Journal: bioRxiv

Article Title: Reconstituted high-density lipoproteins rescue diabetes-impaired endothelial cell metabolic reprograming and angiogenic responses to hypoxia

doi: 10.1101/2024.08.16.608361

Figure Lengend Snippet: Two full-thickness wounds were created on non-diabetic and diabetic C57Bl/6J mice. Mice received daily topical applications of rHDL (50 μg/wound) or PBS (vehicle). Wound tissue was collected from non-diabetic (white and grey circles) and diabetic (red and blue triangles) mice at days 0, 1, 3, and 10 post-wound creation. (A) PDK4 protein expression at days 0, 1 and 3 post-wounding was measured in wound lysates by ELISA (n=4-12) * P <0.05, ** P <0.01 vs controls by two-way ANOVA. (B-C) Phosphorylation of the PDC at days 0, 1, and 3 post-wounding was measured in wounds by western blotting (n=5-10) ** P <0.01 vs. PBS control by two-way ANOVA. (D) Pdk4 mRNA expression was measured in wounds using qRT-PCR. Gene expression was normalized using the ΔΔ Ct method to murine 36B4. (n=8-13) * P <0.05, ** P <0.01 vs. control by two-way ANOVA. * P <0.05 by paired t-test. Data are expressed as mean±SEM.

Article Snippet: For these studies, we used PDK4 inhibitor ‘8c’ (PDK4-IN-1 hydrochloride, HY-135954A, MedChemExpress) with excellent specificity.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Western Blot, Control, Quantitative RT-PCR, Gene Expression

Journal: bioRxiv

Article Title: Reconstituted high-density lipoproteins rescue diabetes-impaired endothelial cell metabolic reprograming and angiogenic responses to hypoxia

doi: 10.1101/2024.08.16.608361

Figure Lengend Snippet: HCAECs were incubated with rHDL (20 µM) or PBS (vehicle control) for 18 h, then exposed to normal (5 mmol/L,) or high (25 mmol/L) glucose conditions for 72 h. Following this, cells were exposed to normoxia or hypoxia (1.2% O 2 ) for 6 h. (A) PDK4 mRNA expression was measured using qRT-PCR and normalized using the ΔΔ Ct method to human B2M, (n=9). (B) PDK4 mRNA expression was measured in HCAECs incubated with rHDL, apoA-I, glycated rHDL (Glyc-rHDL), oxidized rHDL, PLPC or PBS for 18 h then exposed to high glucose and hypoxic conditions (n=6-8). (C) PDK4 protein expression was measured using an ELISA, (n=4-6). (D-E) Phosphorylation of the PDC at serine 293 was measured using western blotting, with data expressed as the ratio of pPDC(S293):Total PDC, and then normalized to α-tubulin, (n=10). * P <0.05, ** P <0.01, *** P <0.001 by two-way ANOVA. (F) Oxygen consumption of treated cells was measured using the Seahorse Bioanalyser system, with data from each well normalized to cell number (n=9-12). * P <0.05, ** P <0.01, *** P <0.001 by two-way ANOVA. ^^^ P <0.001, ^^^^ P <0.0001 vs. normoxia control by two-way ANOVA. (G) Lactate in the media of hypoxia-exposed and rHDL treated cells was measured using a colorimetric assay, (n=5, 6). ** P <0.01 by unpaired t-test. 5 mM glucose, PBS (white circles), 5 mM glucose, rHDL (grey circles), 25 mM glucose, PBS (red triangles), 25 mM glucose, rHDL (blue triangles). Data are expressed as mean±SEM.

Article Snippet: For these studies, we used PDK4 inhibitor ‘8c’ (PDK4-IN-1 hydrochloride, HY-135954A, MedChemExpress) with excellent specificity.

Techniques: Incubation, Control, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Western Blot, Colorimetric Assay

Journal: bioRxiv

Article Title: Reconstituted high-density lipoproteins rescue diabetes-impaired endothelial cell metabolic reprograming and angiogenic responses to hypoxia

doi: 10.1101/2024.08.16.608361

Figure Lengend Snippet: HCAECs were transduced with lentiviral particles containing shRNA for SR-BI (SR-BI) for 24h, then incubated with fresh media and culture for a further 4d. Five days post-transduction, cells were treated with rHDL (20μmol/L) or PBS for 18h then exposed to high glucose (25 mmol/L) for 72h, then hypoxia (1.2% O 2 ) for 6h. (A) SR-BI protein was measured using western blotting densitometry, with data normalized to α-tubulin. **** P <0.0001 by unpaired t-test. (B) PDK4 mRNA expression was measured using qRT-PCR and normalized using the ΔΔ Ct method to human B2M, (n=8). Data are expressed as mean±SEM.

