Mitochondrial stress causes increased succination of proteins in adipocytes in response to glucotoxicity

2SC [S-(2-succino)-cysteine] is a chemical modification formed by a Michael addition reaction of fumarate with cysteine residues in proteins. Formation of 2SC, termed 'succination' of proteins, increases in adipocytes grown in high-glucose medium and in adipose tissues of Type?2 diabetic mice. However, the metabolic mechanisms leading to increased fumarate and succination of protein in the adipocyte are unknown. Treatment of 3T3 cells with high glucose (30?mM compared with 5?mM) caused a significant increase in cellular ATP/ADP, NADH/NAD+ and Δψm (mitochondrial membrane potential). There was also a significant increase in the cellular fumarate concentration and succination of proteins, which may be attributed to the increase in NADH/NAD+ and subsequent inhibition of tricarboxylic acid cycle NAD+-dependent dehydrogenases. Chemical uncouplers, which dissipated Δψm and reduced the NADH/NAD+ ratio, also decreased the fumarate concentration and protein succination. High glucose plus metformin, an inhibitor of complex I in the electron transport chain, caused an increase in fumarate and succination of protein. Thus excess fuel supply (glucotoxicity) appears to create a pseudohypoxic environment (high NADH/NAD+ without hypoxia), which drives the increase in succination of protein. We propose that increased succination of proteins is an early marker of glucotoxicity and mitochondrial stress in adipose tissue in diabetes.     

Succination of proteins in diabetes

Cysteine is arguably the most reactive amino acid in protein. A wide range of cysteine derivatives is formed in vivo, resulting from oxidation, nitrosation, alkylation and acylation reactions. This review describes succination of proteins, an irreversible chemical modification of cysteine by the Krebs cycle intermediate, fumarate, yielding S-(2-succinyl)cysteine (2SC). Intracellular fumarate concentration and succination of proteins are increased by hyperpolarization of the inner mitochondrial membrane and develop in concert with mitochondrial and oxidative stress in diabetes. Increased succination of glyceraldehyde-3-phosphate dehydrogenase explains the loss in specific activity of this enzyme in muscle of streptozotocin-diabetic rats and increased succination of adiponectin may explain the decreased secretion of adiponectin from adipose tissue in type 2 diabetes. In addition to GAPDH and adiponectin, other succinated proteins identified in adipocytes include cytoskeletal proteins (tubulin, actin) and chaperone proteins in the endoplasmic reticulum. Succination of adipocyte protein in vitro is inhibited by uncouplers of oxidative phosphorylation and by inhibitors of ER stress. 2SC serves as a biomarker of mitochondrial stress and recent studies suggest that succination is the mechanistic link between mitochondrial and ER stress in diabetes.     

Succination of protein thiols during adipocyte maturation: a biomarker of mitochondrial stress

Although obesity is a risk factor for development of type 2 diabetes and chemical modification of proteins by advanced glycoxidation and lipoxidation end products is implicated in the development of diabetic complications, little is known about the chemical modification of proteins in adipocytes or adipose tissue. In this study we show that S-(2-succinyl)cysteine (2SC), the product of chemical modification of proteins by the Krebs cycle intermediate, fumarate, is significantly increased during maturation of 3T3-L1 fibroblasts to adipocytes. Fumarate concentration increased > or =5-fold during adipogenesis in medium containing 30 mm glucose, producing a > or =10-fold increase in 2SC-proteins in adipocytes compared with undifferentiated fibroblasts grown in the same high glucose medium. The elevated glucose concentration in the medium during adipocyte maturation correlated with the increase in 2SC, whereas the concentration of the advanced glycoxidation and lipoxidation end products, N(epsilon)-(carboxymethyl)lysine and N(epsilon)-(carboxyethyl)lysine, was unchanged under these conditions. Adipocyte proteins were separated by one- and two-dimensional electrophoresis and approximately 60 2SC-proteins were detected using an anti-2SC polyclonal antibody. Several of the prominent and well resolved proteins were identified by matrix-assisted laser desorption ionization time-of-flight/time-of-flight mass spectrometry. These include cytoskeletal proteins, enzymes, heat shock and chaperone proteins, regulatory proteins, and a fatty acid-binding protein. We propose that the increase in fumarate and 2SC is the result of mitochondrial stress in the adipocyte during adipogenesis and that 2SC may be a useful biomarker of mitochondrial stress in obesity, insulin resistance, and diabetes.     

