scholarly journals PPARα, δ and FOXO1 Gene Silencing Overturns Palmitate-Induced Inhibition of Pyruvate Oxidation Differentially in C2C12 Myotubes

Biology ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 1098
Author(s):  
Hung-Che Chien ◽  
Despina Constantin ◽  
Paul L. Greenhaff ◽  
Dumitru Constantin-Teodosiu
Keyword(s):  
Transcription Factors ◽  
Protein Expression ◽  
Gene Silencing ◽  
Glucose Uptake ◽  
Glucose Oxidation ◽  
Molecular Mechanisms ◽  
C2c12 Myotubes ◽  
Atp Production ◽  
Pyruvate Oxidation ◽  
Metabolic Inflexibility

The molecular mechanisms by which free fatty acids (FFA) inhibit muscle glucose oxidation is still elusive. We recently showed that C2C12 myotubes treated with palmitate (PAL) presented with greater protein expression levels of PDK4 and transcription factors PPARα and PPARδ and lower p-FOXO/t-FOXO protein ratios when compared to control. This was complemented with the hallmarks of metabolic inflexibility (MI), i.e., reduced rates of glucose uptake, PDC activity and maximal pyruvate-derived ATP production rates (MAPR). However, the relative contribution of these transcription factors to the increase in PDK4 and reduced glucose oxidation could not be established. Therefore, by using a similar myotube model, a series of individual siRNA gene silencing experiments, validated at transcriptional and translation levels, were performed in conjunction with measurements of glucose uptake, PDC activity, MAPR and concentrations of metabolites reflecting PDC flux (lactate and acetylcarnitine). Gene silencing of PPARα, δ and FOXO1 individually reduced PAL-mediated inhibition of PDC activity and increased glucose uptake, albeit by different mechanisms as only PPARδ and FOXO1 silencing markedly reduced PDK4 protein content. Additionally, PPARα and FOXO1 silencing, but not PPARδ, increased MAPR with PAL. PPARδ silencing also decreased FOXO1 protein. Since FOXO1 silencing did not alter PPARδ protein, this suggests that FOXO1 might be a PPARδ downstream target. In summary, this study suggests that the molecular mechanisms by which PAL reduces PDC-mediated glucose-derived pyruvate oxidation in muscle occur primarily through increased PPARδ and FOXO1 mediated increases in PDK4 protein expression and secondarily through PPARα mediated allosteric inhibition of PDC flux. Furthermore, since PPARδ seems to control FOXO1 expression, this may reflect an important role for PPARδ in preventing glucose oxidation under conditions of increased lipid availability.

scholarly journals PPARδ and FOXO1 Mediate Palmitate-Induced Inhibition of Muscle Pyruvate Dehydrogenase Complex and CHO Oxidation, Events Reversed by Electrical Pulse Stimulation

2020 ◽  
Vol 21 (16) ◽  
pp. 5942
Author(s):  
Hung-Che Chien ◽  
Paul L. Greenhaff ◽  
Dumitru Constantin-Teodosiu
Keyword(s):  
Transcription Factors ◽  
Glucose Uptake ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Peroxisome Proliferator ◽  
Peroxisome Proliferator Activated Receptor ◽  
Atp Production ◽  
Electrical Pulse Stimulation ◽  
Pulse Stimulation ◽  
Dehydrogenase Complex

The mechanisms behind the reduction in muscle pyruvate dehydrogenase complex (PDC)-controlled carbohydrate (CHO) oxidation during chronic high-fat dietary intake are poorly understood, as is the basis of CHO oxidation restoration during muscle contraction. C2C12 myotubes were treated with (300 μM) palmitate or without (control) for 16 h in the presence and absence of electrical pulse stimulation (EPS, 11.5 V, 1 Hz, 2 ms). Compared to control, palmitate reduced cell glucose uptake (p < 0.05), PDC activity (p < 0.01), acetylcarnitine accumulation (p < 0.05) and glucose-derived mitochondrial ATP production (p < 0.01) and increased pyruvate dehydrogenase kinase isoform 4 (PDK4) (p < 0.01), peroxisome proliferator-activated receptor alpha (PPARα) (p < 0.01) and peroxisome proliferator-activated receptor delta (PPARδ) (p < 0.01) proteins, and reduced the whole-cell p-FOXO1/t-FOXO1 (Forkhead Box O1) ratio (p < 0.01). EPS rescued palmitate-induced inhibition of CHO oxidation, reflected by increased glucose uptake (p < 0.01), PDC activity (p < 0.01) and glucose-derived mitochondrial ATP production (p < 0.01) compared to palmitate alone. EPS was also associated with less PDK4 (p < 0.01) and PPARδ (p < 0.01) proteins, and lower nuclear p-FOXO1/t-FOXO1 ratio normalised to the cytoplasmic ratio, but with no changes in PPARα protein. Collectively, these data suggest PPARδ, and FOXO1 transcription factors increased PDK4 protein in the presence of palmitate, which limited PDC activity and flux, and blunted CHO oxidation and glucose uptake. Conversely, EPS rescued these metabolic events by modulating the same transcription factors.

Abstract 313: Elucidation Of The Fibrotic Transcription Factors Gene Network Leading To Chagas Heart Disease

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Pius N Nde ◽  
Aniekanabassi N Udoko ◽  
Candice A Johnson ◽  
Andrey Dykan ◽  
Girish Rachakonda ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Heart Disease ◽  
Protein Expression ◽  
Molecular Mechanisms ◽  
Cardiac Fibrosis ◽  
P Value ◽  
Expression Levels ◽  
Expression Of Genes ◽  
Chagas Heart Disease ◽  
Whole Transcriptome

Background: Trypanosoma cruzi the causative agent of Chagas heart disease (CHD) remains incurable. The major pathology induced by the parasite is cardiac fibrosis leading to heart failure followed by death. The mechanisms of T. cruzi induced cardio-pathology remains largely unknown. We hypothesize that T. cruzi infection regulates the expression of profibrotic genes in human cardiac myocytes (HCM), tilting the heart towards a profibrotic phenotype seen in CHD patients. Methods and Results: To elucidate the molecular mechanisms of T. cruzi induced cardiac fibrosis, we challenged primary HCM with T. cruzi for two hours and purified total RNA for microarray. We investigated changes at the whole transcriptome level on an affymetrix platform. The arrays were done in triplicates at different time points; changes in gene expression greater than 2-fold and having a Benjamini and Hochburg false discovery rate corrected p-value <0.05 were considered significant. The microarray data was validated using real-time PCR followed by PCR array and immunoblotting, to evaluate changes in the protein expression levels of fibrotic transcription factors. Protein expression levels were evaluated in triplicate and analyzed by ANOVA. The fibrotic interactome induced by T. cruzi in HCM was elucidated using Cytoscape. Our results indicate that exposure of HCM to T. cruzi upregulates the transcript levels of two transcription factors associated with fibrosis, SNAI1 (more than 2 fold up-regulated) and Early Growth Response protein 1, EGR1, (about four fold up-regulated). SNAI1 and EGR1 were increased at the protein level. Furthermore, we identified the first interactome regulating fibrosis in primary HCM induced by T. cruzi . Conclusions: This is the first report showing that T. cruzi upregulates the expression of profibrotic transcription factors in HCM early during the process of cellular infection and the operational fibrotic interactome. Thus, abnormal sustained expression of SNAI1 and EGR1 upregulate the expression of genes essential for conversion of HCM towards a profibrotic phenotype in CHD. Elucidation of the molecular mechanisms by which T. cruzi induces cardiac fibrosis will lead to the identification of new therapeutic targets for CHD.

scholarly journals Exercise Counterbalances Rho/ROCK2 Signaling Impairment in the Skeletal Muscle and Ameliorates Insulin Sensitivity in Obese Mice

2021 ◽  
Vol 12 ◽  
Author(s):  
Vitor R. Muñoz ◽  
Rafael C. Gaspar ◽  
Matheus B. Severino ◽  
Ana P. A. Macêdo ◽  
Fernando M. Simabuco ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Insulin Sensitivity ◽  
Physical Exercise ◽  
Glucose Uptake ◽  
Molecular Mechanisms ◽  
Tyrosine Phosphatase ◽  
C2c12 Myotubes ◽  
Obese Mice ◽  
Skeletal Muscle Glucose Uptake ◽  
The Impact

