Succination of Thiol Groups in Adipose Tissue Proteins in Diabetes

Abstract

S-(2-SuccinylWe show here that succination of protein is also increased in epididymal, mesenteric, and subcutaneous adipose tissue of diabetic (db/db) mice and that adiponectin is a major target for succination in both adipocytes and adipose tissue. Cys-39, which is involved in cross-linking of adiponectin monomers to form trimers, was identified as a key site of succination of adiponectin in adipocytes. 2SC was detected on two of seven monomeric forms of adiponectin immunoprecipitated from adipocytes and epididymal adipose tissue. Based on densitometry, 2SC-adiponectin accounted for ϳ7 and 8% of total intracellular adiponectin in cells and tissue, respectively. 2SC was found only in the intracellular, monomeric forms of adiponectin and was not detectable in polymeric forms of adiponectin in cell culture medium or plasma. We conclude that succination of adiponectin blocks its incorporation into trimeric and higher molecular weight, secreted forms of adiponectin. We propose that succination of proteins is a biomarker of mitochondrial stress and accumulation of Krebs cycle intermediates in adipose tissue in diabetes and that succination of adiponectin may contribute to the decrease in plasma adiponectin in diabetes.The accumulation of sugar and lipid-derived chemical modifications on proteins is associated with the etiology of several age-related diseases, including diabetes and its complications (1, 2). The irreversible adducts formed, termed advanced glycation/lipoxidation end products (AGE/ALEs), 2 accumulate over time on long lived proteins, such as collagens, affecting the solubility, elasticity, and proteolytic digestibility of the protein (3). AGE/ALEs are considered important mediators of the pathogenesis of diabetic complications through engagement of scavenger receptors, such as RAGE (receptor for AGE) and activation of proinflammatory signaling pathways (4). To date, the study of AGE/ALEs has focused mainly on modification of lysine and arginine residues in proteins by reactive carbonyl intermediates formed during metabolism or autoxidation of carbohydrates and lipids (2, 5). However, free cysteine is more abundant on intracellular proteins and, because of its greater nucleophilicity, is a more likely target for chemical modification by intracellular electrophiles.We recently identified S-(2-succinyl)-cysteine (2SC), a cysteine modification formed by a Michael addition reaction between the Krebs cycle intermediate fumarate and free sulfhydryl groups on proteins (6). This reaction, in which a thioether bond is formed, is described as succination of protein in order to distinguish it from succinylation, which leads to formation of amide, ester, or thioester bonds. 2SC was detected in human serum albumin and skin collagen and was increased in skeletal muscle protein and urine of diabetic rats. We also identified glyceraldehyde-3-phosphate dehydrogenase as one protein that is significantly modified by 2SC in skeletal muscle, resulting in the decrease in specific activity of glyceraldehyde-3-phosphate dehydr...

The effect of glucose concentration and sodium phenylbutyrate treatment on mitochondrial bioenergetics and ER stress in 3T3-L1 adipocytes

“…Concomitant with a decrease in p-eIF2α levels, there was a pronounced increase in CHOP, a terminal marker of ER stress, but only in adipocytes matured in 30 mM glucose (Figure 2A&E). Consistent with previous results (Nagai 2007, Frizzell 2009), protein succination increases in adipocytes matured in 30 mM glucose for up to 8 days (M2-M8, lanes 8-23) and interestingly appears to increase in parallel with CHOP levels (Figure 2A). In addition to the observed decrease in intracellular adiponectin, the adipocytes matured in 30 mM glucose secreted increased levels of pro-inflammatory cytokines including TNFα, VEGF and MCP-1 (Figure 2F-G).…”

“…We have identified ∼40 succinated proteins in adipocytes (Merkley 2013, Nagai 2007, Piroli 2014,) and we have demonstrated that succination appears to impair protein function or regulation, e.g. increased succination of adiponectin in the presence of high glucose can reduce the polymerization and secretion of this adipokine (Frizzell 2009). Interestingly we have observed that several endoplasmic reticulum (ER) proteins, including the oxidoreductase protein disulfide isomerase (PDI), are succinated in the adipocyte during diabetes (Nagai 2007).…”

Identification of protein succination as a novel modification of tubulin

“…Isoelectric focusing on pI 4-7 11 cm and 17 cm strips or narrow range 4.7-5.9 11 cm strips and two-dimensional (2D) gel electrophoresis was performed as described previously [3]. The 2D gels were stained for protein using Sypro Ruby total protein stain (Invitrogen, Grand Island, NY) [3] or transferred onto PVDF membrane to detect protein succination by western blotting.…”

“…The 2D gels were stained for protein using Sypro Ruby total protein stain (Invitrogen, Grand Island, NY) [3] or transferred onto PVDF membrane to detect protein succination by western blotting.…”

“…Several of the succinated proteins in adipocytes have been identified including cytoskeletal proteins, endoplasmic reticulum chaperones and hormones [2]. The hormone adiponectin has been shown to be succinated both in vitro and in vivo , preventing its incorporation into high molecular weight oligomers and secretion from the adipocyte [3] and demonstrating the functional impact of succination on a target protein. Protein succination is also increased in the gastrocnemius muscle of 6 month old type 1 diabetic rats and results in a reduction in the activity of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [1, 6].…”