Simultaneous determination and validated quantification of human insulin and its synthetic analogues in human blood serum by immunoaffinity purification and liquid chromatography-mass spectrometry

Possible fatal complications of human insulin and its synthetic analogues like hypoglycemia require precise classification and quantitative determination of these drugs both for clinical purposes as well as for forensic toxicologists. A procedure was developed for the identification and quantification of human insulin and different long-acting as well as short-acting synthetic insulins in human blood serum specimens. After an immunoaffinity purification step and separation by liquid chromatography, the insulins were characterized by their five- or sixfold protonated molecule ions and diagnostic product ions. Clinical samples of 207 diabetic and 50 non-diabetic patients after the administration of human insulin or oral antidiabetics and forensic samples were analyzed for human/synthetic insulin concentrations. The method was validated according to international guidelines. Limits of detection of the insulins ranged between 1.3 and 2.8 mu U/ml. Recoveries ranged between 33.2 % and 51.7 %. Precision data was in accordance with international guidelines. Clinical samples showed concentrations of human insulin lower than 301 mu U/ml. Our liquid chromatography tandem mass spectrometry procedure allows unambiguous identification and quantification of the intact human insulin and its intact synthetic analogues HumalogA (R), NovologA (R), ApidraA (R), LantusA (R), and LevemirA (R) in human blood serum in clinical and overdose cases. The assay could be successfully tested in patients with diabetes mellitus on therapy with insulins or oral antidiabetics

USP15 stabilizes MDM2 to mediate cancer-cell survival and inhibit antitumor T cell responses

Deubiquitinases (DUBs) are a new class of drug targets, although the physiological function of only few DUBs has been characterized. Here we identified the DUB USP15 as a crucial negative regulator of T cell activation. USP15 stabilized the E3 ubiquitin ligase MDM2, which in turn negatively regulated T cell activation by targeting the degradation of the transcription factor NFATc2. USP15 deficiency promoted T cell activation in vitro and enhanced T cell responses to bacterial infection and tumor challenge in vivo. USP15 also stabilized MDM2 in cancer cells and regulated p53 function and cancer-cell survival. Our results suggest that inhibition of USP15 may both induce tumor cell apoptosis and boost antitumor T cell responses

Biogenesis of the insulin secretory granule in health and disease

The secretory granules of pancreatic beta cells are specialized organelles responsible for the packaging, storage and secretion of the vital hormone insulin. The insulin secretory granules also contain more than 100 other proteins including the proteases involved in proinsulin-to insulin conversion, other precursor proteins, minor co-secreted peptides, membrane proteins involved in cell trafficking and ion translocation proteins essential for regulation of the intragranular environment. The synthesis, transport and packaging of these proteins into nascent granules must be carried out in a co-ordinated manner to ensure correct functioning of the granule. The process is regulated by many circulating nutrients such as glucose and can change under different physiological states. This chapter discusses the various processes involved in insulin granule biogenesis with a focus on the granule composition in health and disease1134173