GULP1 deficiency reduces adipogenesis and glucose uptake via downregulation of PPAR signaling and disturbing of insulin/ERK signaling in 3T3-L1 cells

Obesity and metabolic disorders caused by alterations in lipid metabolism are major health issues in developed, affluent societies. Adipose tissue is the only organ that stores lipids and prevents lipotoxicity in other organs. Mature adipocytes can affect themselves and distant metabolism-related tissues by producing various adipokines, including adiponectin and leptin. The engulfment adaptor phosphotyrosine-binding domain-containing 1 (GULP1) regulates intracellular trafficking of glycosphingolipids and cholesterol, suggesting its close association with lipid metabolism. However, the role of GULP1 in adipocytes remains unknown. Therefore, this study aimed to investigate the function of GULP1 in adipogenesis, glucose uptake, and the insulin signaling pathway in adipocytes. A 3T3-L1 cell line with Gulp1 knockdown (shGulp1) and a 3T3-L1 control group (U6) were established. Changes in shGulp1 cells due to GULP1 deficiency were examined and compared to those in U6 cells using microarray analysis. Glucose uptake was monitored via insulin stimulation in shGulp1 and U6 cells using a 2-NBDG glucose uptake assay, and the insulin signaling pathway was investigated by western blot analysis. Adipogenesis was significantly delayed, lipid metabolism was altered, and several adipogenesis-related genes were downregulated in shGulp1 cells compared to those in U6 cells. Microarray analysis revealed significant inhibition of peroxisome proliferator-activated receptor signaling in shGulp1 cells compared with U6 cells. The production and secretion of adiponectin as well as the expression of adiponectin receptor were decreased in shGulp1 cells. In particular, compared with U6 cells, glucose uptake via insulin stimulation was significantly decreased in shGulp1 cells through the disturbance of ERK1/2 phosphorylation. This is the first study to identify the role of GULP1 in adipogenesis and insulin-stimulated glucose uptake by adipocytes, thereby providing new insights into the differentiation and functions of adipocytes and the metabolism of lipids and glucose, which can help better understand metabolic diseases.     

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.     

Prohibitin deficiency causes opposing lipid metabolism between 3T3-L1 adipocytes and Clone 9 hepatocytes

Prohibitin (PHB) is a highly conserved protein in eukaryotic cells that are present in multiple cellular compartments and has potential roles as a tumor suppressor, an anti-proliferative protein, a regulator of cell-cycle progression and in apoptosis. In the present study, we generated PHB-deficient 3T3-L1 adipocytes and Clone 9 (C9) hepatocytes by oligonucleotide siRNA and investigated whether PHB affect lipid metabolism. It was revealed that PHB deficiency caused opposing lipid metabolism between the two cell models. PHB deficiency increased expression of adipogenic, lipogenic, and other lipid metabolic proteins in 3T3-L1 adipocytes, whereas significantly decreased the levels of those proteins in C9 cells. Collectively, PHB deficiency promoted lipid metabolism in 3T3-L1 adipocytes while it aggravated lipid metabolism in C9 hepatocytes.     

The action of D-dopachrome tautomerase as an adipokine in adipocyte lipid metabolism

