Summary Although compensatory islet hyperplasia in response to insulin resistance is a recognized feature in diabetes, the factor(s) that promote β-cell proliferation have been elusive. We previously reported that the liver is a source for such factors in the liver insulin receptor knockout (LIRKO) mouse, an insulin resistance model which manifests islet hyperplasia. Using proteomics we show that serpinB1, a protease inhibitor, which is abundant in the hepatocyte secretome and sera derived from LIRKO mice, is the liver-derived secretory protein that regulates β-cell proliferation in humans, mice and zebrafish. Small molecule compounds, that partially mimic serpinB1 effects of inhibiting elastase activity, enhanced proliferation of β-cells, and mice lacking serpinB1 exhibit attenuated β-cell compensation in response to insulin resistance. Finally, SerpinB1-treatment of islets modulated proteins in growth/survival pathways. Together, these data implicate serpinB1 as an endogenous protein that can potentially be harnessed to enhance functional β-cell mass in patients with diabetes.

Summary Circulating branched-chain amino acid (BCAA) levels are elevated in obesity/diabetes and are a sensitive predictor for type 2 diabetes. Here we show in rats that insulin dose-dependently lowers plasma BCAA levels through induction of hepatic protein expression and activity of branched-chain α keto-acid dehydrogenase (BCKDH), the rate-limiting enzyme in the BCAA degradation pathway. Selective induction of hypothalamic insulin signaling in rats and genetic modulation of brain insulin receptors in mice demonstrate that brain insulin signaling is a major regulator of BCAA metabolism by inducing hepatic BCKDH. Short-term overfeeding impairs the ability of brain insulin to lower BCAAs in rats. High-fat feeding in non-human primates and obesity and/or diabetes in humans is associated with reduced BCKDH protein in liver. These findings support the concept that decreased hepatic BCKDH is a major cause of increased plasma BCAAs, and that hypothalamic insulin resistance may account for impaired BCAA metabolism in obesity and diabetes.

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Detergents have been widely used for the solubilization of membrane proteins and the improvement of their digestion. In this paper, we have evaluated the application of sodium deoxycholate (SDC) to the solubilization and digestion of rat hippocampal plasma membrane (PM) proteins. For in-solution digestion, rat hippocampal PM fraction from sucrose-density gradient centrifugation was solubilized by boiling in 1.0% SDC, and directly digested without dilution. During the in-gel digestion of the hippocampal PM proteins separated by SDS-PAGE, 0.1% SDC was added. Before analysis of peptide mixture by liquid chromatography and electrospray mass spectrometry, SDC in the tryptic digests was removed by centrifugation following acidification. Use of 1.0% SDC in solubilization and in-solution digestion of rat PM proteins had led to 77 PM or membrane-associated proteins identified, a more than 2-fold increase over that by use of SDS. The addition of 0.1% SDC to the in-gel digestion of SDS-PAGE-resolved membrane proteins remarkably enhanced the coverage of tryptic peptides and the number of hydrophobic membrane proteins identified. Being a cheaper and more tractable acid-insoluble detergent, SDC could be used at higher concentration in the solubilization and tryptic digestion of proteins including PM proteins with the purpose of enhancing the protein solubility and at the same time making no interference with trypsin activity and subsequent analyses.

Chronic stressful occurrences are documented as a vital cause of both depression and anxiety disorders. However, the stress-induced molecular mechanisms underlying the common and distinct pathophysiology of these disorders remains largely unclear. We utilized a chronic mild stress (CMS) rat model to differentiate and subgroup depression-susceptible, anxiety-susceptible, and insusceptible rats. The hippocampus was analyzed for differential proteomes by combining mass spectrometry and the isobaric tags for relative and absolute quantitation (iTRAQ) labeling technique. Out of 2593 quantified proteins, 367 were aberrantly expressed. These hippocampal protein candidates might be associated with susceptibility to stress-induced depression or anxiety and stress resilience. They provide the potential protein systems involved in various metabolic pathways as novel investigative protein targets. Further, independent immunoblot analysis identified changes in Por, Idh2 and Esd; Glo1, G6pdx, Aldh2, and Dld; Dlat, Ogdhl, Anxal, Tpp2, and Sdha that were specifically associated to depression-susceptible, anxiety-susceptible, or insusceptible groups respectively, suggesting that identical CMS differently impacted the mitochondrial and metabolic processes in the hippocampus. Collectively, the observed alterations to protein abundance profiles of the hippocampus provided significant and novel insights into the stress regulation mechanism in a CMS rat model. This might serve as the molecular basis for further studies that would contributed to a better understanding of the similarities and differences in pathophysiologic mechanisms underlying stress-induced depression or anxiety, and stress resiliency.

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Urea solution is one of the most commonly employed protein denaturants for protease digestion in proteomic studies. However, it has long been recognized that urea solution can cause carbamylation at the N-termini of proteins/peptides and at the side chain amino groups of lysine and arginine residues. Protein/peptide carbamylation blocks protease digestion and affects protein identification and quantification in mass spectrometry analysis by blocking peptide amino groups from isotopic/isobaric labeling and changing peptide charge states, retention times and masses. In addition, protein carbamylation during sample preparation makes it difficult to study in vivo protein carbamylation. In this study, we compared the peptide carbamylation in urea solutions of different buffers and found that ammonium containing buffers were the most effective buffers to inhibit protein carbamylation in urea solution. The possible mechanism of carbamylation inhibition by ammonium containing buffers is discussed, and a revised procedure for the protease digestion of proteins in urea and ammonium containing buffers was developed to facilitate its application in proteomic research.