Journal: The Journal of Neuroscience

Article Title: Metabolic Connection of Inflammatory Pain: Pivotal Role of a Pyruvate Dehydrogenase Kinase-Pyruvate Dehydrogenase-Lactic Acid Axis

doi: 10.1523/JNEUROSCI.1910-15.2015

Figure Lengend Snippet: Expression of PDK2 and PDK4 in hindpaw tissues after CFA injection. A, The expression of Pdk2 and Pdk4 mRNAs in hindpaw tissues at different time points after CFA injection was assessed by real-time RT-PCR. Pdk2 and Pdk4 mRNA levels in CFA-injected hindpaw tissues were significantly upregulated after injection, peaking at 3 d and then subsiding. Results for mRNA expression are displayed as the fold increase of gene expression normalized to GAPDH. B, Protein levels of PDK2 and PDK4 in hindpaw tissues 3 d after CFA injection were assessed by Western blot analysis. Quantifications of the band intensities are presented in the adjacent graphs. C, D, Immunofluorescence analysis detected a strong expression of PDK2 and PDK4 in the hindpaw tissues of CFA-injected mice at 3 d after injection, but not in the vehicle-treated control animals. *p < 0.05 versus the vehicle-treated control animals (Student's t test). n = 3. Data are mean ± SEM. Scale bars, 200 μm. Images show the representative results of at least three independent experiments.

Article Snippet: For immunofluorescence staining, sections were incubated with primary antibodies against PDK2 (rabbit, 1:200; Acris Antibodies), PDK4 (rabbit, 1:200; Atlas Antibodies), phospho-Ser 293 -PDHE1α (pyruvate dehydrogenase E1α) (rabbit, 1:200; Calbiochem), phospho-Ser 300 -PDHE1α (pyruvate dehydrogenase E1α) (rabbit, 1:200; Calbiochem), Iba-1 (rabbit, 1:1000; Wako; or goat, 1:200; Novus Biologicals [for costainings]), GFAP (mouse, 1:500; BD Biosciences), Ly6G (rat, 1:200; eBioscience) or iNOS (rabbit, 1:200; BD Biosciences), MYH1/2/3 (mouse, 1:200; Santa Cruz Biotechnology) and β3 tubulin (2G10) (mouse, 1:200; Santa Cruz Biotechnology), overnight at 4°C, and then incubated with FITC- or Cy3-conjugated secondary antibodies (1:200; Jackson ImmunoResearch Laboratories).

Techniques: Expressing, Injection, Quantitative RT-PCR, Western Blot, Immunofluorescence

Journal: The Journal of Neuroscience

Article Title: Metabolic Connection of Inflammatory Pain: Pivotal Role of a Pyruvate Dehydrogenase Kinase-Pyruvate Dehydrogenase-Lactic Acid Axis

doi: 10.1523/JNEUROSCI.1910-15.2015

Figure Lengend Snippet: Effect of PDK2/4 deficiency on inflammatory cell infiltration and proinflammatory cytokine expression in CFA-injected hindpaws. A, H&E staining revealed an accumulation of inflammatory cells in the ipsilateral hindpaw tissues at 1 d after injection in the WT mice, but this accumulation was attenuated in the DKO mice. Pdk2/4 deficiency considerably lessened the immunoreactivities of Ly6G (green, a neutrophil marker) and Iba-1 (green, a macrophage marker) in CFA-injected hindpaws at 1 d after injection compared with those of WT animals. Similarly, notably reduced immunoreactivities of iNOS (red, a M1 macrophage phenotype marker) were detected in the CFA-injected hindpaws of Pdk2/4 DKO mice compared with those of WT animals at 3 d after injection. B, Iba-1 immunoreactivity in the ipsilateral hindpaws was also detected at days 3 and 7 after CFA injection. Quantifications and statistical analyses of stained images are presented in adjacent graphs. C, D, The relative mRNA expression of TNF-α, IL-1β, and IL-6 in the hindpaw tissues at 6 h (C) and 3 d (D) after injection was evaluated by real-time RT-PCR. Results for mRNA expression are displayed as the fold increase of gene expression normalized to GAPDH. *p < 0.05 versus the vehicle-treated control animals. #p < 0.05 between indicated groups (Student's t test). n = 3. Data are mean ± SEM. IR, Immunoreactivity; ND, not detected. Scale bars, 200 μm. Images show the representative results of at least three independent experiments.

Article Snippet: For immunofluorescence staining, sections were incubated with primary antibodies against PDK2 (rabbit, 1:200; Acris Antibodies), PDK4 (rabbit, 1:200; Atlas Antibodies), phospho-Ser 293 -PDHE1α (pyruvate dehydrogenase E1α) (rabbit, 1:200; Calbiochem), phospho-Ser 300 -PDHE1α (pyruvate dehydrogenase E1α) (rabbit, 1:200; Calbiochem), Iba-1 (rabbit, 1:1000; Wako; or goat, 1:200; Novus Biologicals [for costainings]), GFAP (mouse, 1:500; BD Biosciences), Ly6G (rat, 1:200; eBioscience) or iNOS (rabbit, 1:200; BD Biosciences), MYH1/2/3 (mouse, 1:200; Santa Cruz Biotechnology) and β3 tubulin (2G10) (mouse, 1:200; Santa Cruz Biotechnology), overnight at 4°C, and then incubated with FITC- or Cy3-conjugated secondary antibodies (1:200; Jackson ImmunoResearch Laboratories).

Techniques: Expressing, Injection, Staining, Marker, Quantitative RT-PCR

Journal: The Journal of Neuroscience

Article Title: Metabolic Connection of Inflammatory Pain: Pivotal Role of a Pyruvate Dehydrogenase Kinase-Pyruvate Dehydrogenase-Lactic Acid Axis

doi: 10.1523/JNEUROSCI.1910-15.2015

Figure Lengend Snippet: Role of PDK2/4 in regulating the phenotypes of cultured macrophages. Peritoneal macrophage cultures prepared from WT and Pdk2/4 DKO mice were treated with M1-phenotype inducer mixture [LPS (100 ng/ml) plus IFN-γ (50 U/ml)] for 8 h. A, The mRNA levels of M1-related genes TNF-α, IL-1β, and IL-6 were assessed by real-time RT-PCR (left). Similarly, peritoneal macrophage cultures prepared from WT and Pdk2/4 DKO mice were treated with M2-phenotype inducer [IL-4 (10 ng/ml)] for 8 h, and the mRNA levels of M2-related genes Ym-1, Arg-1, and IL-10 were then assessed by real-time RT-PCR (right). B, The expression of IRF8 or IRF4 mRNAs in the cultured peritoneal macrophages (prepared from WT and Pdk2/4 DKO mice) following stimulation with LPS (100 ng/ml) plus IFN-γ (50 U/ml) or IL-4 (10 ng/ml) for 8 h was assessed by real-time RT-PCR. C, The expression of Pdk2 and Pdk4 mRNAs in the WT peritoneal macrophages following stimulation with LPS (100 ng/ml) plus IFN-γ (50 U/ml) for 8 h was assessed by real-time RT-PCR. Results for mRNA expression are displayed as the fold increase of gene expression normalized to GAPDH. *p < 0.05 versus the control group. #p < 0.05 between indicated groups (Student's t test). n = 3. Data are mean ± SEM.