Adipose tissue is a critical exchange center for complex energy transactions involving triacylglycerol storage and release. It also has an active endocrine role, releasing various adipose-derived cytokines (adipokines) that participate in complex pathways to maintain metabolic and vascular health. Here, we found D-dopachrome tautomerase (DDT) as an adipokine secreted from human adipocytes by a proteomic approach. DDT mRNA levels in human adipocytes were negatively correlated with obesity-related clinical parameters such as BMI, and visceral and subcutaneous fat areas. Experiments using SGBS cells, a human preadipocyte cell line, revealed that DDT mRNA levels were increased in an adipocyte differentiation-dependent manner and DDT was secreted from adipocytes. In DDT knockdown adipocytes differentiated from SGBS cells that were infected with the adenovirus expressing shRNA against the DDT gene, mRNA levels of genes involved in both lipolysis and lipogenesis were slightly but significantly increased. Furthermore, we investigated AMP-activated protein kinase (AMPK) signaling, which phosphorylates and inactivates enzymes involved in lipid metabolism, including hormone-sensitive lipase (HSL) and acetyl-CoA carboxylase (ACC), in DDT knockdown adipocytes. The AMPK phosphorylation of HSL Ser-565 and ACC Ser-79 was inhibited in DDT knockdown cells and recovered in the cells treated with recombinant DDT (rDDT), suggesting that down-regulated DDT in adipocytes brings about a state of active lipid metabolism. Furthermore, administration of rDDT in db/db mice improved glucose intolerance and decreased serum free fatty acids levels. In the adipose tissue from rDDT-treated db/db mice, not only increased levels of HSL phosphorylated by AMPK, but also decreased levels of HSL phosphorylated by protein kinase A (PKA), which phosphorylates HSL to promote its activity, were observed. These results suggested that DDT acts on adipocytes to regulate lipid metabolism through AMPK and/or PKA pathway(s) and improves glucose intolerance caused by obesity.     

Permethrin Alters Adipogenesis in 3T3‐L1 Adipocytes and Causes Insulin Resistance in C2C12 Myotubes

Pyrethroids are a class of insecticides structurally derived from the naturally occurring insecticides called pyrethrins. Along with emerging evidence that exposure to insecticides is linked to altered weight gain and glucose homeostasis, exposure to pyrethroids has been linked to altered blood glucose levels in humans. Thus, the purpose of this study was to determine the role of permethrin on lipid and glucose metabolisms. Permethrin was treated to 3T3‐L1 adipocytes and C2C12 myoblasts to determine its role in lipid and glucose metabolisms, respectively. Permethrin treatment resulted in increased expression of key markers of adipogenesis and lipogenesis in adipocytes. Permethrin significantly reduced insulin‐stimulated glucose uptake in myotubes. This is the first report on the role of permethrin in altered lipid metabolism in adipocytes and impaired glucose homeostasis in myotubes. These results may help elucidate fundamental underlying mechanisms between insecticide exposure, particularly permethrin, and potential risk of developing obesity and its comorbidities.     

Spexin: A novel regulator of adipogenesis and fat tissue metabolism

Spexin (SPX, NPQ) is a novel peptide involved in the regulation of energy metabolism. SPX inhibits food intake and reduces body weight. In obese humans, SPX is the most down-regulated gene in fat. Therefore, SPX might be involved in the regulation of lipid metabolism. Here, we study the effects of SPX on lipolysis, lipogenesis, glucose uptake, adipogenesis, cell proliferation and survival in isolated human adipocytes or murine 3T3-L1 cells. SPX and its receptors, GALR2 and GALR3, are present at mRNA and protein levels in murine 3T3-L1 cells and human adipocytes. SPX inhibits adipogenesis and down-regulates mRNA expression of proadipogenic genes such as Pparγ, C/ebpα, C/ebpβ and Fabp4. SPX stimulates lipolysis by increasing the phosphorylation of hormone sensitive lipase (HSL). Simultaneously, SPX inhibits lipogenesis and glucose uptake in human adipocytes and murine 3T3-L1 cells. SPX has no effect on murine 3T3-L1 cell proliferation and viability. Moreover, our research showed that the SPX effect on adipocytes metabolism is mediated via GALR2 and GALR3 receptors. SPX is a novel regulator of lipid metabolism in murine 3T3-L1 and human adipocytes.     