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).     

Impaired response of mature adipocytes of diabetic mice to hypoxia

Adipose tissue contains various cells such as infiltrated monocytes/macrophages, endothelial cells, preadipocytes, and adipocytes. Adipocytes have an endocrine function by secreting adipokines such as interleukin (IL)-6, tumor necrosis factor (TNF)-α, leptin, and adiponectin. Dysregulation of adipokines in adipose tissues leads to a chronic low-grade inflammation which could result in atherosclerosis, hypertension, and type 2 diabetes. A sustained inflammatory state, which is characterized by prolonged persistence of macrophages and neutrophils, is found in diabetic wounds. In addition, subcutaneous adipocytes are enormously increased in amount clinically in type 2 diabetes. However, the function of subcutaneous adipocytes, which play an important role in injured tissue subjected to hypoxia, has not been well characterized in vitro due to the difficulty of maintaining mature adipocytes in culture using conventional methods because of their buoyancy. In this study, we established a novel in vitro culture method of mature adipocytes by enclosing them in a hyaluronan (HA) based hydrogel to study their role in response to stress such as hypoxia. BrdU labeling and Ki67 immunostaining experiments showed that hydrogel enclosed mature adipocytes proliferate in vitro. Both mRNA and protein expression analyses for hypoxia regulated genes, such as vascular endothelial growth factor (VEGF) and heme oxygenase 1 (HO1), showed that mature adipocytes of wild type mice respond to hypoxia. In contrast, mature adipocytes of diabetic db/db and TallyHo mice did not efficiently respond to hypoxia. Our studies suggest that mature adipocytes are functionally active cells, and their abnormal function to hypoxia can be one of underlining mechanisms in type 2 diabetes.     

Increased lipids in non-lipogenic tissues are indicators of the severity of type 2 diabetes in mice

We hypothesised that the molecular changes triggered in type 2 diabetes might cause phenotypic changes in the lipid fraction of tissues. We compared tissue lipid profiles of inbred lean B6-Bom with those of the obese B6-ob/ob and diabetic BKS-db/db mice and found that genetically diabetic mice significantly accumulate fat (especially monounsaturated fatty acids, MUFA) in non-lipogenic tissues such as the eye (MUFA, 2-fold), skeletal muscle (MUFA, 13-fold) and pancreas (MUFA, 16-fold). In contrast, the B6-ob/ob mice which manifest a milder form of type 2 diabetes use the liver as their predominant lipid depot (MUFA 91-fold increase, as compared to lean mice values). The lipids in the BKS-db/db skeletal muscle and pancreas were also significantly enriched with linoleic acid (LA, (9-fold and 6-fold, respectively); and alpha-linolenic acid (ALA, 8.5-fold and 8-fold, respectively). MUFA, LA and ALA accumulation in the non-lipogenic tissues of BKS-db/db mice was associated with reduced liver stearoyl-CoA desaturase-1 expression.     

Succination is Increased on Select Proteins in the Brainstem of the NADH dehydrogenase (ubiquinone) Fe-S protein 4 (Ndufs4) Knockout Mouse,a Model of Leigh Syndrome