Article Snippet: For these studies, we used PDK4 inhibitor ‘8c’ (PDK4-IN-1 hydrochloride, HY-135954A, MedChemExpress) with excellent specificity.

Techniques: Transduction, shRNA, Incubation, Western Blot, Expressing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Reconstituted high-density lipoproteins rescue diabetes-impaired endothelial cell metabolic reprograming and angiogenic responses to hypoxia

doi: 10.1101/2024.08.16.608361

Figure Lengend Snippet: HCAECs were treated with PDK4-specific siRNA (50 nM) or a scrambled control for 6 h, then incubated with fresh media for 48 h. HCAECs were then incubated with rHDL (20 µM) or PBS (vehicle control) for 18 h, then exposed to normal (5 mmol/L) or high (25 mmol/L) glucose conditions for 72 h. Treated cells were then seeded on Matrigel and exposed to normoxia or hypoxia (1.2% O 2 ) for 6 h. (A) Representative light microscopy images of tubules from the siScrambled and siPDK4 conditions. (B) Matrigel tubules were imaged and quantified using ImageJ software (n=10). * P <0.05, ** P <0.01, **** P <0.0001 vs. controls by two-way ANOVA. ### P <0.001 vs. non-transfected control by two-way ANOVA. ^^ P <0.01 vs. siScrambled control by two-way ANOVA. 5 mM glucose, PBS (white circles), 5 mM glucose, rHDL (grey circles), 25 mM glucose, PBS (red triangles), 25 mM glucose, rHDL (blue triangles). Data are expressed as mean±SEM.

Article Snippet: For these studies, we used PDK4 inhibitor ‘8c’ (PDK4-IN-1 hydrochloride, HY-135954A, MedChemExpress) with excellent specificity.

Techniques: Control, Incubation, Light Microscopy, Software, Transfection

Journal: bioRxiv

Article Title: Reconstituted high-density lipoproteins rescue diabetes-impaired endothelial cell metabolic reprograming and angiogenic responses to hypoxia

doi: 10.1101/2024.08.16.608361

Figure Lengend Snippet: Two full-thickness wounds were created on diabetic C57Bl/6J mice. Mice received topical applications of the chemical inhibitor, PDK4-IN-1 hydrochloride (50μg) in both wounds at the time of surgery then daily until day 5 post-wounding. For each mouse, one wound received topical rHDL (50μg/wound/day) or PBS (control) daily until final endpoint. (A) Wound area was calculated from the average of three daily diameter measurements along the x-, y-, and z-axes. Wound closure is expressed as a percentage of initial wound area at day 0. * P <0.05, ** P <0.01, diabetic rHDL vs. diabetic PBS; # P <0.05, ### P <0.001, #### P <0.0001, diabetic rHDL + PDK4i vs. diabetic rHDL by two-way ANOVA. (n=8-9). Diabetic PBS-treated wounds (red triangles), diabetic rHDL-treated wounds (blue triangles), diabetic PBS + PDK4i-treated wounds (pink circles), diabetic rHDL + PDK4i-treated wounds (light blue circles). Data are expressed as mean±SEM. (B) The rHDL:PBS wound blood flow perfusion ratio was determined using laser Doppler perfusion imaging; images represent high (red) to low (blue) blood flow from day 1-9 in diabetic (blue triangles) and diabetic + PDK4i (light blue circles) mice. * P <0.05 vs. PBS control by paired t-test.

Article Snippet: For these studies, we used PDK4 inhibitor ‘8c’ (PDK4-IN-1 hydrochloride, HY-135954A, MedChemExpress) with excellent specificity.

Techniques: Control, Imaging

Journal: bioRxiv

Article Title: Reconstituted high-density lipoproteins rescue diabetes-impaired endothelial cell metabolic reprograming and angiogenic responses to hypoxia

