Phosphorylation status of fetuin-A is critical for inhibition of insulin action and is correlated with obesity and insulin resistance.

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.

AMPK activation by pterostilbene contributes to suppression of hepatic gluconeogenic gene expression and glucose production in H4IIE cells.

Pterostilbene, a bioactive component of blueberries and grapes, shows structural similarity to resveratrol, and exhibits antioxidant, anti-inflammatory, anti-cancer, hypoglycemic, and cholesterol lowering effects. Recent evidence indicates that pterostilbene is an agonist of the nuclear receptor, peroxisome proliferator-activated receptor-alpha (PPAR-α). Since PPAR-α agonists induce peroxisomal proliferation and fatty acid oxidation, we examined gene expression of acyl CoA oxidase (ACO) and carnitine palmitoyl transferase-1 (CPT-1). Pterostilbene treatment, at concentrations that demonstrated over 75% cell viability (20 µM, 50 µM), significantly increased gene expression of ACO, CPT-1, and PPAR-α. Pterostilbene treatment (50 µM) also demonstrated potent activation of AMP-activated kinase (AMPK), compared to AICAR (0.5 mM) or metformin (2 mM), consistent with upregulation in fatty acid oxidation gene expression. Since AMPK activators mimic the actions of insulin by repressing hepatic gluconeogenesis, we examined pterostilbene's effects on hepatic gluconeogenic gene expression. Pterostilbene treatment significantly repressed dexamethasone-induced phosphoenol pyruvate carboxykinase (PEPCK) and glucose6-phosphatase (G6Pase) gene expression, and decreased glucose production in H4IIE cells. Taken together, our studies demonstrate that pterostilbene, a natural compound and PPAR-α agonist, modulate several AMPK-dependent metabolic functions. The results of the present study suggest that pterostilbene may have beneficial effects in the prevention and management of type 2 diabetes and related disorders. In this study, we found that pterostilbene activated AMP-activated kinase (AMPK) and increased the expression of fatty acid oxidation genes, including acyl CoA oxidase and carnitine palmitoyl transferase-1.

Evaluation of PPARα activation by known blueberry constituents.

BACKGROUND: Anthocyanins are known to have hypolipidemic properties. It was deemed necessary to determine whether major blueberry anthocyanins and catechins are ligands for the transcription factor peroxisome proliferator activated receptor alpha isoform (PPARα), and compare activation with known PPARα agonistic constituents, pterostilbene and resveratrol. It was also considered important to investigate the effect of pterostilbene on PPARα gene expression, and relate results with hepatic mRNA PPARα expression up-regulation observed previously in hamsters fed a diet supplemented with blueberry peels extract (BBX). RESULTS: The anthocyanins and catechins did not activate PPARα. Only pterostilbene exhibited a dose-dependent activation of PPARα in H4IIEC3 cells. The resveratrol responses were lower than those of pterostilbene. Pterostilbene significantly and dose-dependently (at 10, 20 and 50 µmol L(-1) ) increased PPARα gene expression and the effect at 10 µmol L(-1) was greater than 100 and 200 µmol L(-1) of fenofibrate. Analysis of BBX showed levels of pterostilbene and resveratrol at 418 and 2381 ng g(-1), respectively. CONCLUSION: Anthocyanins and catechins do not appear to contribute to the up-regulation of hepatic PPARα expression observed in hamsters. While pterostilbene and resveratrol demonstrated PPARα activation, their levels in BBX do not seem to be at efficacious concentrations. These stilbenes may contribute to the up-regulation of PPARα expression by acting synergistically with each other or with other constituents in BBX.

Blood Glucose-lowering Effect of T. procumbens L.: A Pilot Clinical Study in Individuals with Type 2 Diabetes.