Elevated fumarate concentrations as a result of Krebs cycle inhibition lead to increases in protein succination, an irreversible post-translational modification that occurs when fumarate reacts with cysteine residues to generate S-(2-succino)cysteine (2SC). Metabolic events that reduce NADH re-oxidation can block Krebs cycle activity; therefore we hypothesized that oxidative phosphorylation deficiencies, such as those observed in some mitochondrial diseases, would also lead to increased protein succination. Using the Ndufs4 knockout (Ndufs4 KO) mouse, a model of Leigh syndrome, we demonstrate for the first time that protein succination is increased in the brainstem (BS), particularly in the vestibular nucleus. Importantly, the brainstem is the most affected region exhibiting neurodegeneration and astrocyte and microglial proliferation, and these mice typically die of respiratory failure attributed to vestibular nucleus pathology. In contrast, no increases in protein succination were observed in the skeletal muscle, corresponding with the lack of muscle pathology observed in this model. 2D SDS-PAGE followed by immunoblotting for succinated proteins and MS/MS analysis of BS proteins allowed us to identify the voltage-dependent anion channels 1 and 2 as specific targets of succination in the Ndufs4 knockout. Using targeted mass spectrometry, Cys⁷⁷ and Cys⁴⁸ were identified as endogenous sites of succination in voltage-dependent anion channels 2. Given the important role of voltage-dependent anion channels isoforms in the exchange of ADP/ATP between the cytosol and the mitochondria, and the already decreased capacity for ATP synthesis in the Ndufs4 KO mice, we propose that the increased protein succination observed in the BS of these animals would further decrease the already compromised mitochondrial function. These data suggest that fumarate is a novel biochemical link that may contribute to the progression of the neuropathology in this mitochondrial disease model.We previously identified the formation of S-(2-succino)cysteine (2SC)¹ (protein succination) as a result of the irreversible reaction of fumarate with reactive cysteine thiols (1, 2). Fumarate concentrations are increased during adipogenesis and adipocyte maturation (2, 3), and the excess of glucose and insulin leads to augmented protein succination in the adipose tissue of type 2 diabetic mice (4, 5). Protein succination is also specifically increased in fumarate hydratase deficient hereditary leiomyomatosis and renal cell carcinoma (HLRCC), because of the decreased conversion of fumarate to malate (6, 7). In both cases, intracellular fumarate concentrations are elevated; in fumarate hydratase deficient cells, the fumarate concentration is about 5 mm (8), whereas fumarate levels increase up to fivefold in adipocytes grown in the presence of high (30 mm) versus normal (5 mm) glucose concentrations (2). In the adipocyte the increase in fumarate and succinated proteins develops as a direct result of mitochondrial stress induced by nutrient excess. Mechanistically, excess glucose without increased ATP demand inhibits the electron transport chain resulting in an elevated NADH/NAD⁺ ratio. This inhibits NAD⁺-dependent Krebs cycle enzymes and leads to an increase in fumarate and protein succination (9). In support of this we have also shown that low concentrations of chemical uncouplers of oxidative phosphorylation (OXPHOS) can decrease fumarate concentrations and protein succination (9). The physiological consequences of protein succination include a decrease in the functionality of the target protein (8, 10–12), for example succination of adiponectin prevents the formation of multimeric complexes and reduces plasma adiponectin levels in diabetes (4). Considering the impact of glucotoxicity driven mitochondrial stress in the adipocyte, we predicted that deficiencies in OXPHOS associated with NADH accumulation would also result in increased protein succination.Mitochondrial respiratory chain disorders encompass a broad range of encephalopathies and myopathies associated with the defective assembly, activity or maintenance of the OXPHOS machinery (13), and are estimated to occur in about 1 in 5,000 live births (14). A common feature in most mitochondrial diseases (MD) is a failure to thrive because of reduced mitochondrial energy production; both the brain and muscle are usually affected because of their high dependence on oxidative metabolism (13). Leigh syndrome is one of the most common manifestations of MD and is characterized by progressive neurodegeneration with bilateral necrotizing lesions of the brainstem and basal ganglia, resulting in lactic acidosis, ataxia, seizures, dystonia, and respiratory failure (15, 16). Mutations in genes encoding the five complexes of the OXPHOS machinery can lead to Leigh syndrome; however, the majority of these mutations affect subunits of complexes I and IV (17), and both mitochondrial and nuclear encoded proteins may be affected (17–19). Complex I is a large (980 kDa) l-shaped protein assembly consisting of 45 peptides, with one flavin mononucleotide and eight iron–sulfur clusters (20). One of the first identified mutations of complex I encoded Ndufs4, a small (18 kDa) assembly protein (21–23). Ndufs4 assists in the final stages of complex I assembly, and its absence results in the formation of a smaller ∼830 kDa subcomplex that lacks the NADH dehydrogenase module and has significantly less electron shuttling activity than the intact holoenzyme (24, 25). Ndufs4 mutations are associated with brainstem deterioration in humans (26), and a recently described Ndufs4 knockout mouse (Ndufs4 KO) exhibits many of the clinical and neurological symptoms observed in human Leigh syndrome (27, 28).One of the most common clinical features of MD is lactic acidosis, derived from the accumulation of pyruvate and elevated NADH. Increased lactate or lactate:pyruvate ratios have been measured in the blood, urine, and cerebrospinal fluid of a large number of Leigh syndrome patients (15, 16). Increases in other organic acids in urine have also been reported (16), indicating that metabolic acidosis is a prominent clinical feature. Interestingly, a study designed to find new diagnostic metabolites in MD demonstrated that within certain age ranges the measurement of urinary fumarate and malate was a more useful discriminator of MD than lactate or other organic acids (29). Barshop''s findings support the hypothesis that MD derived from OXPHOS deficiencies may exhibit increased protein succination because of the accumulation of NADH and subsequently fumarate. In this study we report for the first time that protein succination is present in the brain in an animal model of Leigh syndrome, the Ndufs4 KO mouse, suggesting that this modification may be an important biochemical link between the genetic defect and the onset of neuropathology observed in Leigh syndrome.     