doi: 10.1101/2024.08.16.608361

Figure Lengend Snippet: HCAECs were incubated with rHDL (20 µM) or PBS (vehicle control) for 18 h, then exposed to normal (5 mmol/L) or high (25 mmol/L) glucose conditions for 72 h. Following this, cells were exposed to normoxia or hypoxia (1.2% O 2 ) for 6 h. (A) Representative Western blot images of phosphorylated FOXO1 (pFOXO1) and total FOXO1 levels. (B) Graphed densitometry analyses of pFOXO1:Total FOXO1, with data normalized to α-tubulin (n=5). * P <0.05 by two-way ANOVA. (C) A schematic showing the FOXO1 binding site sequence (underlined) in the PDK4 gene promoter region that was targeted by the chromatin immunoprecipitation assay (ChIP). (D) FOXO1 binding to this known site in the PDK4 promoter was measured using ChIP (n=3). * P <0.05, ** P <0.01, **** P <0.0001 vs. controls by two-way ANOVA. ^^^^ P <0.0001 vs 5mM glucose, PBS control by two-way ANOVA. 5 mM glucose, PBS (white circles), 5 mM glucose, rHDL (grey circles), 25 mM glucose, PBS (red triangles), 25 mM glucose, rHDL (blue triangles). Data are expressed as mean±SEM.

Article Snippet: For these studies, we used PDK4 inhibitor ‘8c’ (PDK4-IN-1 hydrochloride, HY-135954A, MedChemExpress) with excellent specificity.

Techniques: Incubation, Control, Western Blot, Binding Assay, Sequencing, Chromatin Immunoprecipitation

Journal: bioRxiv

Article Title: Reconstituted high-density lipoproteins rescue diabetes-impaired endothelial cell metabolic reprograming and angiogenic responses to hypoxia

doi: 10.1101/2024.08.16.608361

Figure Lengend Snippet: We have previously shown that rHDL rescues diabetes-impaired angiogenesis by targeting multiple angiogenic signalling mediators. This includes the activation of the PI3K/Akt pathway, which induces the expression of the E3 ubiquitin ligases, Siah1 and Siah2. Increases in Siahs result in the inhibition of the prolyl hydroxylase domain (PHD) protein family. Suppression of PHD2/3 prevents HIF-1a from degradation, allowing it to translocate to the nucleus and bind to the hypoxia response element (HRE), activating transcription of proangiogenic mediators including VEGFA. VEGFA is released into the circulation, where it binds and phosphorylates VEGFR2, further augmenting angiogenesis by activating ERK1/2 and p38 MAPK signalling. We now show that rHDL rescue diabetes-impaired endothelial cell metabolic reprograming in hypoxia (denoted by red arrows). Diabetes impairs the induction of the PDK4/PDC axis in response to hypoxia, which is associated with an aberrant increase in mitochondrial respiration and an impairment to EC angiogenesis. rHDL decreases expression of the PHD proteins, which may affect phosphorylation of FOXO1. Decreased phosphorylation of FOXO1 leads to increased transcription factor activity and binding of FOXO1 to the PDK4 promoter region, which promotes the transcription of PDK4. Increased PDK4 is associated with an increase in inactivation (phosphorylation) of the PDC, which also occurs with rHDL treatment. Suppression of PDC activity decreases the amount of glucose-derived acetyl CoA available to fuel the TCA cycle and reduces mitochondrial respiration, which rHDL treatment returns to normal glucose levels. This improves cell survival in high glucose and hypoxia and provides an alternate pathway for the proangiogenic effects of rHDL in diabetes. These effects of rHDL are mediated via SR-BI.

Article Snippet: For these studies, we used PDK4 inhibitor ‘8c’ (PDK4-IN-1 hydrochloride, HY-135954A, MedChemExpress) with excellent specificity.

Techniques: Activation Assay, Expressing, Ubiquitin Proteomics, Inhibition, Phospho-proteomics, Activity Assay, Binding Assay, Derivative Assay

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Defining the contribution of skeletal muscle pyruvate dehydrogenase α1 to exercise performance and insulin action

doi: 10.1152/ajpendo.00241.2018

Figure Lengend Snippet: Quantitative PCR primer sequences

Article Snippet: The following antibodies were used: PDHα (cat. no. 3205, Cell Signaling), eukaryotic translation elongation factor 2 (cat. no. 2332, Cell signaling), ATP synthase subunit alpha, ubiquinol-cytochrome C reductase core protein 2, mitochondrially encoded cytochrome C oxidase I, succinate dehydrogenase subunit B, NADH/ubiquinone oxidoreductase subunit B8 (cat. no. MS-604, MitoSciences), acyl-Coenzyme A dehydrogenase, very long-chain; cat. no. ab-155138, Abcam), acyl-Coenzyme A dehydrogenase, long-chain (cat. no. ab-82853, Abcam), hexokinase 2 (HK2; cat. no. 2857, Cell Signaling), lactate dehydrogenase A (cat. no. ABN-896, MilliporeSigma), Akt (cat. no. 2920, Cell Signaling), pAkt S473 (cat. no. 4058, Cell Signaling), GSK3α/β (5676, Cell signaling), pGSK3α/β S21/S9 (9331, Cell signaling), PDK4 (cat. no. NBP1-07047SS, Novus Biologicals), phosphofructokinase 1 (cat. no. sc-377346, Santa Cruz Biotechnology).

