Quantitative secretome and glycome of primary human adipocytes during insulin resistance

Adipose tissue is both an energy storage depot and an endocrine organ. The impaired regulation of the secreted proteins of adipose tissue, known as adipocytokines, observed during obesity contributes to the onset of whole-body insulin resistance and the pathobiology of type 2 diabetes mellitus (T2DM). In addition, the global elevation of the intracellular glycosylation of proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) via either genetic or pharmacological methods is sufficient to induce insulin resistance in both cultured cells and animal models. The elevation of global O-GlcNAc levels is associated with the altered expression of many adipocytokines. We have previously characterized the rodent adipocyte secretome during insulin sensitive and insulin resistant conditions. Here, we characterize and quantify the secretome and glycome of primary human adipocytes during insulin responsive and insulin resistant conditions generated by the classical method of hyperglycemia and hyperinsulinemia or by the pharmacological manipulation of O-GlcNAc levels. Using a proteomic approach, we identify 190 secreted proteins and report a total of 20 up-regulated and 6 down-regulated proteins that are detected in both insulin resistant conditions. Moreover, we apply glycomic techniques to examine (1) the sites of N-glycosylation on secreted proteins, (2) the structures of complex N- and O-glycans, and (3) the relative abundance of complex N- and O-glycans structures in insulin responsive and insulin resistant conditions. We identify 91 N-glycosylation sites derived from 51 secreted proteins, as well as 155 and 29 released N- and O-glycans respectively. We go on to quantify many of the N- and O-glycan structures between insulin responsive and insulin resistance conditions demonstrating no significant changes in complex glycosylation in the time frame for the induction of insulin resistance. Thus, our data support that the O-GlcNAc modification is involved in the regulation of adipocytokine secretion upon the induction of insulin resistance in human adipocytes.     

Nucleocytoplasmic O-glycosylation: O-GlcNAc and functional proteomics

The molecular complexity that defines different cell types and their biological responses occurs at the level of the cell's proteome. The recent increase in availability of genomic sequence information is a valuable tool for the field of proteomics. While most proteomic studies focus on differential expression levels, post-translational modifications such as phosphorylation, glycosylation, and acetylation, provide additional levels of functional complexity to the cell's proteome. The reversible post-translational modification O-linked beta-N-acetylglucosamine (O-GlcNAc) is found on serines and threonines of nuclear and cytoplasmic proteins. It appears to be as widespread as phosphorylation. While phosphorylation is recognized as a fundamental mechanism for controlling protein function, less is known about the specific roles of O-GlcNAc modification. However, evidence is building that O-GlcNAc may compete with phosphate at some sites of attachment. Aberrant O-GlcNAc modification has been linked to several disease states, including diabetes and Alzheimer's disease. Regulated enzymes catalyzing the addition (O-GlcNAc transferase, OGT) and removal (O-GlcNAcase) of the modification have been cloned and OGT is required for life at the single cell level. Here we review the properties of O-GlcNAc that suggest it is a regulatory modification analogous to phosphorylation. We also discuss the use of comparative functional proteomics to elucidate functions for this ubiquitous intracellular carbohydrate modification.     

脂肪细胞因子对脂肪细胞增殖分化的反馈调节作用

肥胖已被证实是胰岛素抵抗、2 型糖尿病、高血压、高脂血症及冠状动脉粥样硬化性心脏病等代谢性疾病发生的重要诱因.肥胖的发生主要是由于体内脂肪细胞的异常分化和增殖,最终导致脂肪细胞异常增多及细胞内脂质过度沉积产生的.脂肪细胞的增殖分化受到多种因素的调控,其中脂肪细胞因子作为脂肪组织分泌的肽类激素,也在脂肪细胞的发育分化过程中起重要的反馈调节作用.大多数肥胖患者体内存在脂肪细胞因子分泌异常及其相应的功能紊乱.本文将对几种主要的脂肪细胞因子在脂肪细胞发育分化中的作用及最新研究进展进行简要综述及讨论.     