Physical exercise is considered a fundamental strategy in improving insulin sensitivity and glucose uptake in skeletal muscle. However, the molecular mechanisms underlying this regulation, primarily on skeletal muscle glucose uptake, are not fully understood. Recent evidence has shown that Rho-kinase (ROCK) isoforms play a pivotal role in regulating skeletal muscle glucose uptake and systemic glucose homeostasis. The current study evaluated the effect of physical exercise on ROCK2 signaling in skeletal muscle of insulin-resistant obese animals. Physiological (ITT) and molecular analysis (immunoblotting, and RT-qPCR) were performed. The contents of RhoA and ROCK2 protein were decreased in skeletal muscle of obese mice compared to control mice but were restored to normal levels in response to physical exercise. The exercised animals also showed higher phosphorylation of insulin receptor substrate 1 (IRS1 Serine 632/635) and protein kinase B (Akt) in the skeletal muscle. However, phosphatase and tensin homolog (PTEN) and protein-tyrosine phosphatase-1B (PTP-1B), both inhibitory regulators for insulin action, were increased in obesity but decreased after exercise. The impact of ROCK2 action on muscle insulin signaling is further underscored by the fact that impaired IRS1 and Akt phosphorylation caused by palmitate in C2C12 myotubes was entirely restored by ROCK2 overexpression. These results suggest that the exercise-induced upregulation of RhoA-ROCK2 signaling in skeletal muscle is associated with increased systemic insulin sensitivity in obese mice and further implicate that muscle ROCK2 could be a potential target for treating obesity-linked metabolic disorders.

scholarly journals TNF-αand IFN-s-Dependent Muscle Decay Is Linked to NF-κB- and STAT-1α-StimulatedAtrogin1andMuRF1Genes in C2C12 Myotubes

2013 ◽  
Vol 2013 ◽  
pp. 1-18 ◽  
Author(s):  
Barbara Pijet ◽  
Maja Pijet ◽  
Anna Litwiniuk ◽  
Małgorzata Gajewska ◽  
Beata Pająk ◽  
...  
Keyword(s):  
Protein Expression ◽  
Signaling Pathways ◽  
Molecular Mechanisms ◽  
C2c12 Cells ◽  
Metabolic Inhibitors ◽  
C2c12 Myotubes ◽  
Protein Expression Level ◽  
C2c12 Myoblasts ◽  
Stat Signaling ◽  
Increased Activity

TNF-αwas shown to stimulate mitogenicity in C2C12 myoblasts. Selected cytokines TNF-α, IFNα, or IFNγreduced the expression of myosin heavy chain (MyHC IIa) when given together. Molecular mechanisms of cytokine activities were controlled by NF-κB and JAK/STAT signaling pathways, as metabolic inhibitors, curcumin and AG490, inhibited some of TNF-αand IFNα/IFNγeffects. Insulin was hardly antagonistic to TNF-α- and IFNα/IFNγ-dependent decrease in MyHC IIa protein expression. Cytokines used individually or together also repressed myogenesis of C2C12 cells. Moreover, TNF-α- and IFNα/IFNγ-dependent effects on C2C12 myotubes were associated with increased activity ofAtrogin1andMuRF1genes, which code ubiquitin ligases.MyHC IIagene activity was unaltered by cytokines. Inhibition of NF-κB or JAK/STAT with specific metabolic inhibitors decreased activity ofAtrogin1andMuRF1but notMyHC IIagene. Overall, these results suggest cooperation between cytokines in the reduction of MyHC IIa protein expression level via NF-κB/JAK/STAT signaling pathways and activation ofAtrogin1andMuRF1genes as their molecular targets. Insulin cotreatment or pretreatment does not protect against muscle decay induced by examined proinflammatory cytokines.

scholarly journals Gossypol from Cottonseeds Ameliorates Glucose Uptake by Mimicking Insulin Signaling and Improves Glucose Homeostasis in Mice with Streptozotocin-Induced Diabetes

2018 ◽  
Vol 2018 ◽  
pp. 1-11 ◽  
Author(s):  
Md Badrul Alam ◽  
Hongyan An ◽  
Jeong-Sic Ra ◽  
Ji-young Lim ◽  
Seung-Hyun Lee ◽  
...  
Keyword(s):  
Glucose Uptake ◽  
Glucose Homeostasis ◽  
Molecular Mechanisms ◽  
Glucose Transporter ◽  
Specific Inhibitor ◽  
Competitive Inhibitor ◽  
Postprandial Blood Glucose ◽  
C2c12 Myotubes

Glucose absorption from the gut and glucose uptake into muscles are vital for the regulation of glucose homeostasis. In the current study, we determined if gossypol (GSP) reduces postprandial hyperglycemia or enhances glucose uptake; we also investigated the molecular mechanisms underlying those processes in vitro and in vivo. GSP strongly and concentration dependently inhibited α-glucosidase by functioning as a competitive inhibitor with IC50 value of 0.67 ± 0.44. GSP activated the insulin receptor substrate 1 (IRS-1)/protein kinase B (Akt) signaling pathways and enhanced glucose uptake through the translocation of glucose transporter 4 (GLUT4) into plasma membrane in C2C12 myotubes. Pretreatment with a specific inhibitor attenuated the in vitro effects of GSP. We used a streptozotocin-induced diabetic mouse model to assess the antidiabetic potential of GSP. Consistent with the in vitro study, a higher dose of GSP (2.5 mg/kg−1) dramatically decreased the postprandial blood glucose levels associated with the upregulated expressions of GLUT4 and the IRS-1/Akt-mediated signaling cascade in skeletal muscle. GSP treatment also significantly boosted antioxidant enzyme expression and mitigated gluconeogenesis in the liver. Collectively, these data imply that GSP has the potential in managing and preventing diabetes by ameliorating glucose uptake and improving glucose homeostasis.

Active components isolated from Trapa natants increase glucose uptake in C2C12 myotubes

Planta Medica ◽  
2016 ◽  
Vol 81 (S 01) ◽  
pp. S1-S381
Author(s):  
HC Huang ◽  
CL Chao ◽  
SY Hwang ◽  
TC Chang ◽  
CH Chao ◽  
...  
Keyword(s):  
Glucose Uptake ◽  
C2c12 Myotubes ◽  
Active Components ◽  
Increase Glucose Uptake

Inhibition of Migration and Invasion of Hepatocarcinoma HepG2 Cells by Dietary Saponins via Targeting HIF-1α

2018 ◽  
Vol 18 (7) ◽  
pp. 1025-1031
Author(s):  
Cheng Luo ◽  
Di Wu ◽  
Meiling Chen ◽  
Wenhua Miao ◽  
Changfeng Xue ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Confocal Microscopy ◽  
Protein Expression ◽  
Mtt Assay ◽  
Hepg2 Cells ◽  
Molecular Mechanisms ◽  
Migration And Invasion ◽  
Rt Pcr ◽  
Gene And Protein Expression ◽  
Simulated Hypoxia

Background: Different saponins from herbs have been used as tonic or functional foods, and for treatment of various diseases including cancers. Although clinical data has supported the function of these saponins, their underlying molecular mechanisms have not been well defined. Methods: With the simulated hypoxia created by 8 hours of Cu++ exposure and following 24 hour incubation with different concentration of saponins in HepG2 cells for MTT assay, migration and invasion assays, and for RT-PCR, and with each group of cells for immunofluorescence observation by confocal microscopy. Results： ZC-4 had the highest rate of inhibition of cell proliferation by MTT assay, and the highest inhibition of migration rate by in vitro scratch assay, while ZC-3 had the highest inhibition of invasion ratio by transwell assay. Under the same simulated hypoxia, the molecular mechanism of saponin function was conducted by measuring the gene expression of Hypoxia Inducible Factor (HIF)-1α through RT-PCR, in which ZC-3 showed a potent inhibition of gene HIF-1α. For the protein expression by immunofluorescence staining with confocal microscopy, HIF-1α was also inhibited by saponins, with the most potent one being ZC-4 after eight hours’ relatively hypoxia incubation. Conclusion: Saponins ZC-4 and ZC-3 have the potential to reduce HepG2 cell proliferation, migration and invasion caused by hypoxia through effectively inhibiting the gene and protein expression of HIF-1α directly and as antioxidant indirectly

scholarly journals Low-density lipoprotein receptor-related protein 1 (LRP1) is a novel receptor for apolipoprotein A4 (APOA4) in adipose tissue

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jie Qu ◽  
Sarah Fourman ◽  
Maureen Fitzgerald ◽  
Min Liu ◽  
Supna Nair ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Glucose Uptake ◽  
Molecular Mechanisms ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Lipoprotein Receptor ◽  
Dependent Manner ◽  
Related Protein ◽  
Density Lipoprotein Receptor ◽  
Lipoprotein Receptor Related Protein

AbstractApolipoprotein A4 (APOA4) is one of the most abundant and versatile apolipoproteins facilitating lipid transport and metabolism. APOA4 is synthesized in the small intestine, packaged onto chylomicrons, secreted into intestinal lymph and transported via circulation to several tissues, including adipose. Since its discovery nearly 4 decades ago, to date, only platelet integrin αIIbβ3 has been identified as APOA4 receptor in the plasma. Using co-immunoprecipitation coupled with mass spectrometry, we probed the APOA4 interactome in mouse gonadal fat tissue, where ApoA4 gene is not transcribed but APOA4 protein is abundant. We demonstrate that lipoprotein receptor-related protein 1 (LRP1) is the cognate receptor for APOA4 in adipose tissue. LRP1 colocalized with APOA4 in adipocytes; it interacted with APOA4 under fasting condition and their interaction was enhanced during lipid feeding concomitant with increased APOA4 levels in plasma. In 3T3-L1 mature adipocytes, APOA4 promoted glucose uptake both in absence and presence of insulin in a dose-dependent manner. Knockdown of LRP1 abrogated APOA4-induced glucose uptake as well as activation of phosphatidylinositol 3 kinase (PI3K)-mediated protein kinase B (AKT). Taken together, we identified LRP1 as a novel receptor for APOA4 in promoting glucose uptake. Considering both APOA4 and LRP1 are multifunctional players in lipid and glucose metabolism, our finding opens up a door to better understand the molecular mechanisms along APOA4-LRP1 axis, whose dysregulation leads to obesity, cardiovascular disease, and diabetes.

scholarly journals The Hormetic Effect of Metformin: “Less Is More”?

2021 ◽  
Vol 22 (12) ◽  
pp. 6297
Author(s):  
Isabella Panfoli ◽  
Alessandra Puddu ◽  
Nadia Bertola ◽  
Silvia Ravera ◽  
Davide Maggi
Keyword(s):  
Complex I ◽  
Molecular Mechanisms ◽  
Hepatic Glucose Production ◽  
Glucose Production ◽  
Target Tissue ◽  
Atp Production ◽  
Less Is More ◽  
First Line Therapy ◽  
Hormetic Effect ◽  
Intracellular Content

Metformin (MTF) is the first-line therapy for type 2 diabetes (T2DM). The euglycemic effect of MTF is due to the inhibition of hepatic glucose production. Literature reports that the principal molecular mechanism of MTF is the activation of 5′-AMP-activated protein kinase (AMPK) due to the decrement of ATP intracellular content consequent to the inhibition of Complex I, although this effect is obtained only at millimolar concentrations. Conversely, micromolar MTF seems to activate the mitochondrial electron transport chain, increasing ATP production and limiting oxidative stress. This evidence sustains the idea that MTF exerts a hormetic effect based on its concentration in the target tissue. Therefore, in this review we describe the effects of MTF on T2DM on the principal target organs, such as liver, gut, adipose tissue, endothelium, heart, and skeletal muscle. In particular, data indicate that all organs, except the gut, accumulate MTF in the micromolar range when administered in therapeutic doses, unmasking molecular mechanisms that do not depend on Complex I inhibition.

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