Techniques: Real-time Polymerase Chain Reaction

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Defining the contribution of skeletal muscle pyruvate dehydrogenase α1 to exercise performance and insulin action

doi: 10.1152/ajpendo.00241.2018

Figure Lengend Snippet: Metabolic and mitochondrial proteins in HFD-fed PDHmKO and WT mice. WT and PDHmKO mice were fed either a chow or a HFD for 12 wk. Transcript abundance of Pdha1, Pdk2, and Pdk4 (A) and Cd36, Cact (Slc25a20), and Acadl (B) in skeletal muscle from chow- or HFD-fed PDHmKO and WT mice, normalized to Ppib (Chow, n = 5/6; HFD, n = 6/7). Representative blot of PDHα, HK2, LDHA, ACADVL (C), and ATP5A, UQCRC2, MTCO1, SDHB, and NDUFB8 (D) protein abundance in skeletal muscle from chow- or HFD-fed PDHmKO and WT mice. Bar graphs show quantification of protein abundance in skeletal muscle relative to ponceau (Chow, n = 6/6; HFD, n = 6/6) (E). Data reported as means ± SE two-way ANOVA, #P < 0.05, main effect of diet, *P < 0.05, main effect of genotype (A–E). HFD, high-fat diet; PDHmKO, tamoxifen-inducible Pdha1 knockout mice; WT, wild-type.

Article Snippet: The following antibodies were used: PDHα (cat. no. 3205, Cell Signaling), eukaryotic translation elongation factor 2 (cat. no. 2332, Cell signaling), ATP synthase subunit alpha, ubiquinol-cytochrome C reductase core protein 2, mitochondrially encoded cytochrome C oxidase I, succinate dehydrogenase subunit B, NADH/ubiquinone oxidoreductase subunit B8 (cat. no. MS-604, MitoSciences), acyl-Coenzyme A dehydrogenase, very long-chain; cat. no. ab-155138, Abcam), acyl-Coenzyme A dehydrogenase, long-chain (cat. no. ab-82853, Abcam), hexokinase 2 (HK2; cat. no. 2857, Cell Signaling), lactate dehydrogenase A (cat. no. ABN-896, MilliporeSigma), Akt (cat. no. 2920, Cell Signaling), pAkt S473 (cat. no. 4058, Cell Signaling), GSK3α/β (5676, Cell signaling), pGSK3α/β S21/S9 (9331, Cell signaling), PDK4 (cat. no. NBP1-07047SS, Novus Biologicals), phosphofructokinase 1 (cat. no. sc-377346, Santa Cruz Biotechnology).

Techniques: Quantitative Proteomics, Knock-Out

Journal: American Journal of Physiology - Endocrinology and Metabolism

Article Title: Defining the contribution of skeletal muscle pyruvate dehydrogenase α1 to exercise performance and insulin action

doi: 10.1152/ajpendo.00241.2018

Figure Lengend Snippet: PDHmKO mice have reduced running speed during voluntary wheel running (VWR). Weekly average speed (km/h) (A), distance run per 24-h period (B), and time/24 h spent running (C) over 21 days VWR (n = 7/6). Representative blots of PDHα, HK2, PFK1, LDH, PDK4, ACADVL, ACADL, and NDUFB8 protein abundance in skeletal muscle from sedentary (SED) or exercise-trained (EXT) PDHmKO and WT mice (D). Bar graphs show quantification of protein abundance in skeletal muscle relative to ponceau (SED, n = 5/5; EXT, n = 7/6) (E). Data reported as means ± SE 2-way ANOVA, Sidak’s post hoc test, *P < 0.05, PDHmKO compared with WT (A–C); 2-way ANOVA, Tukey’s post hoc test, #P < 0.05, EXT compared with SED, *P < 0.05, PDHmKO compared with WT (D–E). PDHmKO, tamoxifen-inducible Pdha1 knockout mice; WT, wild-type.

