Characterization of Signaling Pathways Associated with Pancreatic β-cell Adaptive Flexibility in Compensation of Obesity-linked Diabetes in db/db Mice

Abstract

The onset of obesity-linked type 2 diabetes (T2D) is marked by an eventual failure in pancreatic β-cell function and mass that is no longer able to compensate for the inherent insulin resistance and increased metabolic load intrinsic to obesity. However, in a commonly used model of T2D, the db/db mouse, β-cells have an inbuilt adaptive flexibility enabling them to effectively adjust insulin production rates relative to the metabolic demand. Pancreatic β-cells from these animals have markedly reduced intracellular insulin stores, yet high rates of (pro)insulin secretion, together with a substantial increase in proinsulin biosynthesis highlighted by expanded rough endoplasmic reticulum and Golgi apparatus. However, when the metabolic overload and/or hyperglycemia is normalized, β-cells from db/db mice quickly restore their insulin stores and normalize secretory function. This demonstrates the β-cell's adaptive flexibility and indicates that therapeutic approaches applied to encourage β-cell rest are capable of restoring endogenous β-cell function. However, mechanisms that regulate β-cell adaptive flexibility are essentially unknown. To gain deeper mechanistic insight into the molecular events underlying β-cell adaptive flexibility in db/db β-cells, we conducted a combined proteomic and post-translational modification specific proteomic (PTMomics) approach on islets from db/db mice and wild-type controls (WT) with or without prior exposure to normal glucose levels. We identified differential modifications of proteins involved in redox homeostasis, protein refolding, K48-linked deubiquitination, mRNA/protein export, focal adhesion, ERK1/2 signaling, and renin-angiotensin-aldosterone signaling, as well as sialyltransferase activity, associated with β-cell adaptive flexibility. These proteins are all related to proinsulin biosynthesis and processing, maturation of insulin secretory granules, and vesicular trafficking—core pathways involved in the adaptation of insulin production to meet metabolic demand. Collectively, this study outlines a novel and comprehensive global PTMome signaling map that highlights important molecular mechanisms related to the adaptive flexibility of β-cell function, providing improved insight into disease pathogenesis of T2D.

Divalent Metal Transporter 1 Knock-Down Modulates IL-1β Mediated Pancreatic Beta-Cell Pro-Apoptotic Signaling Pathways through the Autophagic Machinery

“…To characterize the signaling mechanism of the protective effects of DMT1 silencing upon β-cell cytokine exposure, we applied a high-throughput quantification approach by nLC-MS based proteomics and phosphoproteomics in conjunction with TMT labeling, as well as bioinformatics and validation using PRM ( Figure 1 A) [ 16 , 17 , 18 , 19 ]. For consistent results at protein and mRNA levels, we modified our TiSH protocol [ 16 , 18 ] resulting in a sequential mRNA, protein, and phosphopeptide enrichment strategy, which enabled us to simultaneously investigate molecular events derived from the same sample. As expected, our method led to high correlation of DMT1 levels quantified from qPCR and proteome datasets.…”

“…Then we applied a sequential sample preparation and enrichment strategy of mRNA, protein, and phosphopeptides to characterize the DTM1 knock-down, the proteome and the phosphoproteome from the same set of samples. For the quantitative proteomics and phosphoproteomics analysis, we used a combination of our TiO 2 -SIMAC method (enrichment of mono- and multi-phosphorylated peptides), tandem mass tags (TMT) labeling, and a nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS) approach [ 16 , 17 , 18 , 19 ]. Overall, in addition to identifying canonical pathways for IL-1 receptor signaling, NF-κB activation pathway, and inflammatory response, we identified two novel pathways containing apoptotic and anti-apoptotic proteins that are involved in cell survival.…”

Genome-Wide Analysis Reveals Changes in Long Noncoding RNAs in the Differentiation of Canine BMSCs into Insulin-Producing Cells

“…during induction binding, enzyme regulatory activity and signal transduction were the main activities. Because islets are constructed by endocrine cells, the Golgi complex remains highly dynamic and the enzymes involved vary [31,32]. KEGG analysis identified the MAPK signaling pathway, PI3K-Akt signaling pathway, insulin signaling pathway, cell cycle, and Rap1 signaling pathway, all of which are associated with pancreas development [33][34][35].…”

Recent Advances in Mass Spectrometry-Based Glycomic and Glycoproteomic Studies of Pancreatic Diseases

“…Another strategy used more recently relies on deglycosylation of enriched glycopeptides prior to MS analysis (Krishnan et al, 2017;Nigjeh et al, 2017;Kang et al, 2020). This is helpful by decreasing overall analyte complexity and by improving the ionization efficiency of peptides, which are blunted by the increased hydrophilicity contributed by glycan modifications.…”

“…The resulting mass spectra can be searched against a database to identify peptide sequences and glycan moieties. Quantification can also be done using label-free approaches, such as using areaunder-the-curve, or with reporter ion intensities after isobaric tagging (Shih et al, 2019;Kang et al, 2020). A more detailed discussion of glycan and glycopeptide quantification can be found in Delafield and Li (2021).…”