Traditional knowledge, in vitro studies, and studies using animal models suggest that Tridax procumbens L. exhibits blood glucose-lowering properties and antiinflammatory effects. In this study, we evaluated the blood glucose-lowering effect of T. procumbens supplementation in individuals with type 2 diabetes. An extract (asava) of T. procumbens L. was prepared following Ayurveda guidelines. Chemical and microbial analyses indicated presence of phenolics, flavonoids, and carotenoids, and absence of microbial contamination, aflatoxins, heavy metals, and pesticide residues. A chemical fingerprint of T. procumbens L. asava, developed using Ultra high pressure liquid chromatography/electron spray ionization-mass spectrometry (UPLC/ESI-MS) in negative mode, suggest the presence of several compounds including polyphenols. T. procumbens asava demonstrated strong total antioxidant capacity, Fe3+ reducing potential, Fe2+ chelation, H2 O2 scavenging activity, and inhibition of lipid peroxidation. We recruited 20 type 2 diabetic individuals from Kolhapur, India. Participants received 15 mL of T. procumbens asava, twice daily, for 4 weeks, while continuing their prescribed antidiabetic medications. Fasting blood glucose decreased by 11% in men (p < 0.01) and 20% in women (p < 0.05), and post-prandial blood glucose concentrations were lowered by 26% in men (p < 0.001) and 29% in women (p < 0.001) following 4 weeks of asava supplementation. No adverse events or side effects were reported. This is the first clinical study demonstrating a significant blood glucose-lowering effect of T. procumbens asava in type 2 diabetes. Copyright © 2015 John Wiley & Sons, Ltd.

AMP activated kinase negatively regulates hepatic Fetuin-A via p38 MAPK-C/EBPß/E3 Ubiquitin Ligase Signaling pathway.

Fetuin-A (Fet-A) is a liver-secreted phosphorylated protein, known to impair insulin signaling, which has been shown to be associated with obesity, insulin resistance, and incident diabetes. Fet-A interacts with the insulin-stimulated insulin receptor (IR) and inhibits IR tyrosine kinase activity and glucose uptake. It has been shown that high glucose increases Fet-A expression through the ERK1/2 signaling pathway. However, factors that downregulate Fet-A expression and their potential mechanisms are unclear. We examined the effect of AMP-activated protein kinase (AMPK) on high-glucose induced Fet-A expression in HepG2 cells, Hep3B cells and primary rat hepatocytes. High glucose increased Fet-A and phosphorylated (Ser312) fetuin-A (pFet-A) expression, which are known to impair insulin signaling. AICAR-induced AMPK activation significantly down-regulated high glucose-induced Fet-A expression and secretion of pFet-A while treatment with Compound C (AMPK inhibitor), SB202190 (p38 MAPK inhibitor) or p38 MAPK siRNA transfection prevented AICAR-induced downregulation of Fet-A expression. In addition, activation of p38 MAPK, by anisomycin, decreased the hepatic expression of Fet-A. Further, we our studies have shown that short-term effect of AICAR-treatment on Fet-A expression was mediated by proteosomal degradation, and long-term treatment of AICAR was associated with decrease in hepatic expression of C/EBP beta, an important transcription factor involved in the regulation of Fet-A. Taken together, our studies implicate a critical role for AMPK-p38 MAPK-C/EBPb-ubiquitin-proteosomal axis in the regulation of the expression of hepatic Fet-A.

Serum fetuin-A and Ser312 phosphorylated fetuin-A responses and markers of insulin sensitivity after a single bout of moderate intensity exercise.

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.

Selective activation of PPARgamma in skeletal muscle induces endogenous production of adiponectin and protects mice from diet-induced insulin resistance.

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.

Endogenously produced adiponectin protects cardiomyocytes from hypertrophy by a PPARgamma-dependent autocrine mechanism.

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.

Alterations of Serum Ser312-Phosphorylated Fetuin-A from Exercise-Induced Moderate Body Weight Loss in Individuals with Obesity.

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.

Curcumin activates AMPK and suppresses gluconeogenic gene expression in hepatoma cells.

Curcumin, the bioactive component of curry spice turmeric, and its related structures possess potent anti-oxidant and anti-inflammatory properties. Several lines of evidence suggest that curcumin may play a beneficial role in animal models of diabetes, both by lowering blood glucose levels and by ameliorating the long-term complications of diabetes. However, current understanding of the mechanism of curcumin action is rudimentary and is limited to its anti-oxidant and anti-inflammatory effects. In this study we examine potential anti-diabetic mechanisms of curcumin, curcumin C3 complex), and tetrahydrocurcuminoids (THC). Curcuminoids did not exert a direct effect on receptor tyrosine kinase activity, 2-deoxy glucose uptake in L6-GLUT4myc cells, or intestinal glucose metabolism measured by DPP4/alpha-glucosidase inhibitory activity. We demonstrate that curcuminoids effectively suppressed dexamethasone-induced phosphoenol pyruvate carboxy kinase (PEPCK) and glucose6-phosphatase (G6Pase) in H4IIE rat hepatoma and Hep3B human hepatoma cells. Furthermore, curcuminoids increased the phosphorylation of AMP-activated protein kinase (AMPK) and its downstream target acetyl-CoA carboxylase (ACC) in H4IIE and Hep3B cells with 400 times (curcumin) to 100,000 times (THC) the potency of metformin. These results suggest that AMPK mediated suppression of hepatic gluconeogenesis may be a potential mechanism mediating glucose-lowering effects of curcuminoids.