Article Snippet: The following antibodies were used: PDHα (cat. no. 3205, Cell Signaling), eukaryotic translation elongation factor 2 (cat. no. 2332, Cell signaling), ATP synthase subunit alpha, ubiquinol-cytochrome C reductase core protein 2, mitochondrially encoded cytochrome C oxidase I, succinate dehydrogenase subunit B, NADH/ubiquinone oxidoreductase subunit B8 (cat. no. MS-604, MitoSciences), acyl-Coenzyme A dehydrogenase, very long-chain; cat. no. ab-155138, Abcam), acyl-Coenzyme A dehydrogenase, long-chain (cat. no. ab-82853, Abcam), hexokinase 2 (HK2; cat. no. 2857, Cell Signaling), lactate dehydrogenase A (cat. no. ABN-896, MilliporeSigma), Akt (cat. no. 2920, Cell Signaling), pAkt S473 (cat. no. 4058, Cell Signaling), GSK3α/β (5676, Cell signaling), pGSK3α/β S21/S9 (9331, Cell signaling), PDK4 (cat. no. NBP1-07047SS, Novus Biologicals), phosphofructokinase 1 (cat. no. sc-377346, Santa Cruz Biotechnology).

Techniques: Quantitative Proteomics, Knock-Out

Journal: International Journal of Molecular Sciences

Article Title: Anti-Metastatic Effect of Pyruvate Dehydrogenase Kinase 4 Inhibition in Bladder Cancer via the ERK, SRC, and JNK Pathways

doi: 10.3390/ijms232113240

Figure Lengend Snippet: Expression of PDK4 in siRNA-treated bladder cancer cell lines. ( A ) Fold change of PDK4 mRNA expression in T24 and J82 cells. ( B ) Protein expression of PDK4 in T24 and J82 cells. All data represent means ± SD of three independent experiments (* p < 0.05, ** p < 0.01 NC vs. T). M: mock, N: negative control, T: PDK4 siRNA treated.

Article Snippet: Anti-PDK4 antibody was purchased from Novus Biologicals (Littleton, CO, USA).

Techniques: Expressing, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Anti-Metastatic Effect of Pyruvate Dehydrogenase Kinase 4 Inhibition in Bladder Cancer via the ERK, SRC, and JNK Pathways

doi: 10.3390/ijms232113240

Figure Lengend Snippet: Migration and invasion assay of PDK4 knockdown bladder cancer cells. ( A ) Migration assay. ( B ) Invasion assay. All data represent means ± SD of three independent experiments (* p < 0.05, ** p < 0.01 NC vs. T). M: mock, N: negative control, T: PDK4 siRNA treated.

Article Snippet: Anti-PDK4 antibody was purchased from Novus Biologicals (Littleton, CO, USA).

Techniques: Migration, Invasion Assay, Knockdown, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Anti-Metastatic Effect of Pyruvate Dehydrogenase Kinase 4 Inhibition in Bladder Cancer via the ERK, SRC, and JNK Pathways

doi: 10.3390/ijms232113240

Figure Lengend Snippet: PDK4-related protein expression in PDK4 knockdown bladder cancer cells. β-actin is used as an internal control. M: mock, N: negative control, T: PDK4 siRNA treated. All data represent means ± SD of three independent experiments (* p < 0.05, ** p < 0.01 NC vs. T). M: mock, N: negative control, T: PDK4 siRNA treated.

Article Snippet: Anti-PDK4 antibody was purchased from Novus Biologicals (Littleton, CO, USA).

Techniques: Expressing, Knockdown, Control, Negative Control

Journal: International Journal of Molecular Sciences

Article Title: Anti-Metastatic Effect of Pyruvate Dehydrogenase Kinase 4 Inhibition in Bladder Cancer via the ERK, SRC, and JNK Pathways

doi: 10.3390/ijms232113240

Figure Lengend Snippet: Xenograft model of PDK4 knockdown J82 cells. ( A ) Tumor growth. (* p < 0.05, ** p < 0.01 Day 0 vs. Each day). Ctrl: wild type, KD: knockdown. ( B ) Gross appearance. ( C ) Representative image of immunohisto-chemistry. (* p < 0.05, ** p < 0.01 Ctrl vs. KD).

Article Snippet: Anti-PDK4 antibody was purchased from Novus Biologicals (Littleton, CO, USA).

