLH modulates urokinase (uPA)-dependent fibrinolysis. In an in vitro model, turbidity appearance and clearance monitored fibrin polymer formation and lysis, respectively. Fibrin polymer formed upon fibrinogen incubation with thrombin. In the presence of uPA or plasmin, fibrin polymer lysis took place in a dose-dependent manner as a function of time. We demonstrated that PLH significantly downregulated uPA-dependent fibrinolysis. PLH had no effect on plasminogen activation, as evidenced by no inhibitions on either uPA amidolytic activity or plasmin formation derived from its zymogen. Nor did PLH show any inhibition on plasmin amidolytic activity. PLH caused a profound delay of plasmin-dependent fibrinolysis upon pre-incubation of either plasmin or fibrinogen with PLH. The observations taken together suggest that the complex [plasmin-PLH-fibrin] formation significantly delayed plasmin-dependent fibrinolysis.

Traditional knowledge and anecdotal evidence suggest that Tridax procumbens Linn may have anti-inflammatory and blood glucose lowering properties. Recent studies have shown that T.procumbens extract was effective in lowering blood glucose (42-71% blood glucose reduction at 250-500 mg/kg body weight) in alloxan-induced diabetic rats. The goals of this study were to evaluate blood glucose lowering potential of T.procumbens extract in subjects with type 2 diabetes and characterize underlying molecular mechanisms. Twenty individuals with established type 2 diabetes were orally administered (15 ml, twice daily) T.procumbens extract, prepared according to Ayurveda guidelines, for a period of 4 weeks. Fasting blood glucose concentrations were reduced by 11% in men and 20% in women, and post-prandial blood glucose concentrations were decreased by 26% in men and 29% in women. No adverse effects were reported by the participants. Chemical and microbial analyses of T. procumbens extract demonstrated the presence of ...

C-peptide, released by the beta-cells of pancreatic islets, elicits salutary responses in Type I (insulin-dependent) diabetes mellitus but the molecular mechanisms behind these effects are not known. We assessed whether synthetic rat C-peptide stimulates insulin-like cellular effects in a classic insulin target tissue.To clarify the molecular mechanisms involved in several insulinomimetic actions, we investigated the effect of C-peptide on the insulin signalling pathway in rat skeletal muscle cells. We used L6 myoblasts and myocytes to measure the effects of C-peptide or insulin or both on glycogen synthesis and amino acid uptake. We also studied the effects of C-peptide on insulin receptor autophosphorylation, its tyrosine kinase activity, phosphorylation of IRS-1, PI 3-kinase, Akt, p90Rsk, MAPK, and GSK3 in these cells.In L6 cells, physiological concentrations of C-peptide (0.3-3 nmol/l) significantly activated insulin receptor tyrosine kinase, IRS-1 tyrosine phosphorylation, PI 3-kinase activity, MAPK phosphorylation, p90Rsk, and GSK3 phosphorylation. A scrambled C-peptide sequence - the control - showed no effects. Wortmannin blocked C-peptide-induced glycogen synthesis while pertussis toxin had no effect. Only submaximal insulin concentrations (up to 10 nmol/l) combined with submaximal C-peptide concentrations led to additive effects.C-peptide added to the maximal insulin dose (100 nmol/l) did not increase the effect of insulin alone. We thus conclude that the same signalling elements are used by both ligands. However, the lack of Akt activation by C-peptide and the bell-shaped dose response induced by C-peptide indicate that C-peptide has some effects by another distinct mechanism. We speculate that C-peptide could modulate the metabolic effects of insulin by enhancing them at low hormone concentrations and dampening them at high hormone concentrations.

Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is a polyphenolic phytoalexin that exerts cardioprotective, neuroprotective, and antioxidant effects. Recently it has been shown that obesity is associated with an increase in cerebral oxidative stress levels, which may enhance neurodegeneration. The present study evaluates the neuroprotective action of resveratrol in brain of obese (ob/ob) mice. Resveratrol was administered orally at the dose of 25 mg kg−1body weight daily for three weeks to lean and obese mice. Resveratrol had no effect on body weight or blood glucose levels in obese mice. Lipid peroxides were significantly increased in brain of obese mice. The enzymatic antioxidants superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase and nonenzymatic antioxidants tocopherol, ascorbic acid, and glutathione were decreased in obese mice brain. Administration of resveratrol decreased lipid peroxide levels and upregulated the antioxidant activities in obese mice brain. Our findings indicate a neuroprotective effect of resveratrol by preventing oxidative damage in brain tissue of obese mice.