Extended-release niacin decreases serum fetuin-A concentrations in individuals with metabolic syndrome.

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.

Imaging systems for westerns: chemiluminescence vs. infrared detection.

Western blot detection methods have traditionally used X-ray films to capture chemiluminescence. The increasing costs for film, reagents, and maintenance have driven researchers away from darkrooms to more sensitive and technologically advanced digital imaging systems. Cooled charge coupled devices (CCD) cameras capture both chemiluminescence and fluorescence images, with limitations for each detection method. Chemiluminescence detection is highly sensitive and relies on an enzymatic reaction that produces light, which can be detected by a CCD camera that records photons and displays an image based on the amount of light generated. However, the enzymatic reaction is dynamic and changes over time making it necessary to optimize reaction times and imaging. Fluorescent detection with a CCD camera offers a solution to this problem since the signal generated by the proteins on the membrane is measured in a static state. Despite this advantage, many researchers continue to use chemiluminescent detection methods due to the generally poor performance of fluorophores in the visible spectrum. Infrared imaging systems offer a solution to the dynamic reactions of chemiluminescence and the poor performance of fluorophores detected in the visible spectrum by imaging fluorphores in the infrared spectrum. Infrared imaging is equally sensitive to chemiluminescence and more sensitive to visible fluorescence due in part to reduced autofluorescence in the longer infrared wavelength. Furthermore, infrared detection is static, which allows a wider linear detection range than chemiluminescence without a loss of signal. A distinct advantage of infrared imaging is the ability to simultaneously detect proteins on the same blot, which minimizes the need for stripping and reprobing leading to an increase in detection efficiency. Here, we describe the methodology for chemiluminescent (UVP BioChemi) and infrared (LI-COR Odyssey) imaging, and briefly discuss their advantages and disadvantages.

α-Glucosidase inhibitory effect of resveratrol and piceatannol.

Dietary polyphenols have been shown to inhibit α-glucosidase, an enzyme target of some antidiabetic drugs. Resveratrol, a polyphenol found in grapes and wine, has been reported to inhibit the activity of yeast α-glucosidase. This triggered our interest to synthesize analogs and determine their effect on mammalian α-glucosidase activity. Using either sucrose or maltose as substrate resveratrol, piceatannol and 3'-hydroxypterostilbene showed strong inhibition of mammalian α-glucosidase activity; pinostilbene, cis-desoxyrhapontigenin and trans-desoxyrhapontigenin had moderate inhibition. Compared to acarbose (IC50 3-13 µg/ml), piceatannol and resveratrol inhibited mammalian α-glucosidase to a lesser extent (IC50 14-84 and 111-120 µg/ml, respectively). 3'-Hydroxypterostilbene (IC50 105-302 µg/ml) was 23-35-fold less potent than acarbose. We investigated the effect of piceatannol and resveratrol on postprandial blood glucose response in high-fat-fed C57Bl/6 mice. Animals administered resveratrol (30 mg/kg body weight [BW]) or piceatannol (14 mg/kg BW) 60 min prior to sucrose or starch loading had a delayed absorption of carbohydrates, resulting in significant lowering of postprandial blood glucose concentrations, similar to the antidiabetic drug acarbose, while no significant effect was observed with the glucose-loaded animals. Our studies demonstrate that the dietary polyphenols resveratrol and piceatannol lower postprandial hyperglycemia and indicate that inhibition of intestinal α-glucosidase activity may be a potential mechanism contributing to their antidiabetic property.

Hypoglycemic agents and potential anti-inflammatory activity.