Article Snippet: For immunofluorescence staining, sections were incubated with primary antibodies against PDK2 (rabbit, 1:200; Acris Antibodies), PDK4 (rabbit, 1:200; Atlas Antibodies), phospho-Ser 293 -PDHE1α (pyruvate dehydrogenase E1α) (rabbit, 1:200; Calbiochem), phospho-Ser 300 -PDHE1α (pyruvate dehydrogenase E1α) (rabbit, 1:200; Calbiochem), Iba-1 (rabbit, 1:1000; Wako; or goat, 1:200; Novus Biologicals [for costainings]), GFAP (mouse, 1:500; BD Biosciences), Ly6G (rat, 1:200; eBioscience) or iNOS (rabbit, 1:200; BD Biosciences), MYH1/2/3 (mouse, 1:200; Santa Cruz Biotechnology) and β3 tubulin (2G10) (mouse, 1:200; Santa Cruz Biotechnology), overnight at 4°C, and then incubated with FITC- or Cy3-conjugated secondary antibodies (1:200; Jackson ImmunoResearch Laboratories).

Techniques: Cell Culture, Quantitative RT-PCR, Expressing

Journal: The Journal of Neuroscience

Article Title: Metabolic Connection of Inflammatory Pain: Pivotal Role of a Pyruvate Dehydrogenase Kinase-Pyruvate Dehydrogenase-Lactic Acid Axis

doi: 10.1523/JNEUROSCI.1910-15.2015

Figure Lengend Snippet: Role of PDK2/4 in glial activation and expression of proinflammatory cytokines in the spinal cord after intraplantar administration of CFA. A, Iba-1 (red, a microglia marker) immunoreactivity was significantly increased in the ipsilateral, but not in the contralateral, dorsal horn of the lumbar segment of the spinal cord at 1–3 d. The increased Iba-1 immunoreactivity persisted at a low level for 7 d after CFA injection. However, CFA-induced Iba-1 immunoreactivities were markedly attenuated in the Pdk2/4 DKO mice at all the time points. B, GFAP (red, an astrocyte marker) immunoreactivity was significantly increased in the ipsilateral, but not in the contralateral, dorsal horn of the lumbar segment of the spinal cord at 3–7 d after CFA injection. The CFA-induced GFAP immunoreactivities were markedly attenuated in the Pdk2/4 DKO mice at these time points. Insets, Magnified images (original magnification × 200). Quantifications and statistical analyses of stained images are presented in adjacent graphs. C, The relative mRNA expression of TNF-α, IL-1β, and IL-6 in the lumbar segment of the spinal cord at 3 d after injection was evaluated by real-time RT-PCR. Results for mRNA expression are displayed as the fold increase of gene expression normalized to GAPDH. *p < 0.05 versus the vehicle-treated control animals. #p < 0.05 between indicated groups (Student's t test). n = 3. Data are mean ± SEM. Scale bar, 200 μm. Images show the representative results of at least three independent experiments.

Article Snippet: For immunofluorescence staining, sections were incubated with primary antibodies against PDK2 (rabbit, 1:200; Acris Antibodies), PDK4 (rabbit, 1:200; Atlas Antibodies), phospho-Ser 293 -PDHE1α (pyruvate dehydrogenase E1α) (rabbit, 1:200; Calbiochem), phospho-Ser 300 -PDHE1α (pyruvate dehydrogenase E1α) (rabbit, 1:200; Calbiochem), Iba-1 (rabbit, 1:1000; Wako; or goat, 1:200; Novus Biologicals [for costainings]), GFAP (mouse, 1:500; BD Biosciences), Ly6G (rat, 1:200; eBioscience) or iNOS (rabbit, 1:200; BD Biosciences), MYH1/2/3 (mouse, 1:200; Santa Cruz Biotechnology) and β3 tubulin (2G10) (mouse, 1:200; Santa Cruz Biotechnology), overnight at 4°C, and then incubated with FITC- or Cy3-conjugated secondary antibodies (1:200; Jackson ImmunoResearch Laboratories).

Techniques: Activation Assay, Expressing, Marker, Injection, Staining, Quantitative RT-PCR

Journal: The Journal of Neuroscience

Article Title: Metabolic Connection of Inflammatory Pain: Pivotal Role of a Pyruvate Dehydrogenase Kinase-Pyruvate Dehydrogenase-Lactic Acid Axis

doi: 10.1523/JNEUROSCI.1910-15.2015

Figure Lengend Snippet: Impact of Pdk2/4 gene knock-out on motor coordination, mechanical as well as thermal nociception, and peripheral nerve function. A, Open field test was performed to measure the mean velocity and distance traveled. B, Ability of mice to balance on the rotating rod was assessed by RotaRod test. C, E, The frequency of paw withdraw in response to von Frey monofilaments of forces 2.0 g as well as 4.0 g and the time required to withdraw tail from 45°C, 50°C, and 55°C were assessed. D, F, PWT to force and PWL to heat were measured in hindpaws. G, Motor nerve conduction velocity was measured and expressed in m/s. H, Cross-sections of sciatic nerve were stained with H&E. All of these behavioral/basal studies were performed in unmanipulated WT and Pdk2/4 DKO mice. *p < 0.05 (Student's t test). NS, Not significant. n = 5. Data are mean ± SEM. Scale bars, 50 μm. Images show the representative results of at least three independent experiments.

Article Snippet: For immunofluorescence staining, sections were incubated with primary antibodies against PDK2 (rabbit, 1:200; Acris Antibodies), PDK4 (rabbit, 1:200; Atlas Antibodies), phospho-Ser 293 -PDHE1α (pyruvate dehydrogenase E1α) (rabbit, 1:200; Calbiochem), phospho-Ser 300 -PDHE1α (pyruvate dehydrogenase E1α) (rabbit, 1:200; Calbiochem), Iba-1 (rabbit, 1:1000; Wako; or goat, 1:200; Novus Biologicals [for costainings]), GFAP (mouse, 1:500; BD Biosciences), Ly6G (rat, 1:200; eBioscience) or iNOS (rabbit, 1:200; BD Biosciences), MYH1/2/3 (mouse, 1:200; Santa Cruz Biotechnology) and β3 tubulin (2G10) (mouse, 1:200; Santa Cruz Biotechnology), overnight at 4°C, and then incubated with FITC- or Cy3-conjugated secondary antibodies (1:200; Jackson ImmunoResearch Laboratories).

