1147-P: Pseudo Insulin Resistance: Palmitate Inactivates Insulin Signaling but Stimulates Basal Glucose Uptake in 3T3-L1 Adipocytes

Insulin-resistant type 2 diabetic patients have been reported to have impaired skeletal muscle mitochondrial respiratory function. A key question is whether decreased mitochondrial respiration contributes directly to the decreased insulin action. To address this, a model of impaired cellular respiratory function was established by incubating human skeletal muscle cell cultures with the mitochondrial inhibitor sodium azide and examining the effects on insulin action. Incubation of human skeletal muscle cells with 50 and 75 μM azide resulted in 48 ± 3% and 56 ± 1% decreases, respectively, in respiration compared with untreated cells mimicking the level of impairment seen in type 2 diabetes. Under conditions of decreased respiratory chain function, insulin-independent (basal) glucose uptake was significantly increased. Basal glucose uptake was 325 ± 39 pmol/min/mg (mean ± SE) in untreated cells. This increased to 669 ± 69 and 823 ± 83 pmol/min/mg in cells treated with 50 and 75 μM azide, respectively (vs. untreated, both P < 0.0001). Azide treatment was also accompanied by an increase in basal glycogen synthesis and phosphorylation of AMP-activated protein kinase. However, there was no decrease in glucose uptake following insulin exposure, and insulin-stimulated phosphorylation of Akt was normal under these conditions. GLUT1 mRNA expression remained unchanged, whereas GLUT4 mRNA expression increased following azide treatment. In conclusion, under conditions of impaired mitochondrial respiration there was no evidence of impaired insulin signaling or glucose uptake following insulin exposure in this model system.

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Phenylalanine Impairs Insulin Signaling and Inhibits Glucose Uptake by Modifying IRβ

10.21203/rs.3.rs-483980/v1 ◽

2021 ◽

Author(s):

Qian Zhou ◽

Wan-Wan Sun ◽

Jia-Cong Chen ◽

Huilu Zhang ◽

Jie Liu ◽

...

Keyword(s):

Insulin Resistance ◽

Type 2 Diabetes ◽

Amino Acids ◽

Insulin Receptor ◽

Glucose Uptake ◽

Insulin Signaling ◽

Trna Synthetase ◽

Blood Samples ◽

Receptor Beta

Abstract Although elevated circulating amino acids are associated with the onset of type 2 diabetes (T2D), how amino acids act on cell insulin signaling and glucose uptake remains unclear. Herein, we report that phenylalanine modifies insulin receptor beta (IRβ) and inactivates insulin signaling and glucose uptake. Mice fed phenylalanine-rich chow or overexpressing human phenylalanyl-tRNA synthetase (hFARS) developed insulin resistance and symptoms of T2D. Mechanistically, FARS phenylalanylated lysine 1057/1079 of IRβ (F-K1057/1079) inactivated IRβ and prevented insulin from generating insulin signaling to promote glucose uptake by cells. SIRT1 reversed F-K1057/1079 and counteracted the insulin-inactivating effects of hFARS and phenylalanine. F-K1057/1079 and SIRT1 levels of white cells of T2D patients’ blood samples were positively and negatively correlated with T2D onset, respectively. Blocking F-K1057/1079 with phenylalaninol sensitized insulin signaling and relieved T2D symptoms in hFARS-transgenic and db/db mice. We revealed mechanisms of how phenylalanylation inactivates insulin signaling that may be employed to control T2D.

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Alleviative Effect of Ruellia tuberosa L. on Insulin Resistance and Abnormal Lipid Accumulation in TNF-α-Treated FL83B Mouse Hepatocytes

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2021/9967910 ◽

2021 ◽

Vol 2021 ◽

pp. 1-8

Author(s):

Hong-Jie Chen ◽

Chih-Yuan Ko ◽

Jian-Hua Xu ◽

Yu-Chu Huang ◽

James Swi-Bea Wu ◽

...

Keyword(s):

Insulin Resistance ◽

Ethyl Acetate ◽

Glucose Uptake ◽

Insulin Signaling ◽

Lipid Accumulation ◽

Glucose Transporter ◽

Peroxisome Proliferator ◽

Glucose Transporter 2 ◽

Tnf Α ◽

Mouse Hepatocytes

Type 2 diabetes mellitus (T2DM) is a chronic metabolic disease, and most patients with T2DM develop nonalcoholic fatty liver disease (NAFLD). Both diseases are closely linked to insulin resistance (IR). Our previous studies demonstrated that Ruellia tuberosa L. (RTL) extract significantly enhanced glucose uptake in the skeletal muscles and ameliorated hyperglycemia and IR in T2DM rats. We proposed that RTL might be via enhancing hepatic antioxidant capacity. However, the potent RTL bioactivity remains unidentified. In this study, we investigated the effects of RTL on glucose uptake, IR, and lipid accumulation in vitro to mimic the T2DM accompanied by the NAFLD paradigm. FL83B mouse hepatocytes were treated with tumor necrosis factor-α (TNF-α) to induce IR, coincubated with oleic acid (OA) to induce lipid accumulation, and then, treated with RTL fractions, fractionated with n-hexane or ethyl acetate (EA), from column chromatography, and analyzed by thin-layer chromatography. Our results showed that the ethyl acetate fraction (EAf2) from RTL significantly increased glucose uptake and suppressed lipid accumulation in TNF-α plus OA-treated FL83B cells. Western blot analysis showed that EAf2 from RTL ameliorated IR by upregulating the expression of insulin-signaling-related proteins, including protein kinase B, glucose transporter-2, and peroxisome proliferator-activated receptor alpha in TNF-α plus OA-treated FL83B cells. The results of this study suggest that EAf2 from RTL may improve hepatic glucose uptake and alleviate lipid accumulation by ameliorating and suppressing the hepatic insulin signaling and lipogenesis pathways, respectively, in hepatocytes.

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Effect of Subtoxic DDT Exposure on Glucose Uptake and Insulin Signaling in Rat L6 Myoblast-Derived Myotubes

International Journal of Toxicology ◽

10.1177/1091581819850577 ◽

2019 ◽

Vol 38 (4) ◽

pp. 303-311 ◽

Cited By ~ 2

Author(s):

Vijay Kumar Singh ◽

Sajib Kumar Sarkar ◽

Alpana Saxena ◽

Bidhan Chandra Koner

Keyword(s):

Insulin Resistance ◽

Glucose Uptake ◽

Tyrosine Phosphorylation ◽

Insulin Signaling ◽

Messenger Rna ◽

Insulin Receptors ◽

Serine Phosphorylation ◽

Enzyme Linked Immunosorbent Assay ◽

Antioxidant Content ◽

L6 Myoblast

Exposure to persistent organic pollutants including dichlorodiphenyltrichloroethane (DDT) induces insulin resistance. But the mechanism is not clearly known. The present study was designed to explore the effect of subtoxic DDT exposure on (1) insulin-stimulated glucose uptake, (2) malondialdehyde (MDA) level and total antioxidant content, (3) activation of redox sensitive kinases (RSKs), and (4) insulin signaling in rat L6 myoblast-derived myotubes. Exposure to 30 mg/L and 60 mg/L of DDT for 18 hours dose dependently decreased glucose uptake and antioxidant content in myotubes and increased MDA levels. The exposures did not alter tumor necrosis factor α (TNF-α) level as determined by enzyme-linked immunosorbent assay, despite decreased messenger RNA expression following DDT exposures. Phosphorylation of c-Jun N-terminal kinases and IκBα, an inhibitory component of nuclear factor κB (NFκB), was increased, suggesting activation of RSKs. The level of tyrosine phosphorylation of insulin receptor substrate 1 and serine phosphorylation of protein kinase B (Akt) on insulin stimulation decreased in myotubes with exposure to subtoxic concentrations of DDT, but there was no change in tyrosine phosphorylation level of insulin receptors. We conclude that subtoxic DDT exposure impairs insulin signaling and thereby induces insulin resistance in muscle cells. Data show that oxidative stress-induced activation of RSKs is responsible for impairment of insulin signaling on DDT exposure.

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Ginsenoside Re Reduces Insulin Resistance through Inhibition of c-Jun NH2-Terminal Kinase and Nuclear Factor-κB

Molecular Endocrinology ◽

10.1210/me.2007-0119 ◽

2008 ◽

Vol 22 (1) ◽

pp. 186-195 ◽

Cited By ~ 47

Author(s):

Zhiguo Zhang ◽

Xiaoying Li ◽

Wenshan Lv ◽

Yisheng Yang ◽

Hong Gao ◽

...