Techniques: Knockdown, Immunohistochemistry

Journal: International Journal of Molecular Sciences

Article Title: Anti-Metastatic Effect of Pyruvate Dehydrogenase Kinase 4 Inhibition in Bladder Cancer via the ERK, SRC, and JNK Pathways

doi: 10.3390/ijms232113240

Figure Lengend Snippet: PDK4 expression in human bladder cancer specimen. ( A ) Relative PDK4 mRNA expression in normal and bladder cancer specimen. ( B ) Representative image of immunohisto-chemistry of PDK4 protein. All data represent means ± SD of three independent experiments (** p < 0.01 normal vs. T stages).

Article Snippet: Anti-PDK4 antibody was purchased from Novus Biologicals (Littleton, CO, USA).

Techniques: Expressing, Immunohistochemistry

Journal: International Journal of Molecular Sciences

Article Title: Anti-Metastatic Effect of Pyruvate Dehydrogenase Kinase 4 Inhibition in Bladder Cancer via the ERK, SRC, and JNK Pathways

doi: 10.3390/ijms232113240

Figure Lengend Snippet: MS-based quantitative proteomic profiling of phosphorylation in bladder cancer cell lines. ( A ) Experimental design of proteomic analysis. J82 and J82 KD cells were lysed, subjected to in-solution digestion and 18 O labeling, and then mixed at equal protein amounts. Phosphorylated peptides were enriched by TiO 2 . Eluted peptides were analysis using LC-MS/MS. Mass spectrum data were searched in the MaxQuant (version 1.5) database. ( B ) Venn diagrams showing overlap between J82 knock down and J82 control. ( C ) Volcano plot for 209 differentially phosphorylated protein (DRPs) between PDK4 knockdown and controls.

Article Snippet: Anti-PDK4 antibody was purchased from Novus Biologicals (Littleton, CO, USA).

Techniques: Phospho-proteomics, Labeling, Liquid Chromatography with Mass Spectroscopy, Knockdown, Control

Journal: Cell Death & Disease

Article Title: Mitochondrial complex II is a source of the reserve respiratory capacity that is regulated by metabolic sensors and promotes cell survival

doi: 10.1038/cddis.2015.202

Figure Lengend Snippet: The RRC is regulated by pyruvate dehydrogenase kinases. ( a – c ) Neonatal rat cardiac myocytes were incubated for 24 h in complete growth medium under normoxic (atmospheric O 2 ) ( a ) or hypoxic (<1% O 2 ) ( b and c ) conditions. After this period, RNA was either immediately extracted and subjected to qPCR for the indicated genes ( b ), or the medium was replaced with one that is glucose and fatty acid free, or one containing 17.5 mM glucose, 100 μ M palmitate-BSA, or glucose+palmitate-BSA, as indicated, in normoxic conditions, for another 24 h ( a and c ). The results were averaged plotted as relative values to those from cells incubated in base medium in normoxic conditions adjusted to 1 ( a and c ) or control normoxia adjusted to 1 ( b ), n =3. Error bars represent standard error of the mean (S.E.M.), * P <0.05 versus control. ( d ) Neonatal cardiac myocyte was incubated for 24 h in complete growth medium and then infected with a control or adenoviruses (Ad) harboring PDK1, PDK4 or Hif-1 α , for 24 h. The medium was then replaced with serum-free XF medium containing 17.5 mM glucose plus 100 μ M palmitate for 1 h. The mitochondrial stress test was then performed as described in Materials and Methods, n =4–6, each experiment was performed twice. Error bars represent S.E.M., * P <0.05 max OCR versus basal OCR for control at the time point indicated; # P <0.05 max OCR for control versus max OCR of Pdk1 or Hif-1 α treated. ( e ) Neonatal cardiac myocytes were cultured for 24 h in complete growth medium containing vehicle or 1 μ M etomoxir, a Cpt1 inhibitor. The medium was then replaced with serum-free XF medium containing 17.5 mM glucose plus 100 μ M palmitate containing vehicle or 1 μ M etomoxir, for 1 h. The mitochondrial stress test was then performed as described in Materials and Methods, n =4–6, each experiment was performed twice. Error bars represent S.E.M., * P <0.05 max OCR versus basal OCR for control at the time point indicated; # P <0.05 max OCR for control versus max OCR for etomoxir treated, at the time point indicated ( f and g ). Neonatal rat cardiac myocytes were cultured in complete growth medium containing vehicle or 1 mM DCA and either remained in normoxic conditions (atmospheric O 2 ) ( f ) or were exposed to hypoxia (<1% O 2 ) ( g ), for 24 h. At the end of this period, the medium was changed to base medium containing 17.5 mM glucose or 17.5 mM glucose plus 100 μ M palmitate-BSA containing vehicle or DCA, as indicated, for an additional 24 h. The medium was then replaced with serum-free XF medium containing 17.5 mM glucose or 17.5 mM glucose plus 100 μ M palmitate-BSA containing vehicle or 1 mM DCA, for 1 h. The mitochondrial stress test was then performed as described in Materials and Methods, n =4–6, each experiment was performed three times. Error bars represent S.E.M., * P <0.05 max OCR versus basal OCR for DCA-treated cells, at the time point indicated; # P <0.05 max OCR for DCA-treated versus max OCR for untreated, at the time point indicated

