MS screening strategies: investigating the glycomes of knockout and myodystrophic mice and leukodystrophic human brains

The implementation of highly sensitive and rapid mass spectrometric screening strategies for defining the glycosylation repertoires of organs in knockout mice is helping to reveal the roles that glycans play in health and disease. Thus novel glycosylation pathways have been uncovered in two such knockouts, namely alpha-mannosidase II null mice and UDP-N-acetylglucosamine: alpha 6-D-mannoside beta 1,2-N-acetylglucosaminyltransferase II null mice. This chapter documents the glycosylation profiles of a wide range of organs from the normal mouse which should facilitate future glycomics studies of knockout mice. Furthermore, we report applications of our screening technology in studies of the myodystrophy mouse and a human leukodystrophy.     

Metabolic syndrome in mice induced by expressing a transcriptional activator in adipose tissue

Metabolic syndrome is a combination of medical disorders that increases the risk of developing cardiovascular disease and diabetes. Constitutive overexpression of 11β-HSD1 in adipose tissue in mice leads to metabolic syndrome. In the process of generating transgenic mice overexpressing 11β-HSD1 in an inducible manner, we found a metabolic syndrome phenotype in control, transgenic mice, expressing the reverse tetracycline-transactivator (rtTA) in adipose tissue. The control mice exhibited all four sequelae of metabolic syndrome (visceral obesity, insulin resistance, dyslipidemia, and hypertension), a pro-inflammatory state and marked hepatic steatosis. Gene expression profiling of the adipose tissue, muscle and liver of these mice revealed changes in expression of genes involved in lipid metabolism, insulin resistance, and inflammation. Transient transfection of rtTA, but not tTS, into 3T3-L1 cells resulted in lipid accumulation. We conclude that expression of rtTA in adipose tissue causes metabolic syndrome in mice.     

Integrated mass spectrometric strategy for characterizing the glycans from glycosphingolipids and glycoproteins: direct identification of sialyl Le(x) in mice

The current interest in applying systems biology approaches to studying an organism's form or function promises to reveal further insights into the role of glycosylation in cells and whole organisms. This has prompted the development of a rapid, sensitive method of profiling the glycan component of both glycosphingolipids and glycoproteins from a single sample. Here we report a new mass spectrometric screening strategy for characterizing glycosphingolipid-derived oligosaccharides, which can be integrated into an existing highly sensitive glycoprotein glycomics strategy. Using ceramide glycanase to release the glycans from glycosphingolipids, this method provides a reliable profile of the glycosphingolipid-derived glycans present in a sample and has revealed new glycan structures. Glycoproteins are also efficiently recovered using this method, allowing the subsequent analysis of glycoprotein-derived glycans by mass spectrometry. The high sensitivity of this glycomic screening method allowed us to directly characterize the sialyl Le(x) epitope from mouse brain for the first time, where it was observed on an O-mannose structure. Thus, we present a mass spectrometric method that allows glycomic screening of N- and O-glycans as well as glycosphingolipid-derived glycans from a single tissue.     

灵芝多糖对3T3-L1胰岛素抵抗细胞模型PI-3K p85和GLUT4蛋白表达的影响

目的 研究灵芝多糖对3T3-L1胰岛素抵抗细胞模型PI-3K p85和GLUT4蛋白表达的影响,探讨灵芝多糖改善胰岛素抵抗的分子机制.方法 3T3-L1前脂肪细胞经1-甲基-3-异丁基-黄嘌呤、地塞米松、胰岛素诱导分化成3T3-L1脂肪细胞,以葡萄糖氧化酶法测定培养液中残余的葡萄糖含量.比较二甲双胍组,检测培养液中葡萄糖含量及PI-3K p85和GLUT4蛋白表达变化.结果 地塞米松联合胰岛素诱导3T3-L1脂肪细胞产生胰岛素抵抗,细胞对葡萄糖的摄取量减少.灵芝多糖可改善3T3-L1脂肪细胞胰岛素抵抗.胰岛素抵抗细胞的PI-3K p85和GLUT4蛋白表达明显减少;应用灵芝多糖后,相关蛋白表达增加.结论 灵芝多糖通过提高PI-3K p85和GLUT4蛋白的表达,参与胰岛素抵抗状态下3T3-L1细胞的葡萄糖代谢.     

