Succination of Thiol Groups in Adipose Tissue Proteins in Diabetes: SUCCINATION INHIBITS POLYMERIZATION AND SECRETION OF ADIPONECTIN*

S-(2-Succinyl)cysteine (2SC) is formed by reaction of the Krebs cycle intermediate fumarate with cysteine residues in protein, a process termed succination of protein. Both fumarate and succination of proteins are increased in adipocytes cultured in high glucose medium (Nagai, R., Brock, J. W., Blatnik, M., Baatz, J. E., Bethard, J., Walla, M. D., Thorpe, S. R., Baynes, J. W., and Frizzell, N. (2007) J. Biol. Chem. 282, 34219–34228). We 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 ～7 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 dehydrogenase in muscle of diabetic rats (7). We have proposed that 2SC may accumulate as a result of mitochondrial nutrient “flooding” because of an excess of carbohydrate and lipid fuels in diabetes and may be a biomarker of mitochondrial stress in disease.To gain further insight into the role of succination in the regulation of metabolism, we studied the maturation of 3T3-L1 fibroblasts to adipocytes, an in vitro system in which fumarate and other Krebs cycle intermediates increase severalfold during adipogenesis in high (30 mm) glucose) medium (8, 9). Adipogenesis under these conditions is associated with a substantial increase in oxidative stress as a result of mitochondrial superoxide production (10). We also observed a ≥5-fold increase in fumarate and a ≥10-fold increase in intracellular 2SC accumulation during adipogenesis and identified several of the major proteins modified by 2SC (9). This set of proteins included cytoskeletal proteins, enzymes, heat shock and chaperone proteins, regulatory proteins, and a fatty acid-binding protein, suggesting that succination may have wide ranging effects on the structure of the cytoskeleton and the regulation of metabolism.The adipocyte is increasingly recognized not only for its role in triglyceride storage but also as an active endocrine organ, secreting hormones and cytokines that orchestrate key metabolic processes in tissues, such as heart, liver, and muscle. All of the adipokines work as part of a greater metabolic regulatory network. Adiponectin and leptin are considered positive regulators of energy intake and expenditure, whereas resistin, interleukin-6, tumor necrosis factor-α, and PAI-1 are implicated in the development of inflammation and insulin resistance. Imbalances in adipokine metabolism are central to adipocyte dysfunction and the ensuing events leading to insulin resistance and diabetes (11, 12).Adiponectin has received particular attention as the most abundant adipokine, circulating at high levels in human blood. It is an ～30-kDa glycoprotein that associates intracellularly into trimeric, hexameric (also known as low molecular weight (LMW)), and other high molecular weight (HMW) complexes consisting of 18–36 monomers (13, 14). The various molecular weight species differentially stimulate their target tissues; trimeric adiponectin stimulates muscle fatty acid oxidation through activation of AMP-activated protein kinase, whereas HMW forms act to enhance insulin-mediated inhibition of gluconeogenesis in the liver (15, 16). Plasma adiponectin concentration is reduced in diabetes, in general, as is the ratio of HMW forms to total adiponectin (16).The N-terminal hypervariable domain of adiponectin contains a single cysteine residue followed by a collagenous region containing several conserved lysine and proline residues. Several of these lysines and prolines are subject to modification by hydroxylation and/or glycosylation (17, 18). The cysteine at position 39 in mouse adiponectin is involved in the formation of the oligomeric species of adiponectin through disulfide bonding of monomers and trimers (Fig. 1). Cys-39 is critical for the generation of all higher order complexes, since its mutation to serine inhibits the formation of both trimer and larger species. The only other cysteine present in adiponectin is located in the C-terminal globular domain, and crystallographic studies indicate that it is unlikely to be involved in disulfide bonding of oligomers (14). In this study, we show that adiponectin is a major target of succination in both 3T3-L1 adipocytes and adipose tissue of diabetic (db/db) mice, that Cys-39 is a major site of cysteine succination, and that succinated adiponectin is neither incorporated into polymeric forms in the cell nor secreted from the cell. We propose that succination of adiponectin may contribute to the decrease in plasma adiponectin in diabetes.Open in a separate windowFIGURE 1.Structure of adiponectin. Two cysteines are highly conserved in adiponectin monomer: one in the hypervariable region adjacent to the N terminus (Cys-39) and the other in the C-terminal globular head domain (Cys-155) (A). Adiponectin monomers associate into trimers through disulfide bonding, and trimers associate through disulfide bonds to form LMW and HMW multimers, which are secreted from the adipocyte. Succination of Cys-39 blocks incorporation of adiponectin monomer into trimer and higher molecular weight secreted forms of the protein (B).