Keyword(s):

Insulin Resistance ◽

Glucose Uptake ◽

Insulin Signaling ◽

Infusion Rate ◽

Glucose Infusion ◽

Signaling Cascades ◽

Glucose Infusion Rate ◽

Nuclear Factor ◽

Phosphatidylinositol 3 Kinase ◽

Ginsenoside Re

Abstract Ginsenoside Re (Re), a compound derived from Panax ginseng, shows an antidiabetic effect. However, the molecular basis of its action remains unknown. We investigated insulin signaling and the antiinflammatory effect by Re in 3T3-L1 adipocytes and in high-fat diet (HFD) rats to dissect its anti-hyperglycemic mechanism. Glucose uptake was measured in 3T3-L1 cells and glucose infusion rate determined by clamp in HFD rats. The insulin signaling cascade, including insulin receptor (IR) β-subunit, IR substrate-1, phosphatidylinositol 3-kinase, Akt and Akt substrate of 160 kDa, and glucose transporter-4 translocation are examined. Furthermore, c-Jun NH2-terminal kinase (JNK), MAPK, and nuclear factor (NF)-κB signaling cascades were also assessed. The results show Re increases glucose uptake in 3T3-L1 cells and glucose infusion rate in HFD rats. The activation of insulin signaling by Re is initiated at IR substrate-1 and further passes on through phosphatidylinositol 3-kinase and downstream signaling cascades. Moreover, Re demonstrates an impressive suppression of JNK and NF-κB activation and inhibitor of NF-κBα degradation. In conclusion, Re reduces insulin resistance in 3T3-L1 adipocytes and HFD rats through inhibition of JNK and NF-κB activation.

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Glucose infusion causes insulin resistance in skeletal muscle of rats without changes in Akt and AS160 phosphorylation

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00133.2007 ◽

2007 ◽

Vol 293 (5) ◽

pp. E1358-E1364 ◽

Cited By ~ 34

Author(s):

Andrew J. Hoy ◽

Clinton R. Bruce ◽

Anna Cederberg ◽

Nigel Turner ◽

David E. James ◽

...

Keyword(s):

Insulin Resistance ◽

Skeletal Muscle ◽

Glucose Uptake ◽

Insulin Signaling ◽

Glycogen Synthase ◽

Glycogen Synthesis ◽

Glucose Infusion ◽

Metabolic Effects ◽

Synthesis Rate ◽

Muscle Sample

Hyperglycemia is a defining feature of Type 1 and 2 diabetes. Hyperglycemia also causes insulin resistance, and our group (Kraegen EW, Saha AK, Preston E, Wilks D, Hoy AJ, Cooney GJ, Ruderman NB. Am J Physiol Endocrinol Metab Endocrinol Metab 290: E471–E479, 2006) has recently demonstrated that hyperglycemia generated by glucose infusion results in insulin resistance after 5 h but not after 3 h. The aim of this study was to investigate possible mechanism(s) by which glucose infusion causes insulin resistance in skeletal muscle and in particular to examine whether this was associated with changes in insulin signaling. Hyperglycemia (∼10 mM) was produced in cannulated male Wistar rats for up to 5 h. The glucose infusion rate required to maintain this hyperglycemia progressively lessened over 5 h (by 25%, P < 0.0001 at 5 h) without any alteration in plasma insulin levels consistent with the development of insulin resistance. Muscle glucose uptake in vivo (44%; P < 0.05) and glycogen synthesis rate (52%; P < 0.001) were reduced after 5 h compared with after 3 h of infusion. Despite these changes, there was no decrease in the phosphorylation state of multiple insulin signaling intermediates [insulin receptor, Akt, AS160 (Akt substrate of 160 kDa), glycogen synthase kinase-3β] over the same time course. In isolated soleus strips taken from control or 1- or 5-h glucose-infused animals, insulin-stimulated 2-deoxyglucose transport was similar, but glycogen synthesis was significantly reduced in the 5-h muscle sample (68% vs. 1-h sample; P < 0.001). These results suggest that the reduced muscle glucose uptake in rats after 5 h of acute hyperglycemia is due more to the metabolic effects of excess glycogen storage than to a defect in insulin signaling or glucose transport.