小鼠MKP4基因组织、细胞表达谱研究

为了探讨MKP4基因mRNA在正常雄性C57小鼠的组织表达分布及其在3T3-L1脂肪细胞诱导进程中的表达谱。采用经典诱导分化方案诱导3T3-L1前脂肪细胞,分别收集前脂肪细胞、诱导分化day0、day2、day5、day8和day9的细胞,同时取正常C57雄性小鼠(鼠龄12周,数量n=3)多处组织如心、胰、肝、脂肪、脑、肾及睾丸等,并提取组织、细胞总RNA,RT-QPCR(荧光染料法)检测MKP4基因mRNA表达水平。结果显示MKP4基因在C57小鼠体内存在较广的表达谱,在肝脏、脂肪的表达量最高,其次为脑组织,其它组织存在低度表达;在3T3-L1前脂肪细胞、诱导分化day0、day2中表达极低,在day5中表达骤然升高,随后表达相对增高,于day9表达水平趋于平稳。MKP4基因组织表达谱结果表明其在维持正常生理功能可能发挥一定的作用,在胰岛素敏感相关组织肝脏、脂肪中表达量较高,提示可能参与胰岛素抵抗(IR)的发生与发展过程。此外,MKP4在3T3-L1前脂肪细胞诱导进程中表达上调,表明MKP4可能参与了脂质积累、肥胖等代谢疾病的发生与发展。     

NOD1 activation induces proinflammatory gene expression and insulin resistance in 3T3-L1 adipocytes

Chronic inflammation is associated with obesity and insulin resistance; however, the underlying mechanisms are not fully understood. Pattern recognition receptors Toll-like receptors and nucleotide-oligomerization domain-containing proteins play critical roles in innate immune response. Here, we report that activation of nucleotide binding oligomerization domain-containing protein-1 (NOD1) in adipocytes induces proinflammatory response and impairs insulin signaling and insulin-induced glucose uptake. NOD1 and NOD2 mRNA are markedly increased in differentiated murine 3T3-L1 adipocytes and human primary adipocyte culture upon adipocyte conversion. Moreover, NOD1 mRNA is markedly increased only in the fat tissues in diet-induced obese mice, but not in genetically obese ob/ob mice. Stimulation of NOD1 with a synthetic ligand Tri-DAP induces proinflammatory chemokine MCP-1, RANTES, and cytokine TNF-α and MIP-2 (human IL-8 homolog) and IL-6 mRNA expression in 3T3-L1 adipocytes in a time- and dose-dependent manner. Similar proinflammatory profiles are observed in human primary adipocyte culture stimulated with Tri-DAP. Furthermore, NOD1 activation suppresses insulin signaling, as revealed by attenuated tyrosine phosphorylation and increased inhibitory serine phosphorylation, of IRS-1 and attenuated phosphorylation of Akt and downstream target GSK3α/3β, resulting in decreased insulin-induced glucose uptake in 3T3-L1 adipocytes. Together, our results suggest that NOD1 may play an important role in adipose inflammation and insulin resistance in diet-induced obesity.     

Glycomics of bone marrow-derived mesenchymal stem cells can be used to evaluate their cellular differentiation stage

Human mesenchymal stem cells (MSCs) are adult multipotent progenitor cells. They hold an enormous therapeutic potential, but at the moment there is little information on the properties of MSCs, including their surface structures. In the present study, we analyzed the mesenchymal stem cell glycome by using mass spectrometric profiling as well as a panel of glycan binding proteins. Structural verifications were obtained by nuclear magnetic resonance spectroscopy, mass spectrometric fragmentation, and glycosidase digestions. The MSC glycome was compared to the glycome of corresponding osteogenically differentiated cells. More than one hundred glycan signals were detected in mesenchymal stem cells and osteoblasts differentiated from them. The glycan profiles of MSCs and osteoblasts were consistently different in biological replicates, indicating that stem cells and osteoblasts have characteristic glycosylation features. Glycosylation features associated with MSCs rather than differentiated cells included high-mannose type N-glycans, linear poly-N-acetyllactosamine chains and α2-3-sialylation. Mesenchymal stem cells expressed SSEA-4 and sialyl Lewis x epitopes. Characteristic glycosylation features that appeared in differentiated osteoblasts included abundant sulfate ester modifications. The results show that glycosylation analysis can be used to evaluate MSC differentiation state.     

