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.

Ultrasensitive protein detection and imaging: comparison of Lumitein™, ProteoSilver™, SYPRO(®) Ruby, and Coomassie (®) Brilliant Blue gel stains.

Following sodium dodecyl sulfate polyacrylamide gel electrophoresis, proteins can be visualized by various methods of detection and imaging. Traditional methods of protein gel detection and imaging have been improved and expanded through technological advancement. Today, the detection of proteins, resolved on gels, can be accomplished with a variety of stains with various sensitivities. Digital cameras used in the imaging of protein gels are not only more sensitive than their film precursors, but they can be used in combination with imaging software that offers a host of useful applications. Here we describe the UVP BioImaging System in combination with LabWorks Image and Acquisition software to provide a comparison of four different protein gel stains: Lumitein™, ProteoSilver™, SYPRO(®) Ruby, and Coomassie(®) Brilliant Blue. We demonstrate that the detection sensitivity limit appears to be between 100 and 500 ng/protein band of protein with Coomassie(®) Brilliant Blue, 10-50 ng/protein band with Lumitein™ and SYPRO(®) Ruby, and as little as 5 ng/protein band with the ProteoSilver™ stain.