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

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00709.2010 ◽

2011 ◽

Vol 301 (4) ◽

pp. E587-E598 ◽

Cited By ~ 50

Author(s):

Ling Zhao ◽

Pan Hu ◽

Yijun Zhou ◽

Jaanki Purohit ◽

Daniel Hwang

Keyword(s):

Insulin Resistance ◽

Glucose Uptake ◽

Insulin Signaling ◽

Serine Phosphorylation ◽

Dependent Manner ◽

Proinflammatory Response ◽

Downstream Target ◽

Synthetic Ligand ◽

Underlying Mechanisms ◽

Oligomerization Domain

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.

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Interleukin-1β-Induced Insulin Resistance in Adipocytes through Down-Regulation of Insulin Receptor Substrate-1 Expression

Endocrinology ◽

10.1210/en.2006-0692 ◽

2007 ◽

Vol 148 (1) ◽

pp. 241-251 ◽

Cited By ~ 389

Author(s):

Jennifer Jager ◽

Thierry Grémeaux ◽

Mireille Cormont ◽

Yannick Le Marchand-Brustel ◽

Jean-François Tanti

Keyword(s):

Insulin Resistance ◽

Protein Kinase ◽

Insulin Receptor ◽

Glucose Uptake ◽

Insulin Signaling ◽

Protein Kinase B ◽

Transcriptional Level ◽

Insulin Receptor Substrate ◽

Down Regulation ◽

Glut 4

Inflammation is associated with obesity and insulin resistance. Proinflammatory cytokines produced by adipose tissue in obesity could alter insulin signaling and action. Recent studies have shown a relationship between IL-1β level and metabolic syndrome or type 2 diabetes. However, the ability of IL-1β to alter insulin signaling and action remains to be explored. We demonstrated that IL-1β slightly increased Glut 1 translocation and basal glucose uptake in 3T3-L1 adipocytes. Importantly, we found that prolonged IL-1β treatment reduced the insulin-induced glucose uptake, whereas an acute treatment had no effect. Chronic treatment with IL-1β slightly decreased the expression of Glut 4 and markedly inhibited its translocation to the plasma membrane in response to insulin. This inhibitory effect was due to a decrease in the amount of insulin receptor substrate (IRS)-1 but not IRS-2 expression in both 3T3-L1 and human adipocytes. The decrease in IRS-1 amount resulted in a reduction in its tyrosine phosphorylation and the alteration of insulin-induced protein kinase B activation and AS160 phosphorylation. Pharmacological inhibition of ERK totally inhibited IL-1β-induced down-regulation of IRS-1 mRNA. Moreover, IRS-1 protein expression and insulin-induced protein kinase B activation, AS160 phosphorylation, and Glut 4 translocation were partially recovered after treatment with the ERK inhibitor. These results demonstrate that IL-1β reduces IRS-1 expression at a transcriptional level through a mechanism that is ERK dependent and at a posttranscriptional level independently of ERK activation. By targeting IRS-1, IL-1β is capable of impairing insulin signaling and action, and could thus participate in concert with other cytokines, in the development of insulin resistance in adipocytes.

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Abstract 308: Rgs2 Is An Endogenous Inhibitor Of Insulin Signaling

Circulation ◽

10.1161/circ.118.suppl_18.s_282 ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Prem Sharma ◽

Jennie Bever ◽

Scott Heximer ◽

Carmen Dessauer ◽

Jerrold M Olefsky

Keyword(s):