In experimental animal and cell culture models, activation of peroxisome proliferator-activated receptor (PPAR) gamma in heart has been shown to have beneficial effects on cardiac function and cardiomyocyte physiology. The goal of this study was to identify the signaling pathway by which PPARgamma activation protects cardiomyocytes from the deleterious effects of hypertrophic stimuli. In primary cardiomyocyte cultures, we found that genetic or pharmacological activation of PPARgamma protected cells from cardiac hypertrophy induced by alpha-adrenergic stimulation. Examination of gene expression in these cells revealed a surprising increase in the expression of adiponectin in cardiomyocytes and secretion of the high-molecular-weight form of the hormone into media. Using RNAi to block PPARgamma-induced adiponectin production or adiponectin receptor gene expression, we found that the PPARgamma-mediated anti-hypertrophic effect required cardiomyocyte-produced adiponectin, as well as an intact adiponectin signaling pathway. Furthermore, mice expressing constitutive-active PPARgamma and cardiomyocyte specific adiponectin expression were protected from high-fat diet-induced cardiac hypertrophy and remodeling. These findings demonstrate that functional adiponectin hormone can be produced from the heart and raise the possibility that beneficial effects of PPARgamma activation in heart could be due in part to local production of adiponectin that acts on cardiomyocytes in an autocrine manner.

alpha2-HS glycoprotein (AHSG), also known as fetuin-A, inhibits insulin receptor autophosphorylation and tyrosine kinase activity in vitro and in vivo. Earlier we have shown that fetuin-null (KO) mice demonstrate improved insulin sensitivity and resistance to diet-induced obesity. Since aging is associated with insulin resistance and impaired glucose handling, we tested the hypothesis that fetuin-null (KO) mice are resilient to changes in insulin sensitivity associated with aging. Aged (80-week-old) fetuin-null mice were leaner and demonstrated significantly lower body weights compared to age- and sex-matched wild-type (WT) littermates. Leanness in aged fetuin KO mice was accompanied by a significant increase in dark-onset energy expenditure, without marked alteration of respiratory quotient. In comparison to WT mice, fetuin KO mice demonstrated a lower fasting insulin resistance index, and significantly lower blood glucose and insulin levels, following a 4h fast. Interestingly, despite significantly decreased insulin levels during a glucose tolerance test, aged fetuin-null mice demonstrated a similar glucose excursion as WT mice, indicative of improved insulin sensitivity. Analysis of aldehyde-fuchsin stained pancreas from aged fetuin KO mice indicated no difference in islet beta-cell size or number. An insulin tolerance test confirmed the increased insulin sensitivity of aged fetuin KO mice. Further, compared to WT mice, aged fetuin-null mice demonstrated increased skeletal muscle and liver IR autophosphorylation and TK activity. Taken together, this study suggests that the absence of fetuin may contribute to the improvement of insulin sensitivity associated with aging.

Autosomal dominant familial partial lipodystrophy (FPLD) due to mutant LMNA encoding nuclear lamin A/C is characterized by adipose tissue repartitioning together with multiple metabolic disturbances, including insulin resistance and dyslipidemia. There is emerging evidence that some rare mutations in peroxisome proliferator-activated receptor-γ (PPAR-γ), encoded by PPARG, might be associated with human lipodystrophy. We report a three-generation Canadian kindred ascertained based upon partial lipodystrophy, with a normal LMNA gene sequence. Candidate gene sequencing showed that all four affected subjects were heterozygous for a novel T→A mutation at PPARG nucleotide 1164 in exon 5 that predicted substitution of phenylalanine at codon 388 by leucine (F388L). The mutation was absent from normal family members and normal unrelated subjects, and altered a highly conserved residue within helix 8 of the predicted ligand-binding pocket of PPAR-γ. The mutant receptor had significantly decreased basal transcriptional activity and impaired stimulation by a synthetic ligand. The germline transmission of a transactivation-deficient mutation in PPARG suggests that autosomal dominant partial lipodystrophy is genetically heterogeneous. Our findings are consistent with the idea that mutant PPARG can underlie the partial lipodystrophy phenotype.

Fetuin inhibits insulin-induced insulin receptor (IR) autophosphorylation and tyrosine kinase activity in vitro, in intact cells, and in vivo. The fetuin gene (AHSG) is located on human chromosome 3q27, recently identified as a susceptibility locus for type 2 diabetes and the metabolic syndrome. Here, we explore insulin signaling, glucose homeostasis, and the effect of a high-fat diet on weight gain, body fat composition, and glucose disposal in mice carrying two null alleles for the gene encoding fetuin, Ahsg (B6, 129-Ahsgtm1Mbl). Fetuin knockout (KO) mice demonstrate increased basal and insulin-stimulated phosphorylation of IR and the downstream signaling molecules mitogen-activated protein kinase (MAPK) and Akt in liver and skeletal muscle. Glucose and insulin tolerance tests in fetuin KO mice indicate significantly enhanced glucose clearance and insulin sensitivity. Fetuin KO mice subjected to euglycemic-hyperinsulinemic clamp show augmented sensitivity to insulin, evidenced by increased glucose infusion rate (P = 0.077) and significantly increased skeletal muscle glycogen content (P < 0.05). When fed a high-fat diet, fetuin KO mice are resistant to weight gain, demonstrate significantly decreased body fat, and remain insulin sensitive. These data suggest that fetuin may play a significant role in regulating postprandial glucose disposal, insulin sensitivity, weight gain, and fat accumulation and may be a novel therapeutic target in the treatment of type 2 diabetes, obesity, and other insulin-resistant conditions.

