Proteomic Analysis of GLUT4 Storage Vesicles Reveals Tumor Suppressor Candidate 5 (TUSC5) as a Novel Regulator of Insulin Action in Adipocytes.

Insulin signaling augments glucose transport by regulating glucose transporter 4 (GLUT4) trafficking from specialized intracellular compartments, termed GLUT4 storage vesicles (GSVs), to the plasma membrane. Proteomic analysis of GSVs by mass spectrometry revealed enrichment of 59 proteins in these vesicles. We measured reduced abundance of 23 of these proteins following insulin stimulation and assigned these as high confidence GSV proteins. These included established GSV proteins such as GLUT4 and insulin-responsive aminopeptidase, as well as six proteins not previously reported to be localized to GSVs. Tumor suppressor candidate 5 (TUSC5) was shown to be a novel GSV protein that underwent a 3.7-fold increase in abundance at the plasma membrane in response to insulin. siRNA-mediated knockdown of TUSC5 decreased insulin-stimulated glucose uptake, although overexpression of TUSC5 had the opposite effect, implicating TUSC5 as a positive regulator of insulin-stimulated glucose transport in adipocytes. Incubation of adipocytes with TNFα caused insulin resistance and a concomitant reduction in TUSC5. Consistent with previous studies, peroxisome proliferator-activated receptor (PPAR) γ agonism reversed TNFα-induced insulin resistance. TUSC5 expression was necessary but insufficient for PPARγ-mediated reversal of insulin resistance. These findings functionally link TUSC5 to GLUT4 trafficking, insulin action, insulin resistance, and PPARγ action in the adipocyte. Further studies are required to establish the exact role of TUSC5 in adipocytes

Proteomic Pathways to Type 2 Diabetes in the Pancreatic Islet

Unveiling proteomic changes that occur through stages of pathogenesis can provide unparalleled insights into the mechanisms underpinning disease. Using fluorescence-activated cell sorting (FACS) and liquid chromatography mass spectrometry/mass spectrometry (LC-MS/MS), I aimed to characterise the deep proteome of both whole islets and individual islet cell types (alpha, beta, gamma, delta and epsilon) from mice and humans in variable states of health and disease. Acquisition of individual islet cell type proteomes will serve as a window of enquiry into islet cell regulation and intra-cellular communication providing insight into how these factors affect systemic glucose regulation. A panel of diverse mice were phenotypically characterised to observe their responses to diet or genetically induced metabolic stress. Characterisation of the islet proteomes of these mice revealed background strain as the major determinant of islet capacity to maintain function under increased metabolic stress. Isolation of the individual islet cell types using FACS requires the targeting of specific markers for each cell type. Currently, specific surface markers for several islet cell types are not known and/or antibodies are not available, and the alternative of using internal markers requires fixation. Standardised methods of protein fixation have a variable extent of modifications, for example paraformaldehyde, and hence are incompatible with peptide identification after MS analysis. I have overcome these obstacles by using a reversible crosslinker, dithiobis(succinimidyl propionate), which specifically targets primary amines in proteins. Upon reversal of the crosslinker, it leaves modifications of a standard size, which can be incorporated into the peptide identification analysis as a protein modification. In doing so, internal markers specific to each islet cell type can be utilized for isolation and subsequent proteomic analysis, whilst retaining greater than 96% protein identification compared to non-fixed cells. Preliminary MS analysis of isolated human β-cells revealed 4,561 proteins representing the largest human β-cell proteome to date