Current literature shows an association of diabetes and secondary complications with chronic inflammation. Evidence of these immunological changes include altered levels of cytokines and chemokines, changes in the numbers and activation states of various leukocyte populations, apoptosis, and fibrosis during diabetes. Therefore, treatment of diabetes and its complications may include pharmacological strategies to reduce inflammation. Apart from anti-inflammatory drugs, various hypoglycemic agents have also been found to reduce inflammation that could contribute to improved outcomes. Extensive studies have been carried out with thiazolidinediones (peroxisome proliferator-activated receptor-γ agonist), dipeptidyl peptidase-4 inhibitors, and metformin (AMP-activated protein kinase activator) with each of these classes of compounds showing moderate-to-strong anti-inflammatory action. Sulfonylureas and alpha glucosidase inhibitors appeared to exert modest effects, while the injectable agents, insulin and glucagon-like peptide-1 receptor agonists, may improve secondary complications due to their anti-inflammatory potential. Currently, there is a lack of clinical data on anti-inflammatory effects of sodium-glucose cotransporter type 2 inhibitors. Nevertheless, for all these glucose-lowering agents, it is essential to distinguish between anti-inflammatory effects resulting from better glucose control and effects related to intrinsic anti-inflammatory actions of the pharmacological class of compounds.

PPARG F388L, a transactivation-deficient mutant, in familial partial lipodystrophy.

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-gamma (PPAR-gamma), 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-gamma. 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.

Improved insulin sensitivity and resistance to weight gain in mice null for the Ahsg gene.

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-Ahsg(tm1Mbl)). 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.

Detection of Blotted Proteins: Not All Blockers Are Created Equal.

Western blotting is a standard analytical technique for detection of proteins. It is dependent on a number of components; from the specificity of the primary antibody to the reduction of competing biomolecules present in the assay. Blocking agents are a critical component for western blotting protocols as these diminish nonspecific binding by blocking off-target sites on the membrane. A variety of blocking agents are available and these are selected in an empirical manner, as no single blocker is compatible with every system. The best blocking agent and method for any particular assay will be an optimized but not absolute choice. Here, we describe characteristics of the most common blocking agents used in western blotting and discuss their advantages and disadvantages.

Comparison of Chemiluminescence vs. Infrared Techniques for Detection of Fetuin-A in Saliva.

The western blotting technique for transfer and detection of proteins, named following the discovery of southern and northern blotting for DNA- and RNA-blotting, respectively, has traditionally relied on the use of X-ray films to capture chemiluminescence. Recent advancements use super-cooled charge coupled devices (CCD) cameras to capture both chemiluminescence and fluorescence images, which exhibit a greater dynamic range compared to traditional X-ray film. Chemiluminescence detected by a CCD camera records photons and displays an image based on the amount of light generated as a result of a dynamic chemical reaction. Fluorescent detection with a CCD camera, on the other hand, is measured in a static state. Despite this advantage, researchers continue to widely use chemiluminescent detection methods due to the generally poor performance of fluorophores in the visible spectrum. Infrared imaging systems offer a solution to the dynamic reactions of chemiluminescence and the poor performance of fluorophores detected in the visible spectrum, by imaging fluorophores in the infrared spectrum. Infrared imaging is static, has a wide linear range, high sensitivity, and reduced autofluorescence and light scatter. A distinct advantage of infrared imaging is the ability to detect two target proteins simultaneously on the same blot which increases accuracy of quantification and comparison, while minimizing the need for stripping and reprobing. Here, we compare the methodology for chemiluminescent (UVP BioChemi) and infrared (UVP Odyssey) detection of salivary total and phosphorylated fetuin-A, a multifunctional protein associated with cardio-metabolic risk, and discuss the advantages and disadvantages of these methodologies.

Plasma alpha2-HS glycoprotein concentrations in patients with acute myocardial infarction quantified by a modified ELISA.

BACKGROUND: Human alpha2-HS glycoprotein (alpha2-HSG) is synthesized and secreted by the liver into circulation. Plasma concentrations of alpha2-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 alpha2-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 alpha2-HSG concentrations in plasma. RESULTS: In AMI patients, plasma alpha2-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 alpha2-HSG concentrations below 200 mg/l compared to none in the healthy control group. During the recovery period, alpha2-HSG concentrations begin to increase, with a mean+/-SEM of 290.1+/-22.1 mg/l. Regression analysis comparing plasma alpha2-HSG concentrations on admission to concentrations on discharge showed a significant positive correlation in matched-pair patient samples (P<0.01, r=0.45). CONCLUSIONS: We conclude that, in contrast to C-reactive protein, alpha2-HSG functions as a negative acute phase protein in AMI patients. Plasma alpha2-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).

Poly-L-histidine downregulates fibrinolysis.

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.