Techniques: Knock-Out, Staining

Journal: The Journal of Neuroscience

Article Title: Metabolic Connection of Inflammatory Pain: Pivotal Role of a Pyruvate Dehydrogenase Kinase-Pyruvate Dehydrogenase-Lactic Acid Axis

doi: 10.1523/JNEUROSCI.1910-15.2015

Figure Lengend Snippet: Pdk2/4 DKO mice displayed attenuated paw edema and pain responses to chronic inflammatory insult. To induce local inflammation, CFA was injected into the plantar surface of the left hindpaws (ipsilateral side). A, Paw thicknesses were assessed after injection and were found to be significantly greater in the ipsilateral sides than contralateral sides. The paw edema persisted for >1 week. No change in thickness was observed in the contralateral sides. Pdk2/4 deficiency significantly reduced the CFA-induced increase in paw thickness compared with that of WT animals. PWT to force and PWL to heat were measured in contralateral and ipsilateral sides. In the ipsilateral sides, CFA injection reduced PWT to force (B) and PWL to heat (C). The chronic inflammation-induced pain hypersensitivities were attenuated in Pdk2/4 DKO mice compared with WT animals. No significant change in pain-related behavior was observed in the contralateral sides. *p < 0.05 between ipsilateral sides of WT and DKO mice (one-way ANOVA with Dunnett's procedure for paw thickness and PWL, Mann–Whitney test for PWT). n = 7. Data are mean ± SEM.

Article Snippet: For immunofluorescence staining, sections were incubated with primary antibodies against PDK2 (rabbit, 1:200; Acris Antibodies), PDK4 (rabbit, 1:200; Atlas Antibodies), phospho-Ser 293 -PDHE1α (pyruvate dehydrogenase E1α) (rabbit, 1:200; Calbiochem), phospho-Ser 300 -PDHE1α (pyruvate dehydrogenase E1α) (rabbit, 1:200; Calbiochem), Iba-1 (rabbit, 1:1000; Wako; or goat, 1:200; Novus Biologicals [for costainings]), GFAP (mouse, 1:500; BD Biosciences), Ly6G (rat, 1:200; eBioscience) or iNOS (rabbit, 1:200; BD Biosciences), MYH1/2/3 (mouse, 1:200; Santa Cruz Biotechnology) and β3 tubulin (2G10) (mouse, 1:200; Santa Cruz Biotechnology), overnight at 4°C, and then incubated with FITC- or Cy3-conjugated secondary antibodies (1:200; Jackson ImmunoResearch Laboratories).

Techniques: Injection, MANN-WHITNEY

Journal: The Journal of Neuroscience

Article Title: Metabolic Connection of Inflammatory Pain: Pivotal Role of a Pyruvate Dehydrogenase Kinase-Pyruvate Dehydrogenase-Lactic Acid Axis

doi: 10.1523/JNEUROSCI.1910-15.2015

Figure Lengend Snippet: Pdk2 or Pdk4 single knock-out mice showed attenuated paw edema and pain responses to chronic inflammatory insult. A, CFA was injected into the plantar surface of the left hindpaws (ipsilateral side) to induce local inflammation. Paw thicknesses were significantly greater in the ipsilateral sides than contralateral sides. Pdk2 or Pdk4 single-gene deficiency significantly reduced the CFA-induced increase in paw thickness compared with that of WT animals. PWT to force and PWL to heat were measured in contralateral and ipsilateral sides. In the ipsilateral sides, CFA injection reduced PWT to force (B) and PWL to heat (C). The chronic inflammation-induced pain hypersensitivities were attenuated in Pdk2 or Pdk4 single-gene KO mice compared with WT animals. No significant change in pain-related behavior was observed in the contralateral sides. *p < 0.05 between ipsilateral sides of WT and Pdk2 KO mice. #p < 0.05 between ipsilateral sides of WT and Pdk4 KO mice (one-way ANOVA with Dunnett's procedure for paw thickness and PWL, Mann–Whitney test for PWT). n = 7. Data are mean ± SEM.

Article Snippet: For immunofluorescence staining, sections were incubated with primary antibodies against PDK2 (rabbit, 1:200; Acris Antibodies), PDK4 (rabbit, 1:200; Atlas Antibodies), phospho-Ser 293 -PDHE1α (pyruvate dehydrogenase E1α) (rabbit, 1:200; Calbiochem), phospho-Ser 300 -PDHE1α (pyruvate dehydrogenase E1α) (rabbit, 1:200; Calbiochem), Iba-1 (rabbit, 1:1000; Wako; or goat, 1:200; Novus Biologicals [for costainings]), GFAP (mouse, 1:500; BD Biosciences), Ly6G (rat, 1:200; eBioscience) or iNOS (rabbit, 1:200; BD Biosciences), MYH1/2/3 (mouse, 1:200; Santa Cruz Biotechnology) and β3 tubulin (2G10) (mouse, 1:200; Santa Cruz Biotechnology), overnight at 4°C, and then incubated with FITC- or Cy3-conjugated secondary antibodies (1:200; Jackson ImmunoResearch Laboratories).

Techniques: Knock-Out, Injection, MANN-WHITNEY

Journal: The Journal of Neuroscience

Article Title: Metabolic Connection of Inflammatory Pain: Pivotal Role of a Pyruvate Dehydrogenase Kinase-Pyruvate Dehydrogenase-Lactic Acid Axis

doi: 10.1523/JNEUROSCI.1910-15.2015

Figure Lengend Snippet: Role of lactic acid in the CFA-induced paw edema and pain hypersensitivities. A, The lactate assay was performed to compare the relative accumulation of lactate in the hindpaw tissues of WT and Pdk2/4 DKO mice at 3 d after CFA injection. B, The lactate assay was performed to measure the relative accumulation of lactate in the hindpaw tissues at 3 d after CFA injection (following either DCA or clodronate administration). The results presented are the fold change relative to control. Control animals were injected with either DCA alone (left) or vehicle (right). C, ECAR was recorded for RAW264.7 cells during LPS (100 ng/ml) stimulation and is presented as percentage change to unstimulated control. D, E, FX11 (a small-molecule inhibitor of lactate dehydrogenase A) (2 mg/kg body weight) or vehicle (2% [v/v] DMSO) was administered intraplantar daily to CFA-injected mice for 2 d. CFA-induced increase in paw area (D), PWT to force, and PWL to heat (E) was assessed up to 7 d after CFA injection. *p < 0.05 versus the vehicle-treated control animals, between CFA+Vehicle and CFA+FX11 groups. #p < 0.05 between indicated groups (Student's t test). A, B, n = 3; C, n = 5; D, E, n = 4. Data are mean ± SEM. Arrows indicate the time points of FX11 or vehicle administration.

Article Snippet: For immunofluorescence staining, sections were incubated with primary antibodies against PDK2 (rabbit, 1:200; Acris Antibodies), PDK4 (rabbit, 1:200; Atlas Antibodies), phospho-Ser 293 -PDHE1α (pyruvate dehydrogenase E1α) (rabbit, 1:200; Calbiochem), phospho-Ser 300 -PDHE1α (pyruvate dehydrogenase E1α) (rabbit, 1:200; Calbiochem), Iba-1 (rabbit, 1:1000; Wako; or goat, 1:200; Novus Biologicals [for costainings]), GFAP (mouse, 1:500; BD Biosciences), Ly6G (rat, 1:200; eBioscience) or iNOS (rabbit, 1:200; BD Biosciences), MYH1/2/3 (mouse, 1:200; Santa Cruz Biotechnology) and β3 tubulin (2G10) (mouse, 1:200; Santa Cruz Biotechnology), overnight at 4°C, and then incubated with FITC- or Cy3-conjugated secondary antibodies (1:200; Jackson ImmunoResearch Laboratories).