Dynamic interplay between O-GlcNAc and O-phosphate: the sweet side of protein regulation

Beta-O-linked N-acetylglucosamine (O-GlcNAc) is an abundant modification of cytosolic and nuclear proteins that occurs in metazoans. O-GlcNAc is dynamically processed by a unique set of enzymes that actively add and remove the modification. Functionally, O-GlcNAc appears to regulate protein stability, subcellular localization and protein-protein interactions. The modification often acts in a reciprocal manner to O-phosphate modifications of proteins and together they can synergistically control the activity of many cellular processes. Recently, O-GlcNAc has been demonstrated to play a significant role in diseases such as diabetes, cancer and neurodegeneration. For example, the increased levels of O-GlcNAc that occur in diabetes are associated with decreased insulin responsiveness in adipocytes.     

Elevation of the post-translational modification of proteins by O-linked N-acetylglucosamine leads to deterioration of the glucose-stimulated insulin secretion in the pancreas of diabetic Goto-Kakizaki rats

Many nuclear and cytoplasmic proteins are O-glycosylated on serine or threonine residues with the monosaccharide beta-N-acetylglucosamine, which is then termed O-linked N-acetylglucosamine (O-GlcNAc). It has been shown that abnormal O-GlcNAc modification (O-GlcNAcylation) of proteins is one of the causes of insulin resistance and diabetic complications. In this study, in order to examine the relationship between O-GlcNAcylation of proteins and glucose-stimulated insulin secretion in noninsulin-dependent type (type 2) diabetes, we investigated the level of O-GlcNAcylation of proteins, especially that of PDX-1, and the expression of O-GlcNAc transferase in Goto-Kakizaki (GK) rats, which are an animal model of type-2 diabetes. By immunoblot and immunohistochemical analyses, the expression of O-GlcNAc transferase protein and O-GlcNAc-modified proteins in whole pancreas and islets of Langerhans of 15-week-old diabetic GK rats and nondiabetic Wistar rats was examined. The expression of O-GlcNAc transferase at the protein level and O-GlcNAc transferase activity were increased significantly in the diabetic pancreas and islets. The diabetic pancreas and islets also showed an increase in total cellular O-GlcNAc-modified proteins. O-GlcNAcylation of PDX-1 was also increased. In the diabetic GK rats, significant increases in the immunoreactivities of both O-GlcNAc and O-GlcNAc transferase were observed. PUGNAc, an inhibitor of O-GlcNAcase, induced an elevation of O-GlcNAc level and a decrease of glucose-stimulated insulin secretion in isolated islets. These results indicate that elevation of the O-GlcNAcylation of proteins leads to deterioration of insulin secretion in the pancreas of diabetic GK rats, further providing evidence for the role of O-GlcNAc in the insulin secretion.     

Elevation of global O-GlcNAc levels in 3T3-L1 adipocytes by selective inhibition of O-GlcNAcase does not induce insulin resistance

The O-GlcNAc post-translational modification is considered to act as a sensor of nutrient flux through the hexosamine biosynthetic pathway. A cornerstone of this hypothesis is that global elevation of protein O-GlcNAc levels, typically induced with the non-selective O-GlcNAcase inhibitor PUGNAc (O-(2-acetamido-2-deoxy-D-glycopyranosylidene) amino-N-phenylcarbamate), causes insulin resistance in adipocytes. Here we address the potential link between elevated O-GlcNAc and insulin resistance by using a potent and selective inhibitor of O-GlcNAcase (NButGT (1,2-dideoxy-2'-propyl-alpha-D-glucopyranoso-[2,1-D]-Delta 2'-thiazoline), 1200-fold selectivity). A comparison of the structures of a bacterial homologue of O-GlcNAcase in complex with PUGNAc or NButGT reveals that these inhibitors bind to the same region of the active site, underscoring the competitive nature of their inhibition of O-GlcNAcase and the molecular basis of selectivity. Treating 3T3-L1 adipocytes with NButGT induces rapid increases in global O-GlcNAc levels, but strikingly, NButGT treatment does not replicate the insulin desensitizing effects of the non-selective O-GlcNAcase inhibitor PUGNAc. Consistent with these observations, NButGT also does not recapitulate the impaired insulin-mediated phosphorylation of Akt that is induced by treatment with PUGNAc. Collectively, these results suggest that increases in global levels of O-GlcNAc-modified proteins of cultured adipocytes do not, on their own, cause insulin resistance.     