Background : Human α 2 -HS glycoprotein (α 2 -HSG) is synthesized and secreted by the liver into circulation. Plasma concentrations of α 2 -HSG decrease significantly following infection, inflammation and malignancy. Since increased plasma concentrations of C-reactive protein are observed in patients with acute myocardial infarction (AMI), we hypothesized that plasma concentrations of α 2 -HSG would decrease during the initial phase of AMI and begin to increase in the recovery phase. Methods : Twenty patients diagnosed with AMI were recruited for the study. A sensitive and specific ELISA was developed to assay α 2 -HSG concentrations in plasma. Results : In AMI patients, plasma α 2 -HSG concentrations were decreased (281.3±25.8 mg/l, ranging from 132 to 489 mg/l on admission) compared to healthy individuals (312.3±9.9 mg/l, ranging from 210 to 450 mg/l) ( P = 0.142). Interestingly, 40% of AMI patients demonstrated α 2 -HSG concentrations below 200 mg/l compared to none in the healthy control group. During the recovery period, α 2 -HSG concentrations begin to increase, with a mean±SEM of 290.1±22.1 mg/l. Regression analysis comparing plasma α 2 -HSG concentrations on admission to concentrations on discharge showed a significant positive correlation in matched-pair patient samples ( P r =0.45). Conclusions : We conclude that, in contrast to C-reactive protein, α 2 -HSG functions as a negative acute phase protein in AMI patients. Plasma α 2 -HSG concentrations start to decrease within a few hours after the onset of AMI and return to near normal concentrations during the recovery period (5–7 days after AMI).

The enhanced extrinsic coagulation in response to inflammation could contribute to disseminated intravascular coagulation, often manifesting cardiovascular complications. The complex mechanism remains unclear. Nor is the effective anticoagulation well established. The search for arresting hypercoagulation is of antithrombotic relevance. The ability of polybrene (PB) to inhibit tissue factor (TF)-initiated extrinsic blood coagulation was demonstrated at the protein and cellular levels as well as in human plasma samples. In a single-stage clotting assay, PB dose-dependently offset bacterial endotoxin (lipopolysaccharide)-induced monocytic TF (mTF) hypercoagulation and inhibited rabbit brain thromboplastin (rbTF) procoagulation. Consistent with these findings, the significantly prolonged prothrombin time indicated the depressed extrinsic coagulation by PB. However, PB showed no effect on thrombin time. We dissected the extrinsic pathway to further determine the inhibitory site(s) of PB. A two-stage chromogenic assay monitoring S-2288 hydrolysis showed that PB readily blocked mTF-dependent or rbTF-dependent FVII activation, which was verified by the diminished activated factor VII (FVIIa) formation derived from the proteolytic cleavage of its zymogen factor VII on Western blotting analyses. PB had no effect on FVIIa and activated factor X amidolytic activity. Nor was the dissected TF/ FVIIa-catalyzed factor X activation affected. In conclusion, the preferential downregulation of factor VII activation was responsible for the depressed extrinsic coagulation. PB could present a novel anticoagulant antagonizing the extrinsic hypercoagulation for the prevention of thrombotic complication following sepsis and inflammations.

Human fetuin, [alpha2-Heremans Schmid Glycoprotein (alpha2-HSG)], is a natural inhibitor of insulin receptor tyrosine kinase activity (IR-TKA). Previously, we have demonstrated that alpha2-HSG inhibits the mitogenic pathway without affecting the metabolic arm of insulin signal transduction. In this study, we demonstrate the time-course and specificity of inhibition, its interaction with IR and probable physiological role. In intact rat1 fibroblasts overexpressing the human insulin receptor (HIRc B), incubation of recombinant human alpha2-HSGbac (1.8 microM) inhibited insulin-induced IR autophosphorylation by over 80%. This inhibitory effect of alpha2-HSGbac on insulin-induced IR autophosphorylation was blunted by half in 60 min. Interestingly, alpha2-HSGbac at similar concentrations (0.9 or 1.8 microM), had no effect on EGF- or IGF-I-induced cognate receptor autophosphorylation. Anti-alpha2-HSG immunoprecipitates of alpha2-HSGbac-treated HIRc B cell lysates demonstrated the presence of IR. Our data suggest that alpha2-HSGbac preferentially interacts with the activated IR. To further characterize the site(s) of interaction, the effect of alpha2-HSGbac on trypsin-treated IR autophosphorylation was studied. Trypsin-treatment of intact HIRc B cells results in proteolysis of the IR alpha-chain and constitutive activation of IR-TKA. We demonstrate that alpha2-HSGbac (0.1 microM) completely inhibited trypsin-activated IR autophosphorylation and TKA in vitro indicating that this effect was not mediated by its interaction with the proximal 576 amino acid residues of the IR alpha-subunit. The physiological relevance of these observations was explored by characterizing the effects of alpha2-HSG injection in rats. Alpha2-HSGbac (2 microM), acutely injected through the portal vein of normal rats, inhibited insulin-stimulated IR autophosphorylation and IRS-1 phosphorylation in liver and hindlimb muscle. Taken together our results suggest that alpha2-HSG, by interacting with IR, specifically inhibits insulin-stimulated IR autophosphorylation and may play a physiological role in the regulation of insulin signaling.