Obese and diabetic db/db mice develop marked liver fibrosis in a model of nonalcoholic steatohepatitis: role of short-form leptin receptors and osteopontin

Obesity and type 2 diabetes are associated with nonalcoholic steatohepatitis (NASH), but an obese/diabetic animal model that mimics human NASH remains undefined. We examined the induction of steatohepatitis and liver fibrosis in obese and type 2 diabetic db/db mice in a nutritional model of NASH and determined the relationship of the expressions of osteopontin (OPN) and leptin receptors to the pathogenesis of NASH. db/db mice and the corresponding lean and nondiabetic db/m mice were fed a diet deficient in methionine and choline (MCD diet) or control diet for 4 wk. Leptin-deficient obese and diabetic ob/ob mice fed similar diets were used for comparison. MCD diet-fed db/db mice exhibited significantly greater histological inflammation and higher serum alanine aminotransferase levels than db/m and ob/ob mice. Trichrome staining showed marked pericellular fibrosis in MCD diet-fed db/db mice but no significant fibrosis in db/m or ob/ob mice. Collagen I mRNA expression was increased 10-fold in db/db mice, 4-fold in db/m mice, and was unchanged in ob/ob mice. mRNA expressions of OPN, TNF-alpha, TGF-beta, and short-form leptin receptors (Ob-Ra) were significantly increased in db/db mice compared with db/m or ob/ob mice. Parallel increases in OPN and Ob-Ra protein levels were observed in db/db mice. Cultured hepatocytes expressed only Ob-Ra, and leptin stimulated OPN mRNA and protein expression in these cells. In conclusion, our results demonstrate the development of an obese/diabetic experimental model for NASH in db/db mice and suggest an important role for Ob-Ra and OPN in the pathogenesis of NASH.     

C1q/TNF-related protein-12 (CTRP12), a novel adipokine that improves insulin sensitivity and glycemic control in mouse models of obesity and diabetes