Metformin counters the insulin-induced suppression of fatty acid oxidation and stimulation of triacylglycerol storage in rodent skeletal muscle

The present study examined the acute effects of metformin on fatty acid (FA) metabolism in oxidative soleus (SOL) and glycolytic epitrochlearis (EPT) rodent muscle. SOL and EPT were incubated for either 30 or 180 min in the absence or presence of 2 mM metformin and with or without insulin (10 mU/ml). Metformin did not alter basal FA metabolism but countered the effects of insulin on FA oxidation and incorporation into triacylglyerol (TAG). Specifically, metformin prevented the insulin-induced suppression of FA oxidation in SOL but did not alter FA incorporation into lipid pools. In contrast, in EPT metformin blunted the incorporation of FA into TAG when insulin was present but did not alter FA oxidation. In SOL, metformin resulted in a 50% increase in AMP-activated protein kinase alpha2 activity and prevented the insulin-induced increase in malonyl-CoA content. In both fiber types, basal and insulin-stimulated glucose oxidation were not significantly altered by metformin. All effects were similar regardless of whether they were measured after 30 or 180 min. Because increased muscle lipid storage and impaired FA oxidation have been associated with insulin resistance in this tissue, the ability of metformin to reverse these abnormalities in muscle FA metabolism may be a part of the mechanism by which metformin improves glucose clearance and insulin sensitivity. The present data also suggest that increased glucose clearance is not due to its enhanced subsequent oxidation. Additional studies are warranted to determine whether chronic metformin treatment has similar effects on muscle FA metabolism.     

Adipogenesis and lipid production in adipocytes subjected to sustained tensile deformations and elevated glucose concentration: a living cell-scale model system of diabesity

Adipocyte fate commitment is characterized by morphological changes of fibroblastic pre-adipocyte cells, and specifically by accumulation of lipid droplets (LDs) as part of the adipogenesis metabolism. Formation of LDs indicates the production of triglycerides from glucose through an insulin-regulated glucose internalization process. In obesity, adipocytes typically become insulin resistant, and glucose transport into the cells is impaired, resulting in type 2 diabetes. In the present study, we monitored the adipogenesis in 3T3-L1 cultured cells exposed to high (450 mg/dL hyperglycemia) and low (100 mg/dL physiological) glucose concentrations, in a novel cell culture model system of diabesity. In addition to glucose conditions, cells were concurrently exposed to different substrate tensile strains (12% and control) based on our prior work which revealed that adipogenesis is accelerated in cultures subjected to static, chronic substrate tensile deformations. Phase-contrast images were taken throughout the adipogenesis process (3 weeks) and were analyzed by an image processing algorithm which quantitatively monitors cell differentiation and lipid accumulation (number of LDs per cell and their radius as well as cell size and shape). The results indicated that high glucose concentrations and substrate tensile strains delivered to adipocytes accelerated lipid production by 1.7- and 1.4-fold, respectively. In addition, significant changes in average cell projected area and in other morphological attributes were observed during the differentiation process. The importance of this study is in characterizing the adipogenesis parameters as potential read-outs that can predict the occurrence of insulin resistance in the development of diabesity.     

Metformin Decouples Phospholipid Metabolism in Breast Cancer Cells

Introduction

The antidiabetic drug metformin, currently undergoing trials for cancer treatment, modulates lipid and glucose metabolism both crucial in phospholipid synthesis. Here the effect of treatment of breast tumour cells with metformin on phosphatidylcholine (PtdCho) metabolism which plays a key role in membrane synthesis and intracellular signalling has been examined.

Methods

MDA-MB-468, BT474 and SKBr₃ breast cancer cell lines were treated with metformin and [³H-methyl]choline and [¹⁴C(U)]glucose incorporation and lipid accumulation determined in the presence and absence of lipase inhibitors. Activities of choline kinase (CK), CTP:phosphocholine cytidylyl transferase (CCT) and PtdCho-phospholipase C (PLC) were also measured. [³H] Radiolabelled metabolites were determined using thin layer chromatography.

Results

Metformin-treated cells exhibited decreased formation of [³H]phosphocholine but increased accumulation of [³H]choline by PtdCho. CK and PLC activities were decreased and CCT activity increased by metformin-treatment. [¹⁴C] incorporation into fatty acids was decreased and into glycerol was increased in breast cancer cells treated with metformin incubated with [¹⁴C(U)]glucose.