Structure-activity relationship studies for the development of inhibitors of murine adipose triglyceride lipase (ATGL)

High serum fatty acid (FA) levels are causally linked to the development of insulin resistance, which eventually progresses to type 2 diabetes and non-alcoholic fatty liver disease (NAFLD) generalized in the term metabolic syndrome. Adipose triglyceride lipase (ATGL) is the initial enzyme in the hydrolysis of intracellular triacylglycerol (TG) stores, liberating fatty acids that are released from adipocytes into the circulation. Hence, ATGL-specific inhibitors have the potential to lower circulating FA concentrations, and counteract the development of insulin resistance and NAFLD. In this article, we report about structure–activity relationship (SAR) studies of small molecule inhibitors of murine ATGL which led to the development of Atglistatin. Atglistatin is a specific inhibitor of murine ATGL, which has proven useful for the validation of ATGL as a potential drug target.     

Comprehensive proteomic datasets for studying adipocyte–macrophage cell–cell communication

Cellular communication is a fundamental process in biology. The interaction of adipocytes with macrophages is a key event in the development of common diseases such as type 2 diabetes. We applied an established bilayer cell coculture system and comprehensive MS detection to analyse on a proteome‐wide scale the paracrine interaction of murine adipocytes and macrophages. Altogether, we identified 4486 proteins with at least two unique peptides, of which 2392 proteins were informative for 3T3‐L1 adipocytes and 2957 proteins for RAW 264.7 macrophages. Further, we observed over 12000 phosphorylation sites, of which we could assign 3200 informative phosphopeptides with a single phosphosite for adipocytes and 4514 for macrophages. Using protein set enrichment and phosphosite analyses, we deciphered regulatory protein pathways involved in cellular stress and inflammation, which can contribute to metabolic impairment of cells including insulin resistance and other disorders. The generated datasets provide a holistic, molecular pathway‐centric view on the interplay of adipocytes and macrophages in disease processes and a resource for further studies.     

Type 2 diabetes – An autoinflammatory disease driven by metabolic stress

Type 2 diabetes has traditionally been viewed as a metabolic disorder characterised by chronic high glucose levels, insulin resistance, and declining insulin secretion from the pancreas. Modern lifestyle, with abundant nutrient supply and reduced physical activity, has resulted in dramatic increases in the rates of obesity-associated disease conditions, including diabetes. The associated excess of nutrients induces a state of systemic low-grade chronic inflammation that results from production and secretion of inflammatory mediators from the expanded pool of activated adipocytes. Here, we review the mechanisms by which obesity induces adipose tissue dysregulation, detailing the roles of adipose tissue secreted factors and their action upon other cells and tissues central to glucose homeostasis and type 2 diabetes. Furthermore, given the emerging importance of adipokines, cytokines and chemokines in disease progression, we suggest that type 2 diabetes should now be viewed as an autoinflammatory disease, albeit one that is driven by metabolic dysregulation.     

Adiponectin: a biomarker of obesity-induced insulin resistance in adipose tissue and beyond

Adiponectin is one of the most thoroughly studied adipocytokines. Low plasma levels of adiponectin are found to associate with obesity, metabolic syndrome, diabetes and many other human diseases. From animal experiments and human studies, adiponectin has been shown to be a key regulator of insulin sensitivity. In this article, we review the evidence and propose that hypo-adiponectinemia is not a major cause of obesity. Instead, it is the result of obesity-induced insulin resistance in the adipose tissue. Hypo-adiponectinemia then mediates the metabolic effects of obesity on the other peripheral tissues, such as liver and skeletal muscle and may also exert some direct effects on end-organ damage. We propose that deciphering the molecular details governing the adiponectin gene expression and protein secretion will lead us to more comprehensive understanding of the mechanisms of insulin resistance in the adipose tissue and provide us new avenues for the therapeutic intervention of obesity and insulin resistance-related human disorders     

Adipocytokines - novel link between inflammation and vascular function?

Obesity and obesity related diseases are a major public health problem. Recent studies have shown that fat tissue is not a simple energy storage organ, but exerts important endocrine and immune functions. These are achieved predominantly through release of adipocytokines, which include several novel and highly active molecules released abundantly by adipocytes like leptin, resistin, adiponectin or visfatin, as well as some more classical cytokines released possibly by inflammatory cells infiltrating fat, like TNF-alpha, IL-6, MCP-1 (CCL-2), IL-1. All of those molecules may act on immune cells leading to local and generalized inflammation and may also affect vascular (endothelial) function by modulating vascular nitric oxide and superoxide release and mediating obesity related vascular disorders (including hypertension, diabetes, atherosclerosis, and insulin resistance) but also cancer or non-alcoholic fatty liver diseases. Present review, in a concise form, focuses on the effects of major adipocytokines, characteristic for adipose tissue like leptin, adiponectin, resistin and visfatin on the immune system, particularly innate and adaptive immunity as well as on blood vessels. Macrophages and T cells are populating adipose tissue which develops into almost an organized immune organ. Activated T cells further migrate to blood vessels, kidney, brain and other organs surrounded by infiltrated fat leading to their damage, thus providing a link between metabolic syndrome, inflammation and cardiovascular and other associated disorders. Ceretain treatments may lead to significant changes in adipocytokine levels. For example include beta-2 adrenoreceptor agonists, thiazolidinediones as well as androgens lead to decrease of plasma leptin levels. Moreover future treatments of metabolic system associated disorders should focus on the regulation of adipocytokines and their modes of action.     

Reduction of O-GlcNAc protein modification does not prevent insulin resistance in 3T3-L1 adipocytes

3T3-L1 adipocytes develop insulin-resistant glucose transport upon preincubation with high (25 mM) glucose, provided that insulin (0.6 nM) is included, Akt activation is impaired, and high glucose and insulin act synergistically. Considerable evidence suggests that increased glucose flux via the hexosamine biosynthesis pathway enhances the O-GlcNAc modification (O-GlcNAcylation) of some critical protein(s) that may contribute to insulin resistance. However, whether enhanced protein O-GlcNAcylation is necessary for the development of insulin resistance is unknown. We used two strategies to test this hypothesis. The first strategy was the overexpression of O-GlcNAcase, which removes O-GlcNAc from Ser/Thr of proteins. Cells were infected with O-GlcNAcase-expressing adenovirus (or empty virus) 5 days before they were submitted to protocols that elicit (or not) insulin resistance. O-GlcNAcase was highly expressed and functional as assessed by Western blot, O-GlcNAcase assay, and marked reduction of O-GlcNAcylated proteins. The activity was mainly cytosolic. The second strategy was the expression of O-GlcNAc transferase (OGT) being markedly reduced by transfection of OGT siRNA, resulting in an approximately 90% decrease of nuclear and cytosolic OGT protein expression and similar reduction in O-GlcNAcylated proteins. Nontargeting siRNA had no effect. Preincubation in high glucose with low-dose insulin decreased the acute insulin response of glucose transport by at least 50% and impaired Akt activation. None of these parameters were affected by overexpression of O-GlcNAcase or by OGT knockout. Excess O-GlcNAcylation is one of many factors that can cause insulin resistance. It does not seem to be required for the development of glucose/insulin-induced insulin resistance of glucose transport and Akt activation in 3T3-L1 adipocytes.     

Novel aspects of adipocyte-induced skeletal muscle insulin resistance

Insulin resistance in skeletal muscle is an early event in the development of diabetes with obesity being one of the major contributing factors. Conditioned medium (CM) from differentiated human adipocytes impairs insulin signalling in human skeletal muscle cells. Recent data on adipocyte-induced insulin resistance in skeletal muscle cells describes underlying mechanisms of this process. Skeletal muscle insulin resistance involves multiple pathways and irreversible changes in the expression level of critical proteins. Furthermore, the reversibility of insulin resistance could be demonstrated. Several strategies to combat insulin resistance have been developed. One recent approach to treat obesity and the metabolic syndrome is the use of endocannabinoid receptor antagonists such as rimonabant. These compounds might also reduce insulin resistance in type 2 diabetes with effects on adipose tissue and liver and possibly skeletal muscle.     

Hyperglycemia and the O-GlcNAc transferase in rat aortic smooth muscle cells: elevated expression and altered patterns of O-GlcNAcylation

Hyperglycemia leads to vascular disease specific to diabetes mellitus. This pathology, which results from abnormal proliferation of smooth muscle cells in arterial walls, may lead to cataract, renal failure, and atherosclerosis. The hexosamine biosynthetic pathway is exquisitely responsive to glucose concentration and plays an important role in glucose-induced insulin resistance. UDP-GlcNAc: polypeptide O-N-acetylglucosaminyltransferase (O-GlcNAc transferase; OGTase) catalyzes the O-linked attachment of single GlcNAc moieties to serine and threonine residues on many cytosolic or nuclear proteins. Polyclonal antibody against OGTase was used to examine the expression of OGTase in rat aorta and aortic smooth muscle (RASM) cells. OGTase enzymatic activity and expression at the mRNA and protein levels were determined in RASM cells cultured at normal (5 mM) and at high (20 mM) glucose concentrations. OGTase mRNA and protein are expressed in both endothelial cells and smooth muscle cells in the aorta of normal rats. In both cell types, the nucleus is intensely stained, while the cytoplasm stains diffusely. Immunoelectron microscopy shows that OGTase is localized to euchromatin and around the myofilaments of smooth muscle cells. In RASM cells grown in 5 mM glucose, OGTase is also located mainly in the nucleus. Hyperglycemic RASM cells also display a relative increase in OGTase's p78 subunit and an overall increase protein and activity for OGTase. Biochemical analyses show that hyperglycemia qualitatively and quantitatively alters the glycosylation or expression of many O-GlcNAc-modified proteins in the nucleus. These results suggest that the abnormal O-GlcNAc modification of intracellular proteins may be involved in glucose toxicity to vascular tissues.     

Effects of alpha-lipoic acid on chemerin secretion in 3T3-L1 and human adipocytes

Chemerin is a novel adipokine associated with obesity and insulin resistance. α-Lipoic acid (α-LA) has shown beneficial properties on diabetes and obesity. The aim of this study was to examine the effects of α-LA on chemerin production in adipocytes in absence or presence of TNF-α, insulin and AICAR. The potential signaling pathways involved in α-LA effects on chemerin were also analyzed. α-LA actions on chemerin were tested in differentiated 3T3-L1 adipocytes and in some cases in human subcutaneous and omental adipocytes. Chemerin mRNA levels were measured by RT-PCR and the amount of chemerin secreted to culture media was determined by ELISA. α-LA induced a concentration-dependent inhibition on both chemerin secretion and mRNA levels in 3T3-L1 adipocytes. The AMPK activator AICAR and the PI3K inhibitor LY294002 dramatically abrogated both chemerin secretion and gene expression, and further potentiated the inhibitory effect of α-LA on chemerin secretion. Insulin was able to partially reverse the inhibitory action of α-LA on chemerin secretion. α-LA also reduced basal chemerin secretion in both subcutaneous and omental adipocytes from overweight/obese subjects. Moreover, α-LA was able to abolish the stimulatory effects of the pro-inflammatory cytokine TNF-α on chemerin secretion. Our data demonstrated the ability of α-LA to inhibit chemerin production, an adipokine associated to obesity and metabolic syndrome, suggesting that the reduction of chemerin could contribute to the antiobesity/antidiabetic properties described for α-LA.     

