Regulation of glucose metabolism by posttranslational modifications in Type 1 Diabetes

Mark Mamula (Yale University)

Mei-Ling Yang, Cate Speake, Carmella Evans-Molina, Lut Overbergh, Eddi James, Li Wen, Kevan Herold, Mark Mamula

Emerging evidence indicates that autoimmunity often arises to post-translational protein modifications (PTMs), leading to chronic inflammation by infiltrating lymphocytes of target organs such as the pancreas in type 1 diabetes (T1D). While the identification and assessment of autoimmunity to novel PTM biomarkers is important to understanding pathogenic processes in T1D, it is similarly critical to determine if PTM proteins alter key processes in beta-cell biology, namely glucose sensing and insulin production, folding and release. This study specifically addresses these biologic functions of PTM proteins associated to T1D.

Carbonylation is the major protein modification in response to oxidative stress. Citrullination is mediated by calcium-dependent peptidylarginine deiminase (PAD) enzymes, which catalyze deimination, the conversion of arginine into the non-classical amino acid citrulline. Various proteomic analyses, including mass spectrometry, were performed in both murine and human pancreatic islets to identify carbonylated- and citrullinated-islet proteins. Glucose-stimulated proinsulin/insulin secretion was examined in human islets stressed with inflammatory cytokines and physiologic oxidative conditions in the presence or absence of PAD inhibitors. In addition, the effect of citrullination on the enzyme kinetics of glucokinase (Vmax and Km), the first rate-limiting step of glycolysis in the liver and pancreas, was also examined.

Summary of Results
We identify autoantibodies against to beta subunit of prolyl-4-hydroxylase (P4Hb; native and carbonylated form) and glucokinase (GK; native and citrullinated form) in both human T1D and murine models. By mass spectrometry, six carbonyl residues and sixteen citrulline residues were mapped in oxidative P4Hb and PAD-treated GK, respectively. In regards to glucose metabolism, the carbonylated-P4Hb is amplified in stressed human islets coincident with decreased glucose-stimulated insulin secretion and increased proinsulin to insulin ratios. Citrullination alters GK biologic activity (Km) and suppresses glucose-stimulated insulin secretion. Moreover, PAD2/4 inhibitor can partially correct IFNgamma +IL-1beta suppressed glucose stimulated insulin secretion in INS-1E beta cells.

The major function of pancreatic beta cells is to secret insulin in response to glucose uptake in order to maintain the blood glucose level. Glucokinase is expressed in hepatocytes to regulate glycogen synthesis, and in pancreatic beta cells as a glucose sensor to initiate glycolysis and insulin signaling. P4Hb is a critical macromolecule for the accurate folding of insulin. Our studies implicate the crucial enzymes, glucokinase and P4Hb, as the biomarkers, providing new insights into creating autoantigens and define the impact of PTMs on the aberrant beta cell functions of T1D. Targeting glucose metabolism by PTMs, such as PAD inhibitors, may lead to preventing diabetes autoimmunity and restoring beta cell function.