Conclusion

This is the first study to show that treatment of breast cancer cells with metformin induces profound changes in phospholipid metabolism.     

NYGGF4 (PID1) effects on insulin resistance are reversed by metformin in 3T3-L1 adipocytes

NYGGF4 (also called PID1) is a recently discovered gene that is involved in obesity-related insulin resistance (IR). We aimed in the present study to further elucidate the effects of NYGGF4 on IR and the underlying mechanisms through using metformin treatment in 3T3-L1 adipocytes. Our data showed that the metformin pretreatment strikingly enhanced insulin-stimulated glucose uptake through increasing GLUT4 translocation to the PM in NYGGF4 overexpression adipocytes. NYGGF4 overexpression resulted in significant inhibition of tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt, whereas incubation with metformin strongly activated IRS-1 and Akt phosphorylation in NYGGF4 overexpression adipocytes. The reactive oxygen species (ROS) levels in NYGGF4 overexpression adipocytes were strikingly enhanced, which could be decreased by the metformin pretreatment. Our data also showed that metformin increased the expressions of PGC1-??, NRF-1, and TFAM, which were reduced in the NYGGF4 overexpression adipocytes. These results suggest that NYGGF4 plays a role in IR and its effects on IR could be reversed by metformin through activating IRS-1/PI3K/Akt and AMPK-PGC1-?? pathways.     

Effects of chromium picolinate on glucose uptake in insulin-resistant 3T3-L1 adipocytes involve activation of p38 MAPK

Chromium picolinate (CrPic) has been discovered as a supplemental or alternative medication for type 2 diabetes, but its mechanism of action is not well understood. The purpose of this study was to explore the possible anti-diabetic mechanisms of CrPic in insulin-resistant 3T3-L1 adipocytes; the insulin resistance was induced by treatment with high glucose and insulin for 24 h. The effects of CrPic on glucose metabolism and the glucose uptake-inducing activity of CrPic were investigated. Meanwhile, the effects of CrPic on glucose transporter 4 (GLUT4) translocation were visualized by immonofluorescence microscopy. In addition, its effects on insulin signaling pathways and mitogen-activated protein kinase (MAPK) signaling cascades were assessed by immunoblotting analysis and real-time PCR. The results showed that CrPic induced glucose metabolism and uptake, as well as GLUT4 translocation to plasma membrane (PM) in both control and insulin-resistant 3T3-L1 adipocytes without any changes in insulin receptor β (IR-β), protein kinase B (AKt), c-Cbl, extracellular signal-regulated kinase (ERK), c-Jun phosphorylation and c-Cbl-associated protein (CAP) mRNA levels. Interestingly, CrPic was able to increase the basal and insulin-stimulated levels of p38 MAPK activation in the control and insulin-resistant cells. Pretreatment with the specific p38 MAPK inhibitor SB203580 partially inhibited the CrPic-induced glucose transport, but CrPic-activated translocation of GLUT4 was not inhibited by SB203580. This study provides an experimental evidence of the effects of CrPic on glucose uptake through the activation of p38 MAPK and it is independent of the effect on GLUT4 translocation. The findings also suggest exciting new insights into the role of p38 MAPK in glucose uptake and GLUT4 translocation.     

Managing lipid metabolism in proliferating cells: New perspective for metformin usage in cancer therapy

Cancer cells metabolically adapt to undergo cellular proliferation. Lipids, besides their well-known role as energy storage, represent the major building blocks for the synthesis of neo-generated membranes. There is increasing evidence that cancer cells show specific alterations in different aspects of lipid metabolism. The changes of expression and activity of lipid metabolising enzymes are directly regulated by the activity of oncogenic signals. The dependence of tumour cells on the deregulated lipid metabolism suggests that proteins involved in this process could be excellent chemotherapeutic targets for cancer treatment. Due to its rare side effects in non-cancerous cells, metformin has been recently revaluated as a potential anti-tumourigenic drug, which negatively affects lipid biosynthetic pathways. In this review we summarised the emerging molecular events linking the anti-proliferative effect of metformin with lipid metabolism in cancer cells.     