Despite the prevalence of insulin resistance and type 2 diabetes mellitus, their underlying mechanisms remain incompletely understood. Many secreted endocrine factors and the intertissue cross-talk they mediate are known to be dysregulated in type 2 diabetes mellitus. Here, we describe CTRP12, a novel adipokine with anti-diabetic actions. The mRNA and circulating levels of CTRP12 were decreased in a mouse model of obesity, but its expression in adipocytes was increased by the anti-diabetic drug rosiglitazone. A modest rise in circulating levels of CTRP12 by recombinant protein administration was sufficient to lower blood glucose in wild-type, leptin-deficient ob/ob, and diet-induced obese mice. A short term elevation of serum CTRP12 by adenovirus-mediated expression improved glucose tolerance and insulin sensitivity, normalized hyperglycemia and hyperinsulinemia, and lowered postprandial insulin resistance in obese and diabetic mice. CTRP12 improves insulin sensitivity in part by enhancing insulin signaling in the liver and adipose tissue. Further, CTRP12 also acts in an insulin-independent manner; in cultured hepatocytes and adipocytes, CTRP12 directly activated the PI3K-Akt signaling pathway to suppress gluconeogenesis and promote glucose uptake, respectively. Collectively, these data establish CTRP12 as a novel metabolic regulator linking adipose tissue to whole body glucose homeostasis through insulin-dependent and independent mechanisms.     

Age-Related Modulation of the Effects of Obesity on Gene Expression Profiles of Mouse Bone Marrow and Epididymal Adipocytes

This study aimed to characterize and compare the effects of obesity on gene expression profiles in two distinct adipose depots, epididymal and bone marrow, at two different ages in mice. Alterations in gene expression were analyzed in adipocytes isolated from diet-induced obese (DIO) C57BL/6J male mice at 6 and 14 months of age and from leptin deficient mice (ob/ob) at 6 months of age using microarrays. DIO affected gene expression in both depots at 6 and 14 months, but more genes were altered in epididymal than bone marrow adipocytes at each age and younger mice displayed more changes than older animals. In epididymal adipocytes a total of 2789 (9.6%) genes were differentially expressed at 6-months with DIO, whereas 952 (3.3%) were affected at 14-months. In bone marrow adipocytes, 347 (1.2%) genes were differentially expressed at 6-months with DIO, whereas only 189 (0.66%) were changed at 14-months. 133 genes were altered by DIO in both fat depots at 6-months, and 37 genes at 14-months. Only four genes were altered in both depots at both ages with DIO. Bone marrow adipocytes are less responsive to DIO than epididymal adipocytes and the response of both depots to DIO declines with age. This loss of responsiveness with age is likely due to age-associated changes in expression of genes related to adipogenesis, inflammation and mitochondrial function that are similar to and obscure the changes commonly associated with DIO. Patterns of gene expression were generally similar in epididymal adipocytes from ob/ob and DIO mice; however, several genes were differentially expressed in bone marrow adipocytes from ob/ob and DIO mice, perhaps reflecting the importance of leptin signaling for bone metabolism. In conclusion, obesity affects age-associated alterations in gene expression in both epididymal and bone marrow adipocytes regardless of diet or genetic background.     

Selective up-regulation of fatty acid uptake by adipocytes characterizes both genetic and diet-induced obesity in rodents.

Long chain fatty acid transport is selectively up-regulated in adipocytes of Zucker fatty rats, diverting fatty acids from sites of oxidation toward storage in adipose tissue. To determine whether this is a general feature of obesity, we studied [(3)H]oleate uptake by adipocytes and hepatocytes from 1) homozygous male obese (ob), diabetic (db), fat (fat), and tubby (tub) mice and from 2) male Harlan Sprague-Dawley rats fed for 7 weeks a diet containing 55% of calories from fat. V(max) and K(m) were compared with controls of the appropriate background strain (C57BL/6J or C57BLKS) or diet (13% of calories from fat). V(max) for adipocyte fatty acid uptake was increased 5-6-fold in ob, db, fat, and tub mice versus controls (p < 0.001), whereas no differences were seen in the corresponding hepatocytes. Similar changes occurred in fat-fed rats. Of three membrane fatty acid transporters expressed in adipocytes, plasma membrane fatty acid-binding protein mRNA was increased 9-11-fold in ob and db, which lack a competent leptin/leptin receptor system, but was not increased in fat and tub, i.e. in strains with normal leptin signaling capability; fatty acid translocase mRNA was increased 2.2-6.5-fold in tub, ob, and fat adipocytes, but not in db adipocytes; and only marginal changes in fatty acid transport protein 1 mRNA were found in any of the mutant strains. Adipocyte fatty acid uptake is generally increased in murine obesity models, but up-regulation of individual transporters depends on the specific pathophysiology. Leptin may normally down-regulate expression of plasma membrane fatty acid binding protein.     