Differential effects of palmitate on glucose uptake in rat-1 fibroblasts and 3T3-L1 adipocytes.

Non-esterified fatty acids are thought to be one of the causes for insulin resistance. However, the molecular mechanism of fatty acid-induced insulin resistance is not clearly known. In this study, we first examined the effect of palmitate on insulin signaling in 3T3-L1 adipocytes. We found that 1h treatment with 1 mmol/l palmitate had no effect on insulin binding, tyrosine phosphorylation of insulin receptors, 185 kDa proteins and Shc, and PI3 kinase activity in 3T3-L1 adipocytes. Then, the effects of palmitate on MAP kinase activity and glucose uptake in fully differentiated 3T3-L1 adipocytes were compared with those in poorly differentiated 3T3-L1 cells and in HIRc-B cells. Palmitate treatment had no effect on MAP kinase activity in fully differentiated 3T3-L1 adipocytes, while it inhibited MAP kinase in poorly differentiated 3T3-L1 cells and HIRc-B cells. Glucose transport in 3T3-L1 adipocytes treated with palmitate for 1 h, 4 h and 16 h was higher than that in control cells, but palmitate treatment caused a rightward shift of the insulin-dose responsive curve for glucose uptake in HIRc-B cells. Palmitate treatment did not significantly affect basal and insulin-stimulated GLUT4 translocation. When the cells were treated with PD98059, a specific MEK inhibitor, insulin-stimulated glucose uptake was not affected in 3T3-L1 adipocytes, while it was almost completely inhibited in HIRc-B cells. These results suggest the primary effect of palmitate on adipocytes may not involve insulin resistance of adipocytes themselves.     

Increased Aβ production prompts the onset of glucose intolerance and insulin resistance

Type 2 diabetes (T2D) mellitus and Alzheimer's disease (AD) are two prevalent diseases with comparable pathophysiological features and genetic predisposition. Patients with AD are more susceptible to develop T2D. However, the molecular mechanism linking AD and T2D remains elusive. In this study, we have generated a new mouse model to test the hypothesis that AD would prompt the onset of T2D in mice. To test our hypothesis, we crossed Alzheimer APPswe/PS1dE9 (APP/PS1) transgenic mice with mice partially deficient in leptin signaling (db/+). Body weight, plasma glucose, and insulin levels were monitored. Phenotypic characterization of glucose metabolism was performed using glucose and insulin tolerance tests. β-Cell mass, islet volume, and islet number were analyzed by histomorphometry. APP/PS1 coexpression in mice with intact leptin receptor signaling did not show any metabolic perturbations in glucose metabolism or insulin sensitivity. In contrast, APP/PS1 coexpression in db/+ mice resulted in nonfasting hyperglycemia, hyperinsulinemia, and hypercholesterolemia without changes in body weight. Conversely, fasting blood glucose and cholesterol levels remained unchanged. Coinciding with altered glucose metabolism, APP/PS1 coexpression in db/+ mice resulted in glucose intolerance, insulin resistance, and impaired insulin signaling. In addition, histomorphometric analysis of pancreata revealed augmented β-cell mass. Taken together, these findings provide experimental evidence to support the notion that aberrant Aβ production might be a mechanistic link underlying the pathology of insulin resistance and T2D in AD.     

High fat diet induces formation of spontaneous liposarcoma in mouse adipose tissue with overexpression of interleukin 22

Interleukin 22 (IL-22) is a T-cell secreted cytokine that modulates inflammatory response in nonhematopoietic tissues such as epithelium and liver. The function of IL-22 in adipose tissue is currently unknown. We generated a transgenic mouse model with overexpression of IL-22 specifically in adipose tissue. The IL-22 transgenic mice had no apparent changes in obesity and insulin resistance after feeding with high fat diet (HFD). Unexpectedly, all the IL-22 transgenic mice fed with HFD for four months developed spontaneous tumors in epididymal adipose tissue. Histological analysis indicated that the tumors were well-differentiated liposarcomas with infiltration of inflammatory cells. IL-22 overexpression promotes production of inflammatory cytokines such as IL-1β and IL-10 and stimulates ERK phosphorylation in adipose tissue. Furthermore, IL-22 treatment in differentiated 3T3-L1 adipocytes could induce IL-1β and IL-10 expression, together with stimulation of ERK phosphorylation. Taken together, our study not only established a novel mouse model with spontaneous liposarcoma, but also revealed that IL-22 overexpression may collaborate with diet-induced obesity to impact on tumor development in mouse.     

Asb6, an adipocyte-specific ankyrin and SOCS box protein, interacts with APS to enable recruitment of elongins B and C to the insulin receptor signaling complex

The APS adapter protein plays a pivotal role in coupling the insulin receptor to CAP and c-Cbl in the phosphatidylinositol 3-kinase-independent pathway of insulin-stimulated glucose transport. Yeast two-hybrid screening of a 3T3-L1 adipocyte library using APS as a bait identified a 418-amino acid ankyrin and SOCS (suppressor of cytokine signaling) box protein Asb6 as an interactor. Asb6 is an orphan member of a larger family of Asb proteins that are ubiquitously expressed. However, Asb6 expression appears to be restricted to adipose tissue. Asb6 was specifically expressed in 3T3-L1 adipocytes as a 50-kDa protein but not in fibroblasts. In Chinese hamster ovary-insulin receptor (CHO-IR) cells Myc epitope-tagged APS interacted constitutively with FLAG-tagged Asb6 in the presence or absence of insulin stimulation and insulin stimulation did not alter the interaction. In 3T3-L1 adipocytes, insulin receptor activation was accompanied by the APS-dependent recruitment of Asb6. Asb6 did not appear to undergo tyrosine phosphorylation. Immunofluorescence and confocal microscopy studies revealed that Asb6 colocalized with APS in CHO cells and in 3T3-L1 adipocytes. In immunoprecipitation studies in CHO cells or 3T3-L1 adipocytes, the Elongin BC complex was found to be bound to Asb6, and activation of the insulin receptor was required to facilitate Asb6 recruitment along with Elongins B/C. Prolonged insulin stimulation resulted in the degradation of APS when Asb6 was co-expressed but not in the absence of Asb6. We conclude that Asb6 functions to regulate components of the insulin signaling pathway in adipocytes by facilitating degradation by the APS-dependent recruitment of Asb6 and Elongins BC.     

Targeted overexpression of inducible 6-phosphofructo-2-kinase in adipose tissue increases fat deposition but protects against diet-induced insulin resistance and inflammatory responses

Increasing evidence demonstrates the dissociation of fat deposition, the inflammatory response, and insulin resistance in the development of obesity-related metabolic diseases. As a regulatory enzyme of glycolysis, inducible 6-phosphofructo-2-kinase (iPFK2, encoded by PFKFB3) protects against diet-induced adipose tissue inflammatory response and systemic insulin resistance independently of adiposity. Using aP2-PFKFB3 transgenic (Tg) mice, we explored the ability of targeted adipocyte PFKFB3/iPFK2 overexpression to modulate diet-induced inflammatory responses and insulin resistance arising from fat deposition in both adipose and liver tissues. Compared with wild-type littermates (controls) on a high fat diet (HFD), Tg mice exhibited increased adiposity, decreased adipose inflammatory response, and improved insulin sensitivity. In a parallel pattern, HFD-fed Tg mice showed increased hepatic steatosis, decreased liver inflammatory response, and improved liver insulin sensitivity compared with controls. In both adipose and liver tissues, increased fat deposition was associated with lipid profile alterations characterized by an increase in palmitoleate. Additionally, plasma lipid profiles also displayed an increase in palmitoleate in HFD-Tg mice compared with controls. In cultured 3T3-L1 adipocytes, overexpression of PFKFB3/iPFK2 recapitulated metabolic and inflammatory changes observed in adipose tissue of Tg mice. Upon treatment with conditioned medium from iPFK2-overexpressing adipocytes, mouse primary hepatocytes displayed metabolic and inflammatory responses that were similar to those observed in livers of Tg mice. Together, these data demonstrate a unique role for PFKFB3/iPFK2 in adipocytes with regard to diet-induced inflammatory responses in both adipose and liver tissues.     

The farnesoid X receptor modulates adiposity and peripheral insulin sensitivity in mice

The farnesoid X receptor (FXR) is a bile acid (BA)-activated nuclear receptor that plays a major role in the regulation of BA and lipid metabolism. Recently, several studies have suggested a potential role of FXR in the control of hepatic carbohydrate metabolism, but its contribution to the maintenance of peripheral glucose homeostasis remains to be established. FXR-deficient mice display decreased adipose tissue mass, lower serum leptin concentrations, and elevated plasma free fatty acid levels. Glucose and insulin tolerance tests revealed that FXR deficiency is associated with impaired glucose tolerance and insulin resistance. Moreover, whole-body glucose disposal during a hyperinsulinemic euglycemic clamp is decreased in FXR-deficient mice. In parallel, FXR deficiency alters distal insulin signaling, as reflected by decreased insulin-dependent Akt phosphorylation in both white adipose tissue and skeletal muscle. Whereas FXR is not expressed in skeletal muscle, it was detected at a low level in white adipose tissue in vivo and induced during adipocyte differentiation in vitro. Moreover, mouse embryonic fibroblasts derived from FXR-deficient mice displayed impaired adipocyte differentiation, identifying a direct role for FXR in adipocyte function. Treatment of differentiated 3T3-L1 adipocytes with the FXR-specific synthetic agonist GW4064 enhanced insulin signaling and insulin-stimulated glucose uptake. Finally, treatment with GW4064 improved insulin resistance in genetically obese ob/ob mice in vivo. Although the underlying molecular mechanisms remain to be unraveled, these results clearly identify a novel role of FXR in the regulation of peripheral insulin sensitivity and adipocyte function. This unexpected function of FXR opens new perspectives for the treatment of type 2 diabetes.     

Autophagy participants in the dedifferentiation of mouse 3T3-L1 adipocytes triggered by hypofunction of insulin signaling

Our previous data indicate that both insulin and IGF-1 signallings dysfunction promotes the dedifferentiation of primary human and mouse white adipocytes. Based on the fact that insulin activates mTOR and inhibits autophagy, and autophagy deficiency can inhibit the differentiation of white adipocytes, we speculate that autophagy may be related to the dedifferentiation of white adipocytes. We investigated the underlying mechanism of autophagy during dedifferentiation of mouse 3T3-L1 adipocytes. After incomplete inhibition of insulin and IGF-1 signallings, 3T3-L1 adipocytes manifest dedifferentiation accompanied with an increase of autophagy level. If induction only of autophagy in the adipocytes, then the cells also occur somewhat dedifferentiation, and with a slight decrease of insulin signal, while its degree was weaker than insulin signal inhibited cells. Notably, after inhibition of the insulin and IGF-1 signallings and simultaneously inducing autophagy, the dedifferentiation of 3T3-L1 adipocytes was the most obvious compared with other groups, and the insulin and IGF-1 signallings decreases was greater than the cells with inhibition only of insulin signalling. If inhibition of both insulin signal and autophagy simultaneously, the dedifferentiation of the adipocytes reveals similar tendencies to the cells that insulin signal was inhibited. No significant dedifferentiation occurs of 3T3-L1 cells if only inhibition of autophagy. Taken all together, in this study, we proved that autophagy is positively related to the dedifferentiation of 3T3-L1 adipocytes and is regulated through the insulin-PI3K-AKT-mTOCR1-autophagy pathway. Autophagy may also has a certain degree of negative feedback affect on the insulin signalling of 3T3-L1 cells. Our work may help to better understand the biological properties of mature adipocytes and may help formulate anti-obesity strategies by regulating insulin and insulin signaling level.     

Cyclin-dependent kinase inhibitor, p21WAF1/CIP1, is involved in adipocyte differentiation and hypertrophy, linking to obesity, and insulin resistance

Both adipocyte hyperplasia and hypertrophy are determinant factors for adipocyte differentiation during the development of obesity. p21(WAF1/CIP1), a cyclin-dependent kinase inhibitor, is induced during adipocyte differentiation; however, its precise contribution to this process is unknown. Using both in vitro and in vivo systems, we show that p21 is crucial for maintaining adipocyte hypertrophy and obesity-induced insulin resistance. The absence of p21 in 3T3-L1 fibroblasts by RNA-mediated interference knockdown or in embryonic fibroblasts from p21(-/-) mice impaired adipocyte differentiation, resulting in smaller adipocytes. Despite normal adipose tissue mass on a normal diet, p21(-/-) mice fed high energy diets had reduced adipose tissue mass and adipocyte size accompanied by a marked improvement in insulin sensitivity. Knockdown of p21 in enlarged epididymal fat of diet-induced obese mice and also in fully differentiated 3T3-L1 adipocytes caused vigorous apoptosis by activating p53. Thus, p21 is involved in both adipocyte differentiation and in protecting hypertrophied adipocytes against apoptosis. Via both of these mechanisms, p21 promotes adipose tissue expansion during high fat diet feeding, leading to increased downstream pathophysiological consequences such as insulin resistance.     

Macrophage ubiquitin-specific protease 2 modifies insulin sensitivity in obese mice

We previously reported that ubiquitin-specific protease (USP) 2 in macrophages down-regulates genes associated with metabolic diseases, suggesting a putative anti-diabetic role for USP2 in macrophages. In this study, we evaluate this role at both cellular and individual levels. Isolated macrophages forcibly expressing Usp2a, a longer splicing variant of USP2, failed to modulate the insulin sensitivity of 3T3-L1 adipocytes. Similarly, macrophage-selective overexpression of Usp2a in mice (Usp2a transgenic mice) had a negligible effect on insulin sensitivity relative to wild type littermates following a three-month high-fat diet. However, Usp2a transgenic mice exhibited fewer M1 macrophages in their mesenteric adipose tissue. Following a six-month high-fat diet, Usp2a transgenic mice exhibited a retarded progression of insulin resistance in their skeletal muscle and liver, and an improvement in insulin sensitivity at an individual level. Although conditioned media from Usp2a-overexpressing macrophages did not directly affect the insulin sensitivity of C2C12 myotubes compared to media from control macrophages, they did increase the insulin sensitivity of C2C12 cells after subsequent conditioning with 3T3-L1 cells. These results indicate that macrophage USP2A hampers obesity-elicited insulin resistance via an adipocyte-dependent mechanism.     

Obesity causes a shift in metabolic flow of gangliosides in adipose tissues

Obesity is associated with insulin resistance and a mild chronic inflammation in adipose tissues. Recent studies suggested that GM3 ganglioside mediates dysfunction in insulin signaling. However, it has not been determined the ganglioside profiling in adipose tissues of obese animals. Here, we for the first time examined semi-quantitative ganglioside profiles in the adipose tissues of high fat- and high sucrose-induced obese, diabetic C57BL/6J mice by TLC and HPLC/mass spectrometry. In control adipose tissues GM3 dominated with traces of GM1 and GD1a; obesity led to a dramatic increase in GM2, GM1, and GD1a with the GM3 content unchanged. Similar results were obtained in KK and KKAy mice. Adipocytes separated from stromal vascular cells including macrophages contained more of those gangliosides in KKAy mice than in KK mice. These results underscore those gangliosides in the pathophysiology of obesity-related diseases.