Techniques: Lactate Assay, Injection

Journal: The Journal of Neuroscience

Article Title: Metabolic Connection of Inflammatory Pain: Pivotal Role of a Pyruvate Dehydrogenase Kinase-Pyruvate Dehydrogenase-Lactic Acid Axis

doi: 10.1523/JNEUROSCI.1910-15.2015

Figure Lengend Snippet: A proposed schematic outlining the implications of a PDK-PDH-lactic acid axis in the pathogenesis of chronic inflammatory pain. Inflammatory stimulus enhances the expression and activity of PDK2 and PDK4 at the site of inflammation, thereby decreasing the oxidation of pyruvate and increasing its conversion into lactate via phosphorylation/inhibition of PDH. This metabolic shift-associated lactic acid production and resulting acidic microenvironment favor the recruitment of inflammatory cells to the site of inflammation and amplify the local inflammation ensuing the nociceptive responses. Enhanced PDK2/4 skew macrophages toward the M1 (proinflammatory) phenotype via activation of proinflammatory phenotype-determining transcription factor IRF8, resulting in the increased secretion of proinflammatory mediators, such as TNF-α, IL-1β, iNOS, and IL-6 as well as nitric oxide. The proalgesic mediators thus released activate nociceptors and spinal glia to cause peripheral and central sensitizations, respectively. These findings suggest that the PDK-PDH-lactic acid axis plays a key role in the pathogenesis of inflammation-induced chronic pain via the modulation of multifaceted proinflammatory events.

Article Snippet: For immunofluorescence staining, sections were incubated with primary antibodies against PDK2 (rabbit, 1:200; Acris Antibodies), PDK4 (rabbit, 1:200; Atlas Antibodies), phospho-Ser 293 -PDHE1α (pyruvate dehydrogenase E1α) (rabbit, 1:200; Calbiochem), phospho-Ser 300 -PDHE1α (pyruvate dehydrogenase E1α) (rabbit, 1:200; Calbiochem), Iba-1 (rabbit, 1:1000; Wako; or goat, 1:200; Novus Biologicals [for costainings]), GFAP (mouse, 1:500; BD Biosciences), Ly6G (rat, 1:200; eBioscience) or iNOS (rabbit, 1:200; BD Biosciences), MYH1/2/3 (mouse, 1:200; Santa Cruz Biotechnology) and β3 tubulin (2G10) (mouse, 1:200; Santa Cruz Biotechnology), overnight at 4°C, and then incubated with FITC- or Cy3-conjugated secondary antibodies (1:200; Jackson ImmunoResearch Laboratories).

Techniques: Expressing, Activity Assay, Inhibition, Activation Assay

Journal: Nature Communications

Article Title: Astrocytic pyruvate dehydrogenase kinase-2 is involved in hypothalamic inflammation in mouse models of diabetes

doi: 10.1038/s41467-020-19576-1

Figure Lengend Snippet: The expression of Pdk isoform (Pdk1-4) mRNAs in the hypothalamus at 1, 2, 3, and 6 w following STZ injection ( a ) was assessed by real-time RT-PCR. mRNA expression profiles are displayed as the fold increase of gene expression normalized to Gapdh (* p = 0.0463, 1 w; * p = 0.0249, 2 w; ** p = 0.0063, 3 w; and * p = 0.0212, 6 w for Pdk2 ). PDK2 (* p = 0.0442, STZ), phosphorylated-PDH (p-S 293 -PDH: ** p = 0.0048, STZ; and p-S 300 -PDH: *** p = 0.0002, STZ), and PDH-E1 protein levels in the hypothalamus at 3 w post-STZ injection ( b ) were assessed by Western blot analysis. Western blot band quantification for p-PDH was based on normalization to PDH-E1, and PDK2 and PDH-E1 were normalized to α-tubulin. Immunofluorescence analyses show the expression of PDK2 in GFAP-positive astrocytes ( c ), βIII-tubulin-positive neurons ( d ), and Iba-1-positive microglia ( e ) in the hypothalamus at 3 w post-STZ injection. Inserts show the representative double-labeled cells. Quantification of the percentage of PDK2-positive or -negative astrocytes (### p = 1.5E-12, not co-localized with PDK2; and ### p = 1.6E-12, co-localized with PDK2), neurons (# p = 0.0119, not co-localized with PDK2; and # p = 0.0143, co-localized with PDK2), or microglia. Pearson’s correlation coefficients for co-localization per mm 2 (*** p = 1.02E-5, STZ for c ; and *** p = 5.2E-5, STZ for d ) are shown in the adjacent graphs. Results were obtained from three animals for each condition. Microscopic data were evaluated in five randomly selected fields captured at the same magnification. Scale bars indicate 200 µm. * p < 0.05, ** p < 0.01, or *** p < 0.001 versus the vehicle-treated control animals. One-way ANOVA ( a ), two-way ANOVA ( c – e , left) with Tukey’s post hoc test; Student’s t -test ( b – e , right), n = 3 ( a , b ), and n = 5 ( c – e ); mean ± SEM. Value of “n” indicates the number of animals. Source data are provided as a Source data file. w week(s); STZ streptozotocin; ND not detected; ns not significant.

Article Snippet: For immunofluorescence or 3, 3′-diaminobenzidine (DAB) staining, sections were incubated with primary antibodies against PDK2 (rabbit, 1:100; Abgent, San Diego, CA), Iba-1 (goat, 1:200; Novus Biologicals, Littleton, CO) or Iba-1 (rabbit, 1:1000; Wako Pure Chemical Corporation), GFAP (mouse, 1:200; Novus Biologicals, Littleton, CO) or GFAP (rabbit, 1.500; DAKO), βIII-tubulin (mouse, 1:200; Santa Cruz Biotechnology Inc.), NeuN (rabbit, 1:500, Merck Millipore), AGRP (rabbit, 1:200; Phoenix Pharmaceuticals Inc.), POMC (rabbit, 1:200, phoenix Pharmaceutical Inc.), NF-κB p65 (mouse, 1:200, Santa Cruz Biotechnology Inc), phosphorylated (Thr-172) and total forms of AMPKα (rabbit, 1:100; Cell Signaling Technology) overnight at 4°C.