游离脂肪酸对3T3-L1脂肪细胞糖代谢的影响

目的:通过培养3T3-L1前脂肪细胞,并诱导其分化至成熟,研究游离脂肪酸对脂肪细胞糖代谢的影响。方法:培养诱导3T3-L1脂肪细胞,用油红O染色鉴定并比较其形态结构的变化。LPS、EPA、SA、PA干预成熟脂肪细胞,收集不同时间的培养基,葡萄糖氧化酶法算出各组脂肪细胞的葡萄糖消耗量。用Western blot检测不同时间各组干预后细胞AMPK、GLUT4蛋白含量。结果:油红O染色鉴定成熟脂肪细胞胞浆中的脂滴染成红色,并出现戒环样结构;诱导分化第8天,90%以上细胞均分化成熟。含LPS、EPA、SA、PA的培养基作用于成熟脂肪细胞,随着时间的延长,显著抑制脂肪细胞对葡萄糖的吸收(P<0.05),同时,脂肪细胞AMPK、GLUT4蛋白含量在减少(P<0.05)。结论:游离脂肪酸可以诱导胰岛素抵抗的分子机制可能是通过胰岛素信号通路激活蛋白激酶(AMPK),进而影响GLUT4的蛋白表达,使脂肪细胞的葡萄糖吸收率减低,影响脂肪细胞的糖代谢。     

Oncostatin M decreases adiponectin expression and induces dedifferentiation of adipocytes by JAK3- and MEK-dependent pathways

Adiponectin, an adipokine secreted from adipocytes, plays a crucial role in the regulation of glucose and lipid metabolism. In the present study, we examine the role of the IL-6 family of cytokines in the expression of adiponectin in human adipocytes derived from human adipose tissue-derived stromal cells. Oncostatin M (OSM), but not IL-6, attenuated the expression level of adiponectin dose- and time-dependently, and the inhibitory effect of OSM on adiponectin expression was as potent as that of TNF-alpha. The OSM-induced down-regulation of adiponectin expression was correlated with the down-regulation of PPARgamma2 and lipoprotein lipase, markers for adipogenic differentiation, and depletion of intracellular lipid droplets, suggesting dedifferentiation of adipocytes in response to OSM. OSM induced phosphorylation of STAT1, and treatment of adipocytes with JAK3 inhibitor WHI-P131 or MEK inhibitor U0126, but not with JAK2 inhibitor AG490, prevented the activation of STAT1. Furthermore, the OSM-induced suppression of adiponectin expression and dedifferentiation of adipocytes were ameliorated by WHI-P131 or U0126, but not by AG490. These results suggest that OSM inhibits adiponectin expression by inducing dedifferentiation of adipocytes through signaling pathways involving JAK3 and MEK, but not JAK2.     

Comparative proteome analysis using amine-reactive isobaric tagging reagents coupled with 2D LC/MS/MS in 3T3-L1 adipocytes following hypoxia or normoxia

Hypoxia during the expansion of adipocytes is known to contribute both to the secretion of multiple inflammation-related adipokines as well as to obesity. We therefore investigated the nature of protein changes occurring in adipocytes during hypoxia by observation of the intracellular proteins that are expressed in 3T3-L1 adipocytes. Lysates were utilized for quantitative proteome analysis using isobaric tags for relative and absolute quantitation (iTRAQ) combined with peptide separation by multi-dimensional liquid chromatography. Antioxidants and elongation factors, as well as glycolytic enzymes were increased in hypoxic adipocytes. These changes were supported by similar changes suggested by real-time PCR. The proteins showing changes are all potential targets for revering the mechanism behind the phenomenon of induction of obese adipocytes by hypoxia. This study can therefore aid in defining the proteomic changes that occur in adipocytes in response to oxygen stress, and can further characterize adipocyte metabolism and adaptation to low oxygen conditions.     

Apelin stimulates glucose uptake through the PI3K/Akt pathway and improves insulin resistance in 3T3-L1 adipocytes

Apelin, a cytokine mainly secreted by adipocytes, is closely related with insulin resistance. The underlying molecular mechanisms of how apelin affects insulin resistance, however, are poorly understood. This study aimed to investigate the effect of apelin on glucose metabolism and insulin resistance in 3T3-L1 adipocytes. After 10 ng/ml TNF-α treatment for 24 h, insulin-stimulated glucose uptake was reduced by 47% in 3T3-L1 adipocytes. Apelin treatment improved glucose uptake in a time- and dose-dependent manner. Treatment of 1,000 nM apelin for 60 min maximally augmented glucose uptake in insulin-resistant 3T3-L1 adipocytes. Furthermore, apelin pre-incubation also increased adipocytes' insulin-stimulated glucose uptake, and PI3K/Akt pathway were involved in these effects. In addition, immunocytochemistry staining and western blotting analysis indicated that apelin could increase glucose transporter 4 translocation from the cytoplasm to the plasma membrane. Apelin also increased the anti-inflammatory adipokine adiponectin mRNA expression while reducing that of pro-inflammatory adipokine interleukin-6 in insulin-resistant 3T3-L1 adipocytes. These results suggest that apelin stimulates glucose uptake through the PI3K/Akt pathway, promotes GLUT4 translocation from the cytoplasm to the plasma membrane, and modulates inflammatory responses in insulin-resistant 3T3-L1 adipocytes.     

Citrus auraptene acts as an agonist for PPARs and enhances adiponectin production and MCP-1 reduction in 3T3-L1 adipocytes

Citrus fruit compounds have many health-enhancing effects. In this study, using a luciferase ligand assay system, we showed that citrus auraptene activates peroxisome proliferator-activated receptor (PPAR)-α and PPARγ. Auraptene induced up-regulation of adiponectin expression and increased the ratio of the amount of high-molecular-weight multimers of adiponectin to the total adiponectin. In contrast, auraptene suppressed monocyte chemoattractant protein (MCP)-1 expression in 3T3-L1 adipocytes. Experiments using PPARγ antagonist demonstrated that these effects on regulation of adiponectin and MCP-1 expression were caused by PPARγ activations. The results indicate that auraptene activates PPARγ in adipocytes to control adipocytekines such as adiponectin and MCP-1 and suggest that the consumption of citrus fruits, which contain auraptene can lead to a partial prevention of lipid and glucose metabolism abnormalities.     

Effects of oxytocin and vasopressin on the preadipocyte 3T3-F442A cell line

The 3T3-F442A mouse fibroblast cell line, triggered by factors present in fetal calf serum (FCS), converts either spontaneously or, in the simultaneous presence of FCS and insulin, at an accelerated rate into cells exhibiting the adipocyte phenotype. The effects of the neurohypophysial hormones in differentiated cells on glucose metabolism (glucose oxidation and lipogenesis) were compared with the stimulatory actions of insulin, which had its most pronounced effects in cells differentiated spontaneously with FCS in the absence of insulin. The differentiated 3T3-F442A cells were sensitive to physiological levels of insulin and exhibited manyfold increases in glucose metabolism in response to it. This result demonstrated that these cultured cells respond to insulin, in a manner analogous to freshly isolated adipocytes. In contrast to its insulin-like effects in isolated epididymal adipocytes, oxytocin was not reproducibly able to stimulate glucose metabolism in differentiated 3T3-F442A cells. Vasopressin was similarly inactive. In contrast, both oxytocin and vasopressin blocked adipocyte conversion triggered by FCS, either in the presence or absence of insulin; vasopressin was more potent than oxytocin, indicating that a vasopressin receptor was responsible for the observed inhibition of differentiation. Our work suggests that vasopressin could potentially play a role in the regulation of the adipocyte differentiation process.