Importance of diagnosing and treating the metabolic syndrome in reducing cardiovascular risk

Applying the criteria for the metabolic syndrome serves as a simple and inexpensive tool for identifying patients at high risk for diabetes and coronary heart disease, particularly those who do not fall into traditional risk categories. Several independent physiological processes underlie the non-random risk-factor clustering that defines the metabolic syndrome, including insulin resistance, central obesity, dyslipidemia, impaired glucose tolerance, and hypertension. Other non-classic risk factors, such as abnormal oxidized low-density lipoprotein-cholesterol, adiponectin, and C-reactive protein levels, are highly correlated with the metabolic syndrome. Use of the metabolic syndrome criteria for assessment is comparable with other risk-scoring systems in accurately predicting cardiovascular disease risk and is simpler to implement in the clinic. Further research is needed to define the etiology of the metabolic syndrome.     

Insulin resistance and the metabolism of branched-chain amino acids in humans

Peripheral resistance to insulin action is the major mechanism causing the metabolic syndrome and eventually type 2 diabetes mellitus. The metabolic derangement associated with insulin resistance is extensive and not restricted to carbohydrates. The branched-chain amino acids (BCAAs) are particularly responsive to the inhibitory insulin action on amino acid release by skeletal muscle and their metabolism is profoundly altered in conditions featuring insulin resistance, insulin deficiency, or both. Obesity, the metabolic syndrome and diabetes mellitus display a gradual increase in the plasma concentration of BCAAs, from the obesity-related low-grade insulin-resistant state to the severe deficiency of insulin action in diabetes ketoacidosis. Obesity-associated hyperinsulinemia succeeds in maintaining near-normal or slightly elevated plasma concentration of BCAAs, despite the insulin-resistant state. The low circulating levels of insulin and/or the deeper insulin resistance occurring in diabetes mellitus are associated with more marked elevation in the plasma concentration of BCAAs. In diabetes ketoacidosis, the increase in plasma BCAAs is striking, returning to normal when adequate metabolic control is achieved. The metabolism of BCAAs is also disturbed in other situations typically featuring insulin resistance, including kidney and liver dysfunction. However, notwithstanding the insulin-resistant state, the plasma level of BCAAs in these conditions is lower than in healthy subjects, suggesting that these organs are involved in maintaining BCAAs blood concentration. The pathogenesis of the decreased BCAAs plasma level in kidney and liver dysfunction is unclear, but a decreased afflux of these amino acids into the blood stream has been observed.     

Proteomics reveals the effects of sustained weight loss on the human plasma proteome

Sustained weight loss is a preferred intervention in a wide range of metabolic conditions, but the effects on an individual's health state remain ill‐defined. Here, we investigate the plasma proteomes of a cohort of 43 obese individuals that had undergone 8 weeks of 12% body weight loss followed by a year of weight maintenance. Using mass spectrometry‐based plasma proteome profiling, we measured 1,294 plasma proteomes. Longitudinal monitoring of the cohort revealed individual‐specific protein levels with wide‐ranging effects of losing weight on the plasma proteome reflected in 93 significantly affected proteins. The adipocyte‐secreted SERPINF1 and apolipoprotein APOF1 were most significantly regulated with fold changes of ?16% and +37%, respectively (P < 10^?13), and the entire apolipoprotein family showed characteristic differential regulation. Clinical laboratory parameters are reflected in the plasma proteome, and eight plasma proteins correlated better with insulin resistance than the known marker adiponectin. Nearly all study participants benefited from weight loss regarding a ten‐protein inflammation panel defined from the proteomics data. We conclude that plasma proteome profiling broadly evaluates and monitors intervention in metabolic diseases.