Dysregulation of adipose glutathione peroxidase 3 in obesity contributes to local and systemic oxidative stress

Glutathione peroxidase 3 (GPx3) accounts for the major antioxidant activity in the plasma. Here, we demonstrate that down-regulation of GPx3 in the plasma of obese subjects is associated with adipose GPx3 dysregulation, resulting from the increase of inflammatory signals and oxidative stress. Although GPx3 was abundantly expressed in kidney, lung, and adipose tissue, we observed that GPx3 expression was reduced selectively in the adipose tissue of several obese animal models as decreasing plasma GPx3 level. Adipose GPx3 expression was greatly suppressed by prooxidative conditions such as high levels of TNFalpha and hypoxia. In contrast, the antioxidant N-acetyl cysteine and the antidiabetic drug rosiglitazone increased adipose GPx3 expression in obese and diabetic db/db mice. Moreover, GPx3 overexpression in adipocytes improved high glucose-induced insulin resistance and attenuated inflammatory gene expression whereas GPx3 neutralization in adipocytes promoted expression of proinflammatory genes. Taken together, these data suggest that suppression of GPx3 expression in the adipose tissue of obese subjects might constitute a vicious cycle to expand local reactive oxygen species accumulation in adipose tissue potentially into systemic oxidative stress and obesity-related metabolic complications.     

Adipocyte protein modification by Krebs cycle intermediates and fumarate ester-derived succination

Protein succination, the non-enzymatic modification of cysteine residues by fumarate, is distinguishable from succinylation, an enzymatic reaction forming an amide bond between lysine residues and succinyl-CoA. Treatment of adipocytes with 30 mM glucose significantly increases protein succination with only a small change in succinylation. Protein succination may be significantly increased intracellularly after treatment with fumaric acid esters, however, the ester must be removed by saponification to permit 2SC-antibody detection of the fumarate adduct.     

IFN-alpha induces apoptosis of adipose tissue cells

Interferon alpha (IFN-alpha) is produced in response to viral infections and used clinically in the therapy of a variety of cancers and viral infections. IFN-alpha treatment is often associated with severe weight reduction. To elucidate the mechanism of IFN-associated weight loss, we studied its effect on adipocytes in vitro and in vivo. Diet-induced obese (DIO) C57BL/6 mice were treated continuously for 8 days with human IFN-alpha A/D (100 U/g body weight) or with vehicle alone. The body weight and adipose cell size of IFN-alpha A/D-treated DIO mice were significantly lower (P<0.05 and P<0.001, respectively) as compared with those of control DIO mice. PI3K and Bcl-2 were down-regulated whereas Bax expression was elevated in adipose tissue following IFN treatment as compared to adipose tissue of control DIO mice. Treatment of differentiated 3T3-F442A adipocytes with IFN-alpha A/D (250 U/ml, 36 h) significantly increased the number of apoptotic cells from 15.8% in control cells to 56+/-6%. In conclusion, weight loss following IFN-alpha therapy is due at least in part to increased apoptosis of adipocytes.     

Adipocyte Fetuin-A Contributes to Macrophage Migration into Adipose Tissue and Polarization of Macrophages

Macrophage infiltration into adipose tissue during obesity and their phenotypic conversion from anti-inflammatory M2 to proinflammatory M1 subtype significantly contributes to develop a link between inflammation and insulin resistance; signaling molecule(s) for these events, however, remains poorly understood. We demonstrate here that excess lipid in the adipose tissue environment may trigger one such signal. Adipose tissue from obese diabetic db/db mice, high fat diet-fed mice, and obese diabetic patients showed significantly elevated fetuin-A (FetA) levels in respect to their controls; partially hepatectomized high fat diet mice did not show noticeable alteration, indicating adipose tissue to be the source of this alteration. In adipocytes, fatty acid induces FetA gene and protein expressions, resulting in its copious release. We found that FetA could act as a chemoattractant for macrophages. To simulate lipid-induced inflammatory conditions when proinflammatory adipose tissue and macrophages create a niche of an altered microenvironment, we set up a transculture system of macrophages and adipocytes; the addition of fatty acid to adipocytes released FetA into the medium, which polarized M2 macrophages to M1. This was further confirmed by direct FetA addition to macrophages. Taken together, lipid-induced FetA from adipocytes is an efficient chemokine for macrophage migration and polarization. These findings open a new dimension for understanding obesity-induced inflammation.     