Insulin Resistance ◽

Insulin Sensitivity ◽

Glucose Uptake ◽

Insulin Signaling ◽

Regulatory System ◽

Heart Association ◽

Glut4 Translocation ◽

Wild Type ◽

Akt Phosphorylation ◽

Insulin Resistant

Background: Insulin resistance is the hallmark of type 2 diabetes and is a known risk factor for the development of cardiovascular diseases. We have determined that overexpression of a GTPase-activating protein, RGS2 decreases insulin sensitivity. This study describes RGS2 regulation of insulin signaling pathways in order to assess whether this information can be used to reverse insulin insensitivity in diabetes. Hypothesis, Methods and Results: RGS2 protein levels were elevated 3 to 5-fold in white adipose tissues from ob/ob and high fat diet induced Insulin Resistant mice. Further, RGS2 protein is elevated in insulin resistant 3T3-L1 adipocytes treated chronically with either insulin, ET-1, or TNF-aplha. Further, SiRNA knockdown of endogenous RGS2 protein increases basal, insulin independent and insulin-dependent GLUT4 translocation. We hypothesized that the RGS2 regulatory system is defective/overactive in insulin resistance, and that a modulation of this regulatory system by RGS2 inhibition would improve insulin sensitivity. Thus, we determined the mechanisms whereby RGS2 modulates insulin sensitivity in 3T3-L1 adipocytes; focusing on insulin-regulated G-protein/PI3-K pathways leading to GLUT4 translocation and glucose uptake; utilizing adenoviruses over-expressing wild-type and mutants RGS2, as well as by siRNA-mediated knock down of endogenous RGS2. We overexpressed the Wild-Type (WT), GTPase defective (GD), and plasma membrane translocation defective (TD) RGS2 proteins in 3T3-L1 adipocytes. Overexpression of WT RGS2 leads to ~ 50% inhibition of insulin induced 2-DOG uptake, without affecting IR Tyr phosphorylation. RGS2 constitutively associates with Galpha/q11, and prevent its Tyr phosphorylation and activation by insulin. Interestingly, insulin-stimulated PKClambda phosphorylation was completely blocked by RGS2, whereas, AKT phosphorylation was minimally inhibited. Neither the insulin receptor tyrosine phosphorylation nor insulin-stimulated MAPK phosphorylation was affected by RGS2. Conclusion: This study identifies a novel role of RGS2 in cellular insulin resistance by negatively regulating signaling through the Galpha/q11 pathway to glucose uptake. This research has received full or partial funding support from the American Heart Association, AHA Western States Affiliate (California, Nevada & Utah).

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Abstract 1185: Role Of Sphingosine-1-Phosphate (S1P) In Insulin Signaling.

Circulation ◽

10.1161/circ.116.suppl_16.ii_239-d ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

Suzanne M Nicholl ◽

Elisa Roztocil ◽

Mark G Davies

Keyword(s):

Insulin Resistance ◽

Skeletal Muscle ◽

Glucose Uptake ◽

Insulin Signaling ◽

Receptor Expression ◽

Serine Phosphorylation ◽

Glucose Disposal ◽

Sphingosine 1 Phosphate ◽

Low Glucose

A failure to increase glucose disposal into peripheral tissues in response to insulin leads to impaired insulin signaling and an inability to uptake glucose leading to the onset of insulin resistance, a major contributing factor to diabetes. We examined the role of sphingosine-1-phosphate (S1P) in insulin signaling and its ability to regulate glucose uptake in skeletal muscle cells. S1P, a sphingolipid found in abundance in the circulation, has been implicated in not only mediating crosstalk with other signaling pathways but has also been implicated in insulin resistance. We hypothesize that S1P interacts with post-receptor insulin signaling to increase glucose disposal in an in vitro model of insulin resistance using differentiated mouse skeletal C2C12 myotubes. Our data demonstrates that S1P (10μM) increases basal glucose levels similar to that observed in response to insulin (100nM) under conditions of low glucose (** p < 0.005: n = 3). Conversely, high glucose conditions completely inhibit both insulin and S1P stimulated glucose uptake (*p < 0.01:n = 3). Pre-incubation with S1P does not augment insulin-induced glucose uptake (***p < 0.001:n = 3), suggesting that S1P does not act via a separate signaling pathway. This is confirmed by our data demonstrating that S1P-induced glucose uptake is abrogated by Cytochalasin B (*p < 0.001:n = 3). In addition, the PI3-K inhibitors, LY294002 and Wortmannin, the Akt inhibitor, AKT2 and the p38MAPK inhibitor, SB203580 significantly inhibited glucose uptake in response to S1P, demonstrating their importance in S1P-induced glucose uptake (*p < 0.05:n = 3). S1P2 and S1P3 receptor expression were upregulated in response to insulin (~2-fold over basal) under low glucose conditions suggesting that insulin may regulate S1P signaling via one or both of these receptors. S1P increased serine phosphorylation of IRS1, both at serine 307 and serines 636/639 maximally after 15 minutes of stimulation. This data has important clinical implications in patients with metabolic syndrome who have impaired skeletal muscle glucose disposal due to insulin resistance and will help guide present and future therapy for patients who have this rapidly growing disease.

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