Techniques: Expressing, Injection, Quantitative RT-PCR, Western Blot, Immunofluorescence, Labeling

Journal: Nature Communications

Article Title: Astrocytic pyruvate dehydrogenase kinase-2 is involved in hypothalamic inflammation in mouse models of diabetes

doi: 10.1038/s41467-020-19576-1

Figure Lengend Snippet: GFAP (# p = 0.0101, KO + STZ for hypothalamus; ###p = 0.0003, KO + STZ for ARC; and # p = 0.0192, KO + STZ for PVN) and Iba-1 (# p = 0.0170, KO + STZ for hypothalamus, ## p = 0.0090, KO + STZ for ARC; and # p = 0.0277, KO + STZ for PVN) immunofluorescence staining and image analysis were performed using WT and Pdk2 -deficient mice at 3 w post-STZ injection ( a , b ). Microscope data were gathered using five to six randomly selected fields captured at the same magnification. Scale bars indicate 400 µm ( a , b , hypothalamus) and 200 µm ( a , b , ARC and PVN). The relative expression of Tnf-α (#p = 0.0112, KO + STZ), Il-1β (###p = 0.0003, KO + STZ), and Il-6 (##p = 0.0016, KO + STZ) mRNAs in the hypothalamus after 3 w of STZ injection ( c ) was evaluated by real-time RT-PCR. Results for mRNA expression are displayed as the fold increase in gene expression normalized to Gapdh . Lactate concentration in hypothalamic tissues collected from WT and Pdk2 KO mice at 3 w post-STZ/vehicle injection was measured with an HPLC analyzer ( d ). The quantification of lactate concentration (### p = 0.0002, KO + STZ) from HPLC graphs is shown. Food intake (* p = 0.0017, KO + STZ at 22 d) ( e ) and fasting blood (* p = 0.0033, KO + STZ at 23 d) ( f ) and CSF glucose (# p = 0.0101, KO + STZ) ( g ) levels in mice were assessed following STZ injection at the indicated time points. Fasting blood glucose levels were measured in the pair-feeding condition following STZ administration ( h ). The pair-feeding condition was established based on the consumption of food by diabetic KO animals ( p = 0.9997, KO + STZ for PF at 3 w). The relative expression of Npy (## p = 0.0031, KO + STZ), Agrp (### p = 0.0004, KO + STZ), and Pomc (# p = 0.0158, KO + STZ) mRNA in the hypothalamus after 3 w of STZ injection ( i ) was evaluated by real-time RT-PCR. mRNA expression results are displayed as the fold increase of gene expression normalized to Gapdh . * p < 0.05, ** p < 0.01, or *** p < 0.001 versus the WT/KO control animals ( a – d , g – i ) or STZ-injected animals ( e , f ); # p < 0.05 or ## p < 0.01 versus indicated groups. Two-way ANOVA with Tukey’s post hoc test, n = 6 for KO + Control and 5 for other groups ( a , b ), n = 5 for control and 6 for STZ groups ( d ), n = 6 ( e – h ), and n = 3 ( c , i ); mean ± SEM. Value of “ n ” indicates the number of animals. Source data are provided as a Source data file. w week(s); d day(s); WT wild-type; KO knock out; STZ streptozotocin; ARC arcuate nucleus; PVN paraventricular nucleus; IR immunoreactivity; RF regular feeding; PF pair feeding; ns not significant.

Article Snippet: For immunofluorescence or 3, 3′-diaminobenzidine (DAB) staining, sections were incubated with primary antibodies against PDK2 (rabbit, 1:100; Abgent, San Diego, CA), Iba-1 (goat, 1:200; Novus Biologicals, Littleton, CO) or Iba-1 (rabbit, 1:1000; Wako Pure Chemical Corporation), GFAP (mouse, 1:200; Novus Biologicals, Littleton, CO) or GFAP (rabbit, 1.500; DAKO), βIII-tubulin (mouse, 1:200; Santa Cruz Biotechnology Inc.), NeuN (rabbit, 1:500, Merck Millipore), AGRP (rabbit, 1:200; Phoenix Pharmaceuticals Inc.), POMC (rabbit, 1:200, phoenix Pharmaceutical Inc.), NF-κB p65 (mouse, 1:200, Santa Cruz Biotechnology Inc), phosphorylated (Thr-172) and total forms of AMPKα (rabbit, 1:100; Cell Signaling Technology) overnight at 4°C.

Techniques: Immunofluorescence, Staining, Injection, Microscopy, Expressing, Quantitative RT-PCR, Concentration Assay, Knock-Out

Journal: Nature Communications

Article Title: Astrocytic pyruvate dehydrogenase kinase-2 is involved in hypothalamic inflammation in mouse models of diabetes

doi: 10.1038/s41467-020-19576-1

Figure Lengend Snippet: The relative expression of Pdk2 (*** p = 7.6E-6, Gluc 16 mM; and *** p = 3.6E-7, Gluc 25 mM) ( a ), Tnf-α (*** p = 0.0001, Gluc 16 mM; and *** p = 8.6E-6 Gluc 25 mM), Il-1β (*** p = 2.2e-5, Gluc 16 mM; and *** p = 2.7E-9, Gluc 25 mM), and Il-6 (*** p = 1.8E-5, Gluc 16 mM; and *** p = 7.5E-12, Gluc 25 mM) mRNA in cultured astrocytes was assessed after high glucose (16 mM and 25 mM) treatment for 24 h ( c ). Similarly, the expression of Pdk 2 mRNA (### p = 1.7E-5, Gluc 25 mM + GSK) in astrocytes was assessed after co-treatment with high glucose (25 mM) and GSK0660 (a PPARβ/δ antagonist, 1 µM) for 24 h ( b ). The expression of Tnf-α (## p = 0.0022, KO + Gluc 25 mM), Il-1β (### p = 2.3E-9, KO + Gluc 25 mM) and Il-6 (### p = 0.0002, KO + Gluc 25 mM) mRNA was examined in cultured astrocytes isolated from WT and Pdk2 KO mice ( d ). Extracellular lactate was assessed after primary astrocytes isolated from WT and Pdk2 KO mice were exposed to high glucose for 24 hr by HPLC analysis ( e ). ECAR (* p = 0.0331, KO + Gluc 25 mM at 60 min; and ###p = 7.2E-6, KO + Gluc 25 mM at glycolytic capacity) ( f – g ) and OCR (* p = 0.0289, KO + Gluc 25 mM at 60 min) ( h ) were measured after primary astrocytes isolated from WT and Pdk2 KO mice were treated with high glucose (25 mM) for 24 h by an XF 24 Seahorse analyzer. Relative expression of Tnf-α (# p = 0.0340, Gluc 25 mM+Oxamate; and # p = 0.0246, Gluc 25 mM+GSK2837808A), Il-1β (## p = 0.0080, Gluc 25 mM + Oxamate; and ##p = 0.0020, Gluc 25 mM + GSK2837808A) , Il-6 (###p = 2.1E-5, Gluc 25 mM+Oxamate; and ## p = 0.0012, Gluc 25 mM + GSK2837808A) mRNA in astrocytes was assessed after co-treatment with high glucose (25 mM) and Oxamate (5 mM) or GSK2837808A (2 µM) for 24 h ( i ). The schematic diagram represents the timeline and condition for the hypothalamic slice culture experiment ( j ). Levels of TNF-α protein (## p = 0.0099, AAV5-GFAP-mCherry-Cre + Gluc 25 mM) in the culture media ( k ) and expression of Tnf-α (#p = 0.0294, AAV5-GFAP-mCherry-Cre+Gluc 25 mM) and Il-1β (## p = 0.0058, AAV5-GFAP-mCherry-Cre + Gluc 25 mM) mRNAs ( l ) in the cultured hypothalamus isolated from AAV5-GFAP-eYFP or AAV5-GFAP-mCherry-Cre-injected mice were assessed after high glucose treatment. Protein level was measured by ELISA. mRNA expression was assessed by real-time RT-PCR, and results are displayed as the fold increase of gene expression normalized to Gapdh . * p < 0.05, ** p < 0.01, or *** p < 0.001 versus the non-treated group; # p < 0.05, ## p < 0.01, or ### p < 0.001 versus indicated groups. One-way ANOVA ( a – c , i , k , l ) and two-way ANOVA with Tukey’s post hoc test ( d – h ), n = 6 ( a , c , d ), n = 3 ( b , i , k , l ), and n = 3–4 ( e – h ); mean ± SEM. Value of “ n ” indicates the number of sister wells in culture plates. Source data are provided as a Source data file. A diagram showing the hypothetical involvement of the PDK2-lactic acid axis in high glucose-induced inflammatory activation of astrocytes ( m ). Gluc glucose; GSK GSK0660.

