Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: A novel pharmacological strategy by PTEN inhibition for improving metabolic resuscitation and survival after mouse cardiac arrest

doi: 10.1152/ajpheart.00748.2014

Figure Lengend Snippet: 31P NMR of high energy phosphates and energetic status (PCr/ATP). A: representative 31P NMR spectrum indicates the relative high energy phosphate content in an isolated retrograde perfused sham-operated heart. The ratio of the area for the PCr resonance relative to the ATP is used here as a measure of the energetic reserve. B: relative to the shams (n = 4), the NS group (n = 5) showed an expected drop in energetic reserve 2 h after reperfusion (#P < 0.05). VO group (n = 5) recovered energetic reserve (#P < 0.05). Data presented are means ± SE. Piext, inorganic phosphate signal from extracellular buffer; PCr, phosphocreatine; ATP, adenosine triphosphate groups. C: p-pyruvate dehydrogenase (PDH) E1 α Ser293 at R30 in heart was analyzed by Western blot and shows an increase in the active form of PDH with VO treatment. Data presented are means ± SE of 5 mice.

Article Snippet: The protein phosphorylation and expression were detected with antibodies against phosphorylation of Akt Thr308, p-Akt Ser473, and GSK3β Ser9 (Cell Signaling Technology, Danvers, MA), pyruvate dehydrogenase (PDH) E1-α subunit (p Ser293; Novus Biologicals, Littleton, CO), phospholamban (p-PLB Thr17), sarco(endo)plasmic reticulum Ca 2+ -ATPase (SERCA2α; Santa Cruz Biotechnology, Dallas, TX), α-tubulin (NeoMarkers, Fremont, CA), and β-actin (Sigma-Aldrich, St. Louis, MO).

Techniques: Isolation, Western Blot

Journal: American journal of physiology. Endocrinology and metabolism

Article Title: Muscle expression of a malonyl-CoA-insensitive carnitine palmitoyltransferase-1 protects mice against high-fat/high-sucrose diet-induced insulin resistance.

doi: 10.1152/ajpendo.00020.2016

Figure Lengend Snippet: Fig. 7. Impact of muscle CPT1mt expression on muscle glucose metabolism in dbTg-HFHS mice. WT and dbTg mice fed Ctrl or HFHS diet for 16 wk were euthanized in the postprandial state after a 2-h fast. The gastrocnemius protein expression level of Glut4, phosphorylated (pS293PDH), and total pyruvate dehydrogenase (PDH), PDH kinases 1 and 4 (PDK1, PDK4) (A) as well as phosphorylated (pS641GS) and total glycogen synthase (GS; B) was analyzed by Western blotting using GAPDH as loading control. The same membranes as in Fig. 6A were used for Fig. 7B. Quantification and determination of the p-PDH/PDH and p-GS/GS ratios relative to WT-Ctrl mice are shown (n 5–6/group). C: gastrocnemius glycogen content in WT-Ctrl (n 9), WT-HFHS (n 9), and dbTg-HFHS (n 11) mice. Results are means SE. $P 0.05, $$P 0.01, $$$P 0.001, dbTg-HFHS vs. WT-Ctrl mice; *P 0.05, **P 0.01, dbTg-HFHS vs. WT-HFHS mice.

Article Snippet: Primary antibodies were directed against mouse total and phosphorylated (Ser473) Akt (cs-9272 and cs-9271, Cell Signaling Technology), rat CPT1A (30), rat CPT1B (a gift from VA Zammit, Warwick University, Warwick, UK), human CPT2 (4), human total and phosphorylated (Tyr202/204) ERK (cs-9102 and cs-9101, Cell Signaling Technology), mouse GAPDH (sc-25778; Santa Cruz Biotechnology), rabbit total and phosphorylated (Ser641) glycogen synthase (GS) (cs-3886 and cs-3891; Cell Signaling Technology), human total and phosphorylated (Ser9) GSK-3 (cs-9315 and cs-9336, Cell Signaling Technology), rat Glut4 (cs-2213, Cell Signaling Technology), human total PDH E1 subunit (MSP03-SP, MitoSciences), human phosphorylated (Ser293) PDH E1 subunit (ab110330, Abcam), human PDK1 (ab110025, Abcam), human PDK4 (ab71240, Abcam), and sea urchin -tubulin (Tyr5168, Sigma).

Techniques: Expressing, Western Blot, Control

Journal: American journal of physiology. Endocrinology and metabolism

Article Title: Muscle expression of a malonyl-CoA-insensitive carnitine palmitoyltransferase-1 protects mice against high-fat/high-sucrose diet-induced insulin resistance.

doi: 10.1152/ajpendo.00020.2016

Figure Lengend Snippet: Fig. 6. Impact of muscle CPT1mt expression on muscle insulin signaling in dbTg-HFHS mice. After feeding a Ctrl or HFHS diet for 16 wk, mice were euthanized in the postprandial state after a 2-h fast, and skeletal muscles were collected. A: gastrocnemius protein expression level of phosphorylated (pS473Akt, pY202/204ERK, pS9GSK-3) and total Akt, ERK, and GSK-3 was analyzed by Western blotting using GAPDH as loading control. Quantification and determination of the p-Akt/Akt, p-ERK/ERK, and p-GSK-3/GSK-3 ratios relative to WT-Ctrl mice are shown (n 5–6/group). B: ex vivo muscle insulin signaling analysis in isolated WT and dbTg soleus incubated at 30°C for 15 min in the absence (insulin) or presence (insulin) of 100 nM insulin. Representative Western blots of phosphorylated (pS473Akt) and total Akt are shown. Quantification was expressed relative to WT-Ctrl in the absence of insulin (n 5–6/group). Results are means SE. #P 0.05, WT-HFHS vs. WT-Ctrl mice; *P 0.05, dbTg-HFHS vs. WT-HFHS mice.