Amelioration of accelerated diabetic mesangial expansion by treatment with a PKC beta inhibitor in diabetic db/db mice, a rodent model for type 2 diabetes.

Activation of protein kinase C (PKC) is implicated as an important mechanism by which diabetes causes vascular complications. We have recently shown that a PKC beta inhibitor ameliorates not only early diabetes-induced glomerular dysfunction such as glomerular hyperfiltration and albuminuria, but also overexpression of glomerular mRNA for transforming growth factor beta1 (TGF-beta1) and extracellular matrix (ECM) proteins in streptozotocin-induced diabetic rats, a model for type 1 diabetes. In this study, we examined the long-term effects of a PKC beta inhibitor on glomerular histology as well as on biochemical and functional abnormalities in glomeruli of db/db mice, a model for type 2 diabetes. Administration of a PKC beta inhibitor reduced urinary albumin excretion rates and inhibited glomerular PKC activation in diabetic db/db mice. Administration of a PKC beta inhibitor also prevented the mesangial expansion observed in diabetic db/db mice, possibly through attenuation of glomerular expression of TGF-beta and ECM proteins such as fibronectin and type IV collagen. These findings provide the first in vivo evidence that the long-term inhibition of PKC activation in the renal glomeruli can ameliorate glomerular pathologies in diabetic state, and thus suggest that a PKC beta inhibitor might be an useful therapeutic strategy for the treatment of diabetic nephropathy.     

Hepatic glycogen metabolism in the db/db mouse

Summary Knowledge of the metabolic changes that occur in insulin-resistant type 2 diabetes is relatively lacking compared to insulin-deficient type 1 diabetes. This paper summarizes the importance of the C57BL/KsJ-db/db mouse as a model of type 2 diabetes, and illustrates the effects that insulin-deficient and insulin-resistant states have on hepatic glycogen metabolism. A longitudinal study of db/db mice of ages 2–15 weeks revealed that significant changes in certain parameters of hepatic glycogen metabolism occur during this period. The liver glycogen levels were similar between diabetic and control mice. However, glycogen particles from db/db mice were on average smaller in mass and had shorter exterior and interior chain lengths. Total phosphorylase and phosphorylase a activities were elevated in the genetically diabetic mice. This was primarily due to an increase in the amount of enzymic protein apparently the result of a decreased rate of degradation. It was not possible to find a consistent alteration in glycogen synthase activity in the db/db mice. Glycogen synthase and phosphorylase from diabetic liver revealed some changes in kinetic properties in the form of a decrease in V_max, and altered sensitivity to inhibitors like ATP. The altered glycogen structure in db/db mice may have contributed to changes in the activities and properties of glycogen synthase and phosphorylase. The exact role played by hormones (insulin and glucagon) in these changes is not clear but further studies should reveal their contributions. The db/db mouse provides a good model for type 2 diabetes and for fluctuating insulin and glucagon ratios. Its use should clarify the regulation of hepatic glycogen metabolism and other metabolic processes known to be controlled by these hormones. The other animal models of type 2 diabetes, ob/ob mouse and fatty Zucker (fa/fa) rat, show similar impairment of hepatic glycogen metabolism. The concentrations of glycogen metabolizing enzymes are high and in vitro studies indicate enhanced rate of glycogen synthesis and breakdown. However, streptozotocin-induced diabetic animals and BB rats which resemble insulin-deficient type 1 diabetes are characterized by decreased glycogen turnover as a result of reduction in the levels of glycogen metabolizing enzymes.