Article Snippet: The human cDNA clones for Pdk1 (SC321678/NM_002610), Pdk4 (SC118542/NM_002612), Sdhaf1 (NM_001042631) and Sirt3 (NM_012239) were purchased (Origene Technologies, Inc., Rockville, MD, USA) and cloned into recombinant adenovirus vectors, propagated, and titered as previously described by Graham and Prevec.

Techniques: Incubation, Control, Infection, Cell Culture

Journal: The Kurume Medical Journal

Article Title: Effects of Pemafibrate on Reducing Oxidative Stress and Augmenting Angiogenesis in Ischemic Limb Tissue

doi: 10.2739/kurumemedj.ms6934006

Figure Lengend Snippet: Fig. 1. Tissue expression of pyruvate dehydrogenase kinase 4 (PDK4) in the livers of control mice and mice administered pemafibrate. (a) Representative western blotting images of the expression of PDK4 and glyceral- dehyde-3-phosphate dehydrogenase (GAPDH) in liver tissues of control mice and mice administered pemafi- brate. GAPDH was used as a loading control. C = con- trol group; P-05 = P-05 group (mice treated with 0.5 mg/ kg/day pemafibrate); and P-10 = P-10 group (mice treated with 1.0 mg/kg/day pemafibrate). (b) Pooled data for the PDK4/GAPDH expression ratios in the three groups (*p < 0.05 versus the control group; n = 10, each).

Article Snippet: The membranes were blocked with milk buffer (Blocking One; Nacalai Tesque, Inc., Kyoto, Japan) and then incubated overnight at 4 °C with the following primary antibodies: anti-pyruvate dehydrogenase kinase 4 (PDK4) antibody (cat. no. 12949-1-AP; Proteintech, Rosemont, IL, USA), anti-catalase antibody (cat. no. 66765-1-Ig; Proteintech), anti-superoxide dismutase (SOD)-1 and -2 antibodies (cat. nos. sc-101523 and sc-137254, respectively; Santa Cruz Biotechnology, CA, USA), anti-heme oxygenase (HO)-1 antibody (cat. no. 10701- 1-AP; Proteintech), anti-nuclear factor-erythroid 2-related factor 2 (Nrf2) antibody (cat. no. 16396-1-AP; Proteintech), anti-α-actin antibody (cat. no. 23660-1- AP; Proteintech), and anti-glyceraldehyde-3- phosphate dehydrogenase (GAPDH) antibody (cat. no. MAB374; Millipore, Billerica, MA, USA).

Techniques: Expressing, Control, Western Blot

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Pyruvate Dehydrogenase Kinase 4 Underlies the Metabolic Disorder of Cardiomyocytes in Patients With Hypertrophic Cardiomyopathy From Hypertrophy to Heart Failure

doi: 10.1161/JAHA.125.041401

Figure Lengend Snippet: A , Metabolism of 13 C 6 ‐glucose. Horizontal columns indicate the fraction labeled in labeled metabolites in the WT and Mybpc3 −/− mice, total labeling of each metabolite normalized to 13 C 6 ‐glucose. B , The experimental design of Seahorse analysis. C , OCR was measured by Seahorse analysis with the indicated reagents in cardiomyocytes slices from Mybpc3‐KO (6‐week‐old) mice with PBS or pyruvate dehydrogenase kinase 4 inhibitor treatment. D , Basal respiration and glucose oxidation dependency were decreased in knockout mice PDK4‐IN‐1 treatment compared with PBS treatment. The glucose oxidation dependency measured by Seahorse XFe24 analyzer as described in the “ ” section. Statistical significance was calculated by Mann–Whitney U test. * P <0.05. Acetyl‐CoA indicates acetyl‐coenzyme A; BPTES, bis‐2‐(5‐phenylacetamido‐1,3,4‐thiadiazol‐2‐yl)ethyl sulfide; ETO, etomoxir; KO, knockout; Mybpc3 ‐KO, Mybpc3 −/− knockout mice; OCR, oxygen consumption rate; PDK4‐IN‐1, pyruvate dehydrogenase kinase 4 inhibitor; and WT, wild type.