Article Snippet: For immunofluorescence or 3, 3′-diaminobenzidine (DAB) staining, sections were incubated with primary antibodies against PDK2 (rabbit, 1:100; Abgent, San Diego, CA), Iba-1 (goat, 1:200; Novus Biologicals, Littleton, CO) or Iba-1 (rabbit, 1:1000; Wako Pure Chemical Corporation), GFAP (mouse, 1:200; Novus Biologicals, Littleton, CO) or GFAP (rabbit, 1.500; DAKO), βIII-tubulin (mouse, 1:200; Santa Cruz Biotechnology Inc.), NeuN (rabbit, 1:500, Merck Millipore), AGRP (rabbit, 1:200; Phoenix Pharmaceuticals Inc.), POMC (rabbit, 1:200, phoenix Pharmaceutical Inc.), NF-κB p65 (mouse, 1:200, Santa Cruz Biotechnology Inc), phosphorylated (Thr-172) and total forms of AMPKα (rabbit, 1:100; Cell Signaling Technology) overnight at 4°C.

Techniques: Expressing, Cell Culture, Isolation, Injection, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Activation Assay

Journal: Nature Communications

Article Title: Astrocytic pyruvate dehydrogenase kinase-2 is involved in hypothalamic inflammation in mouse models of diabetes

doi: 10.1038/s41467-020-19576-1

Figure Lengend Snippet: To determine the role of hypothalamic PDK2 or lactic acid in the regulation of food intake, we administered AZD7545 (6.4 nM of CSF concentration), GSK2837808A (2 μM of CSF concentration), or oxamate (25 μg) by multiple icv injections into mice starting from 5 d post-STZ injection to the following indicated time points. The schematic diagram represents the timeline of experimentation and route of administration of the inhibitors ( a ). The relative expression of Tnf-α , Il-1β , and Il-6 mRNA in the hypothalamic tissues isolated from AZD7545 or GSK2837808A or oxamate and STZ-injected mice was evaluated by real-time RT-PCR ( b ). Food intake ( c , d ) was assessed following AZD7545, GSK2837808A, or oxamate and STZ administration. Results for mRNA expression are displayed as the fold increase of mRNA expression normalized to Gapdh . * p or § p < 0.05, ** p < 0.01, or *** p < 0.001 versus the vehicle-treated control animals ( b ) or STZ + vehicle-treated animals ( c , d ); # p < 0.05, ## p < 0.01, or ### p < 0.001 versus indicated groups. One-way ANOVA ( b ) and two-way ANOVA with Tukey’s post hoc test ( c , d ), n = 4 ( b ), and n = 9 for STZ and 5 for other groups ( c , d ); mean ± SEM. Value of ‘n’ indicates the number of animals. Source data are provided as a Source data file. d day(s); STZ streptozotocin.

Article Snippet: For immunofluorescence or 3, 3′-diaminobenzidine (DAB) staining, sections were incubated with primary antibodies against PDK2 (rabbit, 1:100; Abgent, San Diego, CA), Iba-1 (goat, 1:200; Novus Biologicals, Littleton, CO) or Iba-1 (rabbit, 1:1000; Wako Pure Chemical Corporation), GFAP (mouse, 1:200; Novus Biologicals, Littleton, CO) or GFAP (rabbit, 1.500; DAKO), βIII-tubulin (mouse, 1:200; Santa Cruz Biotechnology Inc.), NeuN (rabbit, 1:500, Merck Millipore), AGRP (rabbit, 1:200; Phoenix Pharmaceuticals Inc.), POMC (rabbit, 1:200, phoenix Pharmaceutical Inc.), NF-κB p65 (mouse, 1:200, Santa Cruz Biotechnology Inc), phosphorylated (Thr-172) and total forms of AMPKα (rabbit, 1:100; Cell Signaling Technology) overnight at 4°C.

Techniques: Concentration Assay, Injection, Expressing, Isolation, Quantitative RT-PCR

Journal: Nature Communications

Article Title: Astrocytic pyruvate dehydrogenase kinase-2 is involved in hypothalamic inflammation in mouse models of diabetes

doi: 10.1038/s41467-020-19576-1

Figure Lengend Snippet: To further determine the role of hypothalamic PDK2 in diabetes-induced neuroinflammation and dysregulation of feeding behavior, we administered Ad-PDK2-GFP or Ad-GFP (1.0 × 10 9 pfu) via the icv route 1 w before STZ injection. The schematic diagram shows the experimentation timeline and the route of administration ( a ). PDK2 protein levels (### p = 4.5E-5, KO + Ad-PDK2-GFP + STZ) in the hypothalamus after 3 w of STZ and 4 w of adenovirus injection were assessed by Western blot analysis ( b ). Western blot band quantification for PDK2 protein levels normalized to α-tubulin. The relative expression of Tnf-α (## p = 0.0030, KO + Ad-PDK2-GFP + STZ), Il-1β (# p = 0.0380, KO + Ad-PDK2-GFP + STZ), and Il-6 (## p = 0.0020, KO + Ad-PDK2-GFP + STZ) mRNA in the hypothalamic tissues isolated from Ad-PDK2-GFP or Ad-GFP and WT mice was evaluated by real-time RT-PCR ( c ). Food intake (* p = 0.0130, KO + Ad-PDK2-GFP + STZ at 28 d) ( d ) and fasting blood glucose levels (* p = 0.0127, KO + Ad-PDK2-GFP + STZ at 28 d) ( e ) were assessed following Ad-PDK2-GFP or Ad-GFP and STZ administration. Arrows indicate the time points of adenovirus (Ad) and STZ administration. The relative expression of Npy (# p = 0.0489, KO + Ad-PDK2-GFP + STZ), Agrp (## p = 0.0090, KO + Ad-PDK2-GFP + STZ), and Pomc (#p = 0.0270, KO + Ad-PDK2-GFP + STZ) mRNA in the hypothalamic tissues isolated from Ad-PDK2-GFP or Ad-GFP and STZ-injected mice just after completion of behavioral assessments was evaluated by real-time RT-PCR ( f ). Results for mRNA expression are displayed as the fold increase of mRNA expression normalized to Gapdh . * p < 0.05, ** p < 0.01, or *** p < 0.001 versus the vehicle-treated control animals ( b , c , f ) or STZ/Ad-GFP + STZ-treated animals ( d , e ); # p < 0.05, ## p < 0.01 between the indicated groups. One-way ANOVA ( b , c , f ) and two-way ANOVA with Tukey’s post hoc test ( d , e ), n = 3 (b), n = 4 ( c , f ), and n = 6 ( d , e ); mean ± SEM. Value of “ n ” indicates the number of animals. Source data are provided as a Source data file. d day(s); STZ streptozotocin; pfu plaque-forming unit; ns not significant; ND not detected.

Article Snippet: For immunofluorescence or 3, 3′-diaminobenzidine (DAB) staining, sections were incubated with primary antibodies against PDK2 (rabbit, 1:100; Abgent, San Diego, CA), Iba-1 (goat, 1:200; Novus Biologicals, Littleton, CO) or Iba-1 (rabbit, 1:1000; Wako Pure Chemical Corporation), GFAP (mouse, 1:200; Novus Biologicals, Littleton, CO) or GFAP (rabbit, 1.500; DAKO), βIII-tubulin (mouse, 1:200; Santa Cruz Biotechnology Inc.), NeuN (rabbit, 1:500, Merck Millipore), AGRP (rabbit, 1:200; Phoenix Pharmaceuticals Inc.), POMC (rabbit, 1:200, phoenix Pharmaceutical Inc.), NF-κB p65 (mouse, 1:200, Santa Cruz Biotechnology Inc), phosphorylated (Thr-172) and total forms of AMPKα (rabbit, 1:100; Cell Signaling Technology) overnight at 4°C.

Techniques: Injection, Western Blot, Expressing, Isolation, Quantitative RT-PCR

Journal: Nature Communications

Article Title: Astrocytic pyruvate dehydrogenase kinase-2 is involved in hypothalamic inflammation in mouse models of diabetes

doi: 10.1038/s41467-020-19576-1

Figure Lengend Snippet: p-AMPK (# p = 0.0157, KO + STZ) and AMPK protein levels in the hypothalamic tissues isolated from WT and Pdk2 KO mice at 3 w post-STZ/vehicle injection were assessed by Western blot analysis ( a ). Co-immunofluorescence staining of mouse brain tissues showed p-AMPK and βIII-tubulin-positive neurons (### p = 1.4E-7, not co-localized with p-AMPK; and (### p = 3.7E-7, co-localized with p-AMPK) in the ARC at 3 w post-STZ/vehicle injection ( b ). The percentage of p-AMPK-positive neurons was calculated from the total number of cells present per mm 2 using six randomly selected fields. Scale bar indicates 100 µm. The expression of p-AMPK (** p = 0.0051, lactic acid) and AMPK proteins ( c ), as well as Agrp ( d ) in mHypoE-N41 cells, and Pomc mRNA in mHypoE-N43/5 cells ( e ) after treating with lactic acid (7.1 mM) for 72 h was assessed by Western blot and real-time RT-PCR, respectively. The effect of Compound C (16 µM) and metformin (100 µM) on lactic acid-induced increased expression of Agrp mRNA (## p = 0.0011, lactic acid+ComC) in mHypoE-N41 cells was observed ( d ). Results for mRNA expression are displayed as the fold increase of gene expression normalized to Gapdh . The diagram shows the proposed lactic acid-AMPK axis in the regulation of the AgRP ( f ). Representative traces of I NMDA induced by NMDA (100 μM) in the absence or presence of l -lactate (7.1 mM) in NPY/AgRP-expressing neurons (left) and POMC-expressing neurons (right) ( g ). l -lactate-induced changes of the NMDA-induced currents in NPY and POMC neurons (## p = 0.0006, POMC) ( h ). Representative traces of sEPSC NMDA in the absence (left) or presence (right) of l -lactate in NPY neurons ( i ). Cumulative probability distribution for inter-event interval (left) and amplitude (right) of sEPSC NMDA with or without l -lactate ( j ). The plot includes 195 events for control (thin lines) and 143 events for l -lactate (thick lines). Representative traces before, during, and after the application of NMDA in the absence (left) or presence (right) of l -lactate in NPY neurons in the current-clamp condition ( k ). NMDA-induced changes of action potential frequency in the absence or presence of l -lactate in NPY neurons (* p = 0.0220, NMDA + l -lactate) ( l ). Open and closed circles represent the value from individual neurons and their average respectively. Insets represent the l -lactate-induced changes in the frequency (left) and amplitude (right) of sEPSC NMDA . Dotted lines represent the relative control of basal frequency and amplitude of sEPSC NMDA . * p < 0.05, ** p < 0.01, or *** p < 0.001 versus the vehicle-treated control animals/cells. Two-way ANOVA ( a , b ), one-way ANOVA with Tukey’s post hoc test ( d , h ), two-tailed Student’s t -test ( c , e ), two-tailed paired t -test ( h , j , l ), unpaired t -tes t , n = 9 individual neurons ( h ), n = 195 events for control and 143 for l -lactate ( j ), n = 6 individual neurons ( l ) n = 3 animals per group ( a ) or sister wells ( c – e ), and n = 5 animals per group ( b ); mean ± SEM. Source data are provided as a Source data file. ComC Compound C; Met metformin; L-lac l -lactate; Amp Amplitude; Freq Frequency; NC negative control; ns not significant.

Article Snippet: For immunofluorescence or 3, 3′-diaminobenzidine (DAB) staining, sections were incubated with primary antibodies against PDK2 (rabbit, 1:100; Abgent, San Diego, CA), Iba-1 (goat, 1:200; Novus Biologicals, Littleton, CO) or Iba-1 (rabbit, 1:1000; Wako Pure Chemical Corporation), GFAP (mouse, 1:200; Novus Biologicals, Littleton, CO) or GFAP (rabbit, 1.500; DAKO), βIII-tubulin (mouse, 1:200; Santa Cruz Biotechnology Inc.), NeuN (rabbit, 1:500, Merck Millipore), AGRP (rabbit, 1:200; Phoenix Pharmaceuticals Inc.), POMC (rabbit, 1:200, phoenix Pharmaceutical Inc.), NF-κB p65 (mouse, 1:200, Santa Cruz Biotechnology Inc), phosphorylated (Thr-172) and total forms of AMPKα (rabbit, 1:100; Cell Signaling Technology) overnight at 4°C.

Techniques: Isolation, Injection, Western Blot, Immunofluorescence, Staining, Expressing, Quantitative RT-PCR, Two Tailed Test, Negative Control

Journal: Nature Communications

Article Title: Astrocytic pyruvate dehydrogenase kinase-2 is involved in hypothalamic inflammation in mouse models of diabetes

doi: 10.1038/s41467-020-19576-1

Figure Lengend Snippet: To determine the role of hypothalamic PDK2 in the regulation of neuroinflammation and feeding behavior, we administered AAV5-GFAP-mCherry-Cre or AAV5-GFAP-eYFP (4.3 × 10 9 virus particles) into the mediobasal hypothalamus of Pdk2 floxed mice, bilaterally, using a stereotaxic device 1 w before STZ injection. The schematic diagram presents the experiment timeline and route of administration ( a ). The relative expression of Tnf-α (# p = 0.0141, AAV5-GFAP-mCherry-Cre+STZ), IL-1β (# p = 0.0316, AAV5-GFAP-mCherry-Cre+STZ), and IL-6 (## p = 0.0016, AAV5-GFAP-mCherry-Cre+STZ) mRNA in the hypothalamic tissues isolated from AAV5-GFAP-mCherry-Cre or AAV5-GFAP-eYFP and STZ-injected mice was evaluated by real-time RT-PCR ( b ). GFAP (## p = 0.0020, AAV5-GFAP-mCherry-Cre + STZ) and Iba-1 (## p = 0.0053, AAV5-GFAP-mCherry-Cre+STZ) immunofluorescence staining revealed their increased immunoreactivity in the hypothalamus of diabetes Pdk2 floxed mice, but Pdk2 -deficiency in hypothalamic astrocytes attenuated such an increase in the immunoreactivities at 3 w post-STZ injection ( c ). Microscope data were gathered using five randomly selected fields captured at the same magnification. Scale bar indicates 200 µm. Lactate concentration (## p = 0.0030, AAV5-GFAP-mCherry-Cre+STZ) in hypothalamic tissues collected from AAV5-GFAP-mCherry-Cre- or AAV5-GFAP-eYFP- and STZ-injected mice was measured with an HPLC analyzer ( d ). Quantification of the lactate concentration from HPLC graphs is shown. Food intake (* p = 0.0393, AAV5-GFAP-mCherry-Cre+STZ at 4 w) was assessed following AAV5-GFAP-mCherry-Cre or AAV5-GFAP-eYFP and STZ administration ( e ). Arrows indicate the time points of adeno-associated virus (AAV), and STZ administration. The relative expression of Npy (# p = 0.0140, AAV5-GFAP-mCherry-Cre+STZ), Agrp (# p = 0.0380, AAV5-GFAP-mCherry-Cre+STZ) and Pomc (## p = 0.0090, AAV5-GFAP-mCherry-Cre + STZ) mRNA in hypothalamic tissues isolated from AAV5-GFAP-mCherry-Cre- or AAV5-GFAP-eYFP- and STZ-injected mice just after completion of behavioral assessments was evaluated by real-time RT-PCR ( f ). mRNA expression results are displayed as the fold increase of mRNA expression normalized to Gapdh . * p < 0.05, ** p < 0.01, or *** p < 0.001 versus the vehicle-treated control animals ( b – d , f ) or STZ/AAV5-GFAP-eYFP+STZ-treated animals ( e ); # p < 0.05, ## p < 0.01, or ### p < 0.001 versus indicated groups. One-way ANOVA ( b – d , f ) and two-way ANOVA ( e ) with Tukey’s post hoc test, n = 3 ( b , d , f ), n = 5 ( c ), and n = 6 ( e ); mean ± SEM. Value of “ n ” indicates the number of animals. Source data are provided as a Source data file. w week(s); IR immunoreactivity.

Article Snippet: For immunofluorescence or 3, 3′-diaminobenzidine (DAB) staining, sections were incubated with primary antibodies against PDK2 (rabbit, 1:100; Abgent, San Diego, CA), Iba-1 (goat, 1:200; Novus Biologicals, Littleton, CO) or Iba-1 (rabbit, 1:1000; Wako Pure Chemical Corporation), GFAP (mouse, 1:200; Novus Biologicals, Littleton, CO) or GFAP (rabbit, 1.500; DAKO), βIII-tubulin (mouse, 1:200; Santa Cruz Biotechnology Inc.), NeuN (rabbit, 1:500, Merck Millipore), AGRP (rabbit, 1:200; Phoenix Pharmaceuticals Inc.), POMC (rabbit, 1:200, phoenix Pharmaceutical Inc.), NF-κB p65 (mouse, 1:200, Santa Cruz Biotechnology Inc), phosphorylated (Thr-172) and total forms of AMPKα (rabbit, 1:100; Cell Signaling Technology) overnight at 4°C.

Techniques: Injection, Expressing, Isolation, Quantitative RT-PCR, Immunofluorescence, Staining, Microscopy, Concentration Assay

Journal: Journal of Ageing and Longevity

Article Title: Momordica charantia Extract Treatment Extends the Healthy Lifespan of Aging Mice via the Bitter Taste Receptor/mTOR Pathway

doi: 10.3390/jal4040021

Figure Lengend Snippet: Figure 3. Effect of Momordica charantia (MC) extract treatment on signal transduction from T2R1 to mTOR in the skeletal muscle of aged male and female mice. The levels of T2R1 (bitter taste receptor) (A), IP3 (B), PDK1 (C,D), AKT (C,E), and mTOR (F) were measured. Values are expressed as the mean ± SD derived from six animals. Scale bar = 100 µm. The intensity of the signal was calculated from five random visual fields with the same area using ImageJ software. ** p < 0.01; * p < 0.05. ns: not significant.

Article Snippet: After electrophoresis, the membranes were incubated with primary antibodies against pyruvate dehydrogenase kinase isoform 1 (PDK1) (1:1000; Proteintech Group, Rosemont, IL, USA), protein kinase B (Akt) (1:1000; Cell Signaling Technology, Inc.), sterol regulatory element-binding protein (SREBP) (1:1000; Proteintech Group), and β-actin (1:5000; Sigma-Aldrich, St. Louis, MO, USA) for 1 h at room temperature. β-actin was used as a loading control.

Techniques: Transduction, Derivative Assay, Software

Journal: International Journal of Molecular Sciences

Article Title: Suppression of Pyruvate Dehydrogenase Kinase by Dichloroacetate in Cancer and Skeletal Muscle Cells Is Isoform Specific and Partially Independent of HIF-1α

doi: 10.3390/ijms22168610

Figure Lengend Snippet: Overview of the antibodies used for immunoblotting. Abbreviations: Ab—antibody, Mo—mouse, O/N—overnight, Rb—rabbit. Cell Signaling Technology (Danvers, MA, USA), Novus Biologicals (Centennial, CO, USA), Abcam (Cambridge, UK).

Article Snippet: PDK2 , 46 , Novus Biologicals , NBP1-87307 , Rb , 1:800 , O/N , Bio-Rad , 1706515 , 1:8000 , 1 h.

Techniques: Western Blot

Journal: