scholarly journals PDE5A Suppresses Proteasome Activity Leading to Insulin Resistance in C2C12 Myotubes

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Wei Liu ◽  
Xiaojun Tian ◽  
Ti Wu ◽  
Le Liu ◽  
Yanghongyun Guo ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Er Stress ◽  
Insulin Signaling ◽  
Specific Protein ◽  
Proteasome Activity ◽  
C2c12 Myotubes ◽  
Akt Phosphorylation ◽  
Phosphorylated Proteins ◽  
Activity Inhibition ◽  
Phosphodiesterase Type

Objective. The involvement of phosphodiesterase type 5 (PDE5) in the development of insulin resistance has been reported recently. However, the underlying molecular mechanism remains unclear. The present study aims at investigating the potential impacts of PDE5A on insulin signaling in C2C12 skeletal muscle myotubes and uncover the related mechanism. Methods. C2C12 myoblasts were differentiated into myotubes. Western blot was performed to detect the levels of proteins and phosphorylated proteins. Glucose uptake was determined by a colorimetric kit. The overexpression or knockdown of specific protein was carried out by infecting the myotubes with adenoviruses carrying cDNA or shRNA corresponding to the targeted protein, respectively. Results. PDE5A was demonstrated to negatively regulate insulin signaling, evidenced by the opposite effects on the suppression or enhancement of the insulin-stimulated Akt phosphorylation and 2-deoxy-D-glucose (2-DG) uptake in C2C12 myotubes, when PDE5A was overexpressed or knockdown, respectively. Interestingly, PDE5A overexpression led to significantly enhanced, while its knockdown resulted in markedly reduced, endoplasmic reticulum (ER) stress. Inhibition of ER stress improved PDE5A overexpression-induced insulin resistance. In addition, PDE5A was found to suppress proteasome activity. Inhibition of PDE5 by its selective inhibitor icariin restored PDE5A overexpression-reduced proteasome activity and mitigated PDE5A overexpression-induced ER stress. Consistently, icariin administration also markedly attenuated the detrimental impacts of PDE5A overexpression on insulin signaling. Conclusions. These results suggest that PDE5A suppresses proteasome activity, which results in ER stress and subsequent insulin resistance in C2C12 myotubes.

scholarly journals Lipid-Induced Endoplasmic Reticulum Stress in Liver Cells Results in Two Distinct Outcomes: Adaptation with Enhanced Insulin Signaling or Insulin Resistance

Endocrinology ◽  
2012 ◽  
Vol 153 (5) ◽  
pp. 2164-2177 ◽  
Author(s):  
Caroline S. Achard ◽  
D. Ross Laybutt
Keyword(s):  
Insulin Resistance ◽  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Insulin Signaling ◽  
Hepg2 Cells ◽  
Glycogen Synthesis ◽  
Liver Cells ◽  
Dependent Manner ◽  
Akt Phosphorylation ◽  
High Level

Chronically elevated fatty acids contribute to insulin resistance through poorly defined mechanisms. Endoplasmic reticulum (ER) stress and the subsequent unfolded protein response (UPR) have been implicated in lipid-induced insulin resistance. However, the UPR is also a fundamental mechanism required for cell adaptation and survival. We aimed to distinguish the adaptive and deleterious effects of lipid-induced ER stress on hepatic insulin action. Exposure of human hepatoma HepG2 cells or mouse primary hepatocytes to the saturated fatty acid palmitate enhanced ER stress in a dose-dependent manner. Strikingly, exposure of HepG2 cells to prolonged mild ER stress activation induced by low levels of thapsigargin, tunicamycin, or palmitate augmented insulin-stimulated Akt phosphorylation. This chronic mild ER stress subsequently attenuated the acute stress response to high-level palmitate challenge. In contrast, exposure of HepG2 cells or hepatocytes to severe ER stress induced by high levels of palmitate was associated with reduced insulin-stimulated Akt phosphorylation and glycogen synthesis, as well as increased expression of glucose-6-phosphatase. Attenuation of ER stress using chemical chaperones (trimethylamine N-oxide or tauroursodeoxycholic acid) partially protected against the lipid-induced changes in insulin signaling. These findings in liver cells suggest that mild ER stress associated with chronic low-level palmitate exposure induces an adaptive UPR that enhances insulin signaling and protects against the effects of high-level palmitate. However, in the absence of chronic adaptation, severe ER stress induced by high-level palmitate exposure induces deleterious UPR signaling that contributes to insulin resistance and metabolic dysregulation.

scholarly journals In vitro contraction protects against palmitate-induced insulin resistance in C2C12 myotubes

2017 ◽  
Vol 313 (5) ◽  
pp. C575-C583 ◽  
Author(s):  
Stephan Nieuwoudt ◽  
Anny Mulya ◽  
Ciarán E. Fealy ◽  
Elizabeth Martelli ◽  
Srinivasan Dasarathy ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Glucose Uptake ◽  
Insulin Signaling ◽  
Protective Role ◽  
Electrode System ◽  
C2c12 Myotubes ◽  
Noise Mapping ◽  
Whole Cell ◽  
Akt Phosphorylation

We are interested in understanding mechanisms that govern the protective role of exercise against lipid-induced insulin resistance, a key driver of type 2 diabetes. In this context, cell culture models provide a level of abstraction that aid in our understanding of cellular physiology. Here we describe the development of an in vitro myotube contraction system that provides this protective effect, and which we have harnessed to investigate lipid-induced insulin resistance. C2C12 myocytes were differentiated into contractile myotubes. A custom manufactured platinum electrode system and pulse stimulator, with polarity switching, provided an electrical pulse stimulus (EPS) (1 Hz, 6-ms pulse width, 1.5 V/mm, 16 h). Contractility was assessed by optical flow flied spot noise mapping and inhibited by application of ammonium acetate. Following EPS, myotubes were challenged with 0.5 mM palmitate for 4 h. Cells were then treated with or without insulin for glucose uptake (30 min), secondary insulin signaling activation (10 min), and phosphoinositide 3-kinase-α (PI3Kα) activity (5 min). Prolonged EPS increased non-insulin-stimulated glucose uptake (83%, P = 0.002), Akt (Thr308) phosphorylation ( P = 0.005), and insulin receptor substrate-1 (IRS-1)-associated PI3Kα activity ( P = 0.048). Palmitate reduced insulin-specific action on glucose uptake (−49%, P < 0.001) and inhibited insulin-stimulated Akt phosphorylation ( P = 0.049) and whole cell PI3Kα activity ( P = 0.009). The inhibitory effects of palmitate were completely absent with EPS pretreatment at the levels of glucose uptake, insulin responsiveness, Akt phosphorylation, and whole cell PI3Kα activity. This model suggests that muscle contraction alone is a sufficient stimulus to protect against lipid-induced insulin resistance as evidenced by changes in the proximal canonical insulin-signaling pathway.

scholarly journals Imoxin inhibits tunicamycin-induced endoplasmic reticulum stress and restores insulin signaling in C2C12 myotubes.

2021 ◽  
Author(s):  
Hyeyoon Eo ◽  
Rudy J. Valentine
Keyword(s):  
Skeletal Muscle ◽  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Insulin Signaling ◽  
Binding Protein ◽  
C2c12 Myotubes ◽  
Protein Kinase R ◽  
Akt Phosphorylation ◽  
Protein Levels ◽  
Activating Transcription Factor 4

Prolonged endoplasmic reticulum (ER) stress can mediate inflammatory myopathies and insulin signaling pathways. The double stranded RNA (dsRNA) activated protein kinase R (PKR) has been implicated in skeletal muscle dysfunction. However, pathological roles of PKR in ER stress in muscle are not fully understood. The current study aimed to investigate the effect of imoxin (IMX), a selective PKR inhibitor, on tunicamycin (TN)-induced promotion of ER stress and suppression of insulin signaling in C2C12 myotubes. Cells were pre-treated with 5 uM IMX for 1 hr, and exposed to 0.5 µg/ml TN for 23 hr. A subset of cells was stimulated with 100 nM insulin for the last 15 min. mRNA expression and protein levels involved in ER stress were measured by RT-PCR and Western blotting, respectively. TN significantly augmented PKR phosphorylation by 231%, which was prevented by IMX. In addition, IMX reduced mRNA and protein levels of ER stress-related markers including CCAAT-enhancer-binding protein homologous protein (CHOP, mRNA: 95% decrease; protein: 98% decrease), activating transcription factor 4 (ATF4, mRNA: 69% decrease; protein: 99% decrease), cleavage of ATF6, and spliced X-box binding protein 1 (XBP-1s, mRNA: 88% decrease; protein: 79% decrease) which were induced by TN. Furthermore, IMX ameliorated TN-induced suppression of phospho-insulin receptor beta (317% increase) and Akt phosphorylation (by 36% at Ser473 and 30% at Thr308) in myotubes, while augmenting insulin-stimulated AS160 phosphorylation and glucose uptake (by ~30%). These findings suggest that IMX may protect against TN-induced skeletal muscle ER stress and insulin resistance, which are potentially mediated by PKR.

scholarly journals Palmitate Attenuates Insulin Signaling and Induces Endoplasmic Reticulum Stress and Apoptosis in Hypothalamic Neurons: Rescue of Resistance and Apoptosis through Adenosine 5′ Monophosphate-Activated Protein Kinase Activation

Endocrinology ◽  
2010 ◽  
Vol 151 (2) ◽  
pp. 576-585 ◽  
Author(s):  
Christopher M. Mayer ◽  
Denise D. Belsham
Keyword(s):  
Insulin Resistance ◽  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Insulin Signaling ◽  
Caspase 3 ◽  
Saturated Fatty Acids ◽  
Amp Kinase ◽  
Hypothalamic Neurons ◽  
Peripheral Tissues ◽  
Short Term Exposure

Hypothalamic insulin signaling is essential to the maintenance of glucose and energy homeostasis. During pathological states, such as obesity and type 2 diabetes mellitus, insulin signaling is impaired. One key mechanism involved in the development of insulin resistance is lipotoxicity, through increased circulating saturated fatty acids. Although many studies have begun to determine the underlying mechanisms of lipotoxicity in peripheral tissues, little is known about the effects of excess lipids in the brain. We used a hypothalamic, neuronal cell model, mHypoE-44, to understand how the highly prevalent nonesterified fatty acid, palmitate, affects neuronal insulin signaling. Through Western blot analysis, we discerned that prolonged exposure to palmitate impairs insulin activation, as assessed by phosphorylation of Akt. We investigated the role of endoplasmic reticulum (ER) stress, which is known to promote cellular insulin resistance and apoptosis in peripheral tissues. Palmitate treatment induced ER stress through a c-Jun N-terminal kinase (JNK)-dependent pathway because a selective JNK inhibitor blocked palmitate activation of the ER stress pathways eIF2α and X-box binding protein-1. Interestingly, JNK inhibition did not prevent the palmitate-mediated cleaved caspase-3 increase, an apoptotic marker, or insulin signaling attenuation. However, pretreatment with the AMP kinase activator, aminoimidazole carboxamide ribonucleotide, blocked JNK phosphorylation and importantly prevented caspase-3 cleavage and restored insulin signaling during short-term exposure to palmitate. Thus, activation of AMP kinase prevents the deleterious effects of palmitate on hypothalamic neurons by inhibiting the onset of insulin resistance and apoptosis.

scholarly journals Low-dose spironolactone reduces reactive oxygen species generation and improves insulin-stimulated glucose transport in skeletal muscle in the TG(mRen2)27 rat

2008 ◽  
Vol 295 (1) ◽  
pp. E110-E116 ◽  
Author(s):  
Guido Lastra ◽  
Adam Whaley-Connell ◽  
Camila Manrique ◽  
Javad Habibi ◽  
Alex A. Gutweiler ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Nadph Oxidase ◽  
Soleus Muscle ◽  
Insulin Signaling ◽  
Oxidase Activity ◽  
Low Dose ◽  
Ros Production ◽  
Oxygen Species ◽  
Akt Phosphorylation ◽  
Subunit Expression

Renin-angiotensin-aldosterone system (RAAS) activation mediates increases in reactive oxygen species (ROS) and impaired insulin signaling. The transgenic Ren2 rat manifests increased tissue renin-angiotensin system activity, elevated serum aldosterone, hypertension, and insulin resistance. To explore the role of aldosterone in the pathogenesis of insulin resistance, we investigated the impact of in vivo treatment with a mineralocorticoid receptor (MR) antagonist on insulin sensitivity in Ren2 and aged-matched Sprague-Dawley (SD) control rats. Both groups (age 6–8 wk) were implanted with subcutaneous time-release pellets containing spironolactone (0.24 mg/day) or placebo over 21 days. Systolic blood pressure (SBP) and intraperitoneal glucose tolerance test were determined. Soleus muscle insulin receptor substrate-1 (IRS-1), tyrosine phosphorylated IRS-1, protein kinase B (Akt) phosphorylation, GLUT4 levels, and insulin-stimulated 2-deoxyglucose uptake were evaluated in relation to NADPH subunit expression/oxidase activity and ROS production (chemiluminescence and 4-hydroxy-2-nonenal immunostaining). Along with increased soleus muscle NADPH oxidase activity and ROS, there was systemic insulin resistance and reduced muscle IRS-1 tyrosine phosphorylation, Akt phosphorylation/activation, and GLUT4 expression in the Ren2 group (each P < 0.05). Despite not decreasing blood pressure, low-dose spironolactone treatment improved soleus muscle insulin signaling parameters and systemic insulin sensitivity in concert with reductions in NADPH oxidase subunit expression/activity and ROS production (each P < 0.05). Our findings suggest that aldosterone contributes to insulin resistance in the transgenic Ren2, in part, by increasing NADPH oxidase activity in skeletal muscle tissue.

scholarly journals Oleate Blocks Palmitate-Induced Abnormal Lipid Distribution, Endoplasmic Reticulum Expansion and Stress, and Insulin Resistance in Skeletal Muscle

Endocrinology ◽  
2011 ◽  
Vol 152 (6) ◽  
pp. 2206-2218 ◽  
Author(s):  
Gong Peng ◽  
Linghai Li ◽  
Yanbo Liu ◽  
Jing Pu ◽  
Shuyan Zhang ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Skeletal Muscle ◽  
Fatty Acids ◽  
Endoplasmic Reticulum ◽  
Insulin Sensitivity ◽  
Er Stress ◽  
Molecular Mechanisms ◽  
Dietary Fatty Acids ◽  
Akt Phosphorylation ◽  
Stress Relieving

Pathological elevation of plasma fatty acids reduces insulin sensitivity. Although several regulation pathways have been reported, the molecular mechanisms of insulin sensitivity remain elusive, especially in skeletal muscle where most glucose is consumed. This study focuses on how two major dietary fatty acids affect insulin signaling in skeletal muscle cells. Palmitic acid (PA) not only reduced insulin-stimulated phosphorylation of Akt but also induced endoplasmic reticulum (ER) expansion and ER stress. Relieving ER stress using 4-phenyl butyric acid blocked PA-mediated protein kinase R-like ER kinase phosphorylation and ER expansion and reversed the inhibitory effect of PA on insulin-stimulated Akt phosphorylation. Importantly, oleic acid (OA) could also recover PA-reduced Akt phosphorylation and abolish both PA-mediated ER expansion and ER stress. The competition between these two fatty acids was further verified in rat skeletal muscle using venous fatty acid infusion. 3H-labeled PA was converted mainly to active lipids (phospholipids and diacylglycerol) in the absence of OA, but to triacylglycerol in the presence of OA. Subcellular triacylglycerol and adipocyte differentiation-related protein from PA-treated cells cofractionated with the ER in the absence of OA but switched to the low-density fraction in the presence of OA. Taken together, these data suggest that the PA-mediated lipid composition and localization may cause ER expansion and consequently cause ER stress and insulin resistance in skeletal muscle.

Abstract 308: Rgs2 Is An Endogenous Inhibitor Of Insulin Signaling

Circulation ◽  
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).

Hydrogen Peroxide Inhibits Insulin Signaling in Vascular Smooth Muscle Cells

2003 ◽  
Vol 228 (7) ◽  
pp. 836-842 ◽  
Author(s):  
Carla D. Gardner ◽  
Satoru Eguchi ◽  
Cherilynn M. Reynolds ◽  
Kunie Eguchi ◽  
Gerald D. Frank ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Smooth Muscle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Smooth Muscle Cells ◽  
Vascular Smooth Muscle Cells ◽  
Insulin Signaling ◽  
Akt Phosphorylation ◽  
Akt Activation ◽  
Kinase C

Both insulin resistance and reactive oxygen species (ROS) have been reported to play essential pathophysiological roles in cardiovascular diseases, such as hypertension and atherosclerosis. However, the mechanistic link between ROS, such as H2O2 and insulin resistance in the vasculature, remains undetermined. Akt, a Ser/Thr kinase, mediates various biological responses induced by insulin. In this study, we examined the effects of H2O2 on Akt activation in the insulin-signaling pathway in vascular smooth muscle cells (VSMCs). In VSMCs, insulin stimulates Akt phosphorylation at Ser473. Pretreatment with H2O2 concentration- and time-dependently inhibited insulin-induced Akt phosphorylation with significant inhibition observed at 50 μM for 10 min. A ROS inducer, diamide, also inhibited insulin-induced Akt phosphorylation. In addition, H2O2 inhibited insulin receptor binding partially and inhibited insulin receptor autophosphorylation almost completely. However, pretreatment with a protein kinase C inhibitor, GF109203X (2 μM), for 30 min did not block the inhibitory effects of H2O2 on insulin-induced Akt phosphorylation, suggesting that protein kinase C is not involved in the inhibition by H2O2. We conclude that ROS inhibit a critical insulin signal transduction component required for Akt activation in VSMCs, suggesting potential cellular mechanisms of insulin resistance, which would require verification in vivo.

scholarly journals Increased basal level of Akt-dependent insulin signaling may be responsible for the development of insulin resistance

2009 ◽  
Vol 297 (4) ◽  
pp. E898-E906 ◽  
Author(s):  
Hui-Yu Liu ◽  
Tao Hong ◽  
Ge-Bo Wen ◽  
Jianmin Han ◽  
Degen Zuo ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Insulin Resistance ◽  
Insulin Sensitivity ◽  
Insulin Signaling ◽  
Kinase Inhibitor ◽  
Phosphatidylinositol 3 Kinase ◽  
Akt Phosphorylation ◽  
Glucose Levels ◽  
Blood Glucose Levels ◽  
Triglyceride Content

A majority of subjects with insulin resistance and hyperinsulinemia can maintain their blood glucose levels normal for the whole life presumably through protein kinase B (Akt)-dependent insulin signaling. In this study, we found that the basal Akt phosphorylation level was increased in liver and gastrocnemius of mice under the high-fat diet (HFD). Levels of mitochondrial DNA and expression of some mitochondrion-associated genes were decreased by the HFD primarily in liver. Triglyceride content was increased in both liver and gastrocnemius by the HFD. Oxidative stress was induced by the HFD in both liver and gastrocnemius. Insulin sensitivity was decreased by the HFD. All of these changes were largely or completely reversed by treatment of animals with the phosphatidylinositol 3-kinase inhibitor LY-294002 during the time when animals usually do not eat. Consequently, the overall insulin sensitivity was increased by treatment with LY-294002. Together, our results indicate that increased basal Akt-dependent insulin signaling suppresses mitochondrial production, increases ectopic fat accumulation, induces oxidative stress, and desensitizes insulin signaling in subjects with insulin resistance and hyperinsulinemia.

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