Article Snippet: Mice in the Mybpc3 ‐KO+PDK4‐IN‐1 group were administered PDK4‐IN‐1 (100 mg/kg, MedChem Express, HY‐135954A) dissolved in PBS via intragastric injection.

Techniques: Labeling, Knock-Out, MANN-WHITNEY

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Pyruvate Dehydrogenase Kinase 4 Underlies the Metabolic Disorder of Cardiomyocytes in Patients With Hypertrophic Cardiomyopathy From Hypertrophy to Heart Failure

doi: 10.1161/JAHA.125.041401

Figure Lengend Snippet: A , The experimental design to evaluate the effect of PDK4‐IN‐1 on Mybpc3 ‐KO mice. B , The representative short‐axis echocardiography images of different groups and statistical analysis of heart failure and thickness of left ventricle. C , The heart from 3 groups and statistical analysis of heart weight. D , The representative Masson trichrome staining images and statistical analysis. E , The immunostaining of ACTN2 and PDK4 in different groups of mice (n WT =6, n Mybpc3 ‐KO =7, n Mybpc3 ‐KO+PDK4‐IN‐1 =7). Data are represented as mean±SD. Statistical significance was calculated by Kruskal–Wallis test with Dunn correction, * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001. ACTN2 indicates actinin alpha 2; EF, ejection fraction; KO, knockout; LVESD, left ventricular end‐systolic diameter; Mybpc3 ‐KO, Mybpc3 −/− knockout mice; ns, not significant; PDK4‐IN‐1, pyruvate dehydrogenase kinase 4 inhibitor; and WT, wild type.

Article Snippet: Mice in the Mybpc3 ‐KO+PDK4‐IN‐1 group were administered PDK4‐IN‐1 (100 mg/kg, MedChem Express, HY‐135954A) dissolved in PBS via intragastric injection.

Techniques: Staining, Immunostaining, Knock-Out

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Pyruvate Dehydrogenase Kinase 4 Underlies the Metabolic Disorder of Cardiomyocytes in Patients With Hypertrophic Cardiomyopathy From Hypertrophy to Heart Failure

doi: 10.1161/JAHA.125.041401

Figure Lengend Snippet: A , Schematic of the experiments procedure in 4 groups of mice: (1) PDK4‐CKO 4W: intragastric administration of PBS in PDK4‐CKO mice once every 2 days from 2W to 4W; (2) Mybpc3 ‐KO 8W+PDK4‐IN‐1: intragastric administration of PDK4‐IN‐1 in Mybpc3‐KO mice once every 2 days from 6W to 8W; (3) Mybpc3‐KO/Pdk4‐CKO 8W: intragastric administration of PBS in Mybpc3 ‐KO/ Pdk4 ‐CKO mice once every 2 days from 6W to 8W; (4) Mybpc3 ‐KO/ Pdk4 ‐CKO 8W+Tamoxifen: intragastric administration of tamoxifen in Mybpc3 ‐KO/ Pdk4 ‐CKO mice once every 2 days from 6W to 8W. B , M mode echocardiography of mice in each group. C , Statistics of EF of each group. D , H&E and Masson trichrome staining of mice in different treated groups (n=3). E , Quantification of ventricular fibrosis. F , Statistics of HW to BW ratio (HW/BW). G , TEM images of cardiac tissue in each group. Data are represented as mean±SD. Statistical significance was calculated by Kruskal–Wallis test with Dunn correction, * P <0.05, ** P <0.01, *** P <0.001. BW indicates body weight; CKO, conditional knockout; EF, ejection fraction; H&E, hematoxylin and eosin; HW, heart weight; ns, not significant; KO, knockout; Mybpc3 ‐KO, Mybpc3 −/− knockout mice; Mybpc3 ‐KO/ Pdk4 ‐CKO, Mybpc3 and Pdk4 conditional double knockout mice; PDK4, pyruvate dehydrogenase kinase 4; PDK4‐IN‐1, pyruvate dehydrogenase kinase 4 inhibitor; and TEM, transmission electron microscopy.

Article Snippet: Mice in the Mybpc3 ‐KO+PDK4‐IN‐1 group were administered PDK4‐IN‐1 (100 mg/kg, MedChem Express, HY‐135954A) dissolved in PBS via intragastric injection.

Techniques: Staining, Knock-Out, Double Knockout, Transmission Assay, Electron Microscopy