Article Snippet: Primary antibodies were directed against mouse total and phosphorylated (Ser473) Akt (cs-9272 and cs-9271, Cell Signaling Technology), rat CPT1A (30), rat CPT1B (a gift from VA Zammit, Warwick University, Warwick, UK), human CPT2 (4), human total and phosphorylated (Tyr202/204) ERK (cs-9102 and cs-9101, Cell Signaling Technology), mouse GAPDH (sc-25778; Santa Cruz Biotechnology), rabbit total and phosphorylated (Ser641) glycogen synthase (GS) (cs-3886 and cs-3891; Cell Signaling Technology), human total and phosphorylated (Ser9) GSK-3 (cs-9315 and cs-9336, Cell Signaling Technology), rat Glut4 (cs-2213, Cell Signaling Technology), human total PDH E1 subunit (MSP03-SP, MitoSciences), human phosphorylated (Ser293) PDH E1 subunit (ab110330, Abcam), human PDK1 (ab110025, Abcam), human PDK4 (ab71240, Abcam), and sea urchin -tubulin (Tyr5168, Sigma).

Techniques: Expressing, Muscles, Western Blot, Control, Ex Vivo, Isolation, Incubation

Journal: American journal of physiology. Endocrinology and metabolism

Article Title: Muscle expression of a malonyl-CoA-insensitive carnitine palmitoyltransferase-1 protects mice against high-fat/high-sucrose diet-induced insulin resistance.

doi: 10.1152/ajpendo.00020.2016

Figure Lengend Snippet: Fig. 7. Impact of muscle CPT1mt expression on muscle glucose metabolism in dbTg-HFHS mice. WT and dbTg mice fed Ctrl or HFHS diet for 16 wk were euthanized in the postprandial state after a 2-h fast. The gastrocnemius protein expression level of Glut4, phosphorylated (pS293PDH), and total pyruvate dehydrogenase (PDH), PDH kinases 1 and 4 (PDK1, PDK4) (A) as well as phosphorylated (pS641GS) and total glycogen synthase (GS; B) was analyzed by Western blotting using GAPDH as loading control. The same membranes as in Fig. 6A were used for Fig. 7B. Quantification and determination of the p-PDH/PDH and p-GS/GS ratios relative to WT-Ctrl mice are shown (n 5–6/group). C: gastrocnemius glycogen content in WT-Ctrl (n 9), WT-HFHS (n 9), and dbTg-HFHS (n 11) mice. Results are means SE. $P 0.05, $$P 0.01, $$$P 0.001, dbTg-HFHS vs. WT-Ctrl mice; *P 0.05, **P 0.01, dbTg-HFHS vs. WT-HFHS mice.

Article Snippet: Primary antibodies were directed against mouse total and phosphorylated (Ser473) Akt (cs-9272 and cs-9271, Cell Signaling Technology), rat CPT1A (30), rat CPT1B (a gift from VA Zammit, Warwick University, Warwick, UK), human CPT2 (4), human total and phosphorylated (Tyr202/204) ERK (cs-9102 and cs-9101, Cell Signaling Technology), mouse GAPDH (sc-25778; Santa Cruz Biotechnology), rabbit total and phosphorylated (Ser641) glycogen synthase (GS) (cs-3886 and cs-3891; Cell Signaling Technology), human total and phosphorylated (Ser9) GSK-3 (cs-9315 and cs-9336, Cell Signaling Technology), rat Glut4 (cs-2213, Cell Signaling Technology), human total PDH E1 subunit (MSP03-SP, MitoSciences), human phosphorylated (Ser293) PDH E1 subunit (ab110330, Abcam), human PDK1 (ab110025, Abcam), human PDK4 (ab71240, Abcam), and sea urchin -tubulin (Tyr5168, Sigma).

Techniques: Expressing, Western Blot, Control

Journal: PLoS ONE

Article Title: Akt1-mediated CPR cooling protection targets regulators of metabolism, inflammation and contractile function in mouse cardiac arrest

doi: 10.1371/journal.pone.0220604

Figure Lengend Snippet: A , C : IκBα and NF-κB at R30 in heart. α-tubulin was used as loading controls for heart. B , D : IκBα and NF-κB at R30 in brain. β-actin was used as loading controls for brain. * p < 0.05 between sham and NT groups of Akt1 +/+ mice; # p < 0.05 between NT and TH groups of Akt1 +/+ mice. Data presented are means ± SEM of five mice.

Article Snippet: Protein phosphorylation and expression were detected by antibodies against phosphorylation of Akt Thr308 (Cell Signaling Technology #9275, Danvers, MA), Akt Ser473 (Cell Signaling #4058), GSK3β Ser9 (Cell Signaling #9336), phospholamban Ser16/Thr17 (Cell Signaling #8496) and pyruvate dehydrogenase (PDH) E1-alpha subunit p-Ser293 (Novus Biologicals, NB110-93479, Littleton, CO), Akt1 (Cell Signaling #2967), Akt (Cell Signaling #9272), IκBα (Cell Signaling #9242), α-tubulin (used as a loading control in heart, NeoMarkers, MS581-P, Fremont, CA) and β-actin (used as a loading control in brain, Sigma-Aldrich, A5441 St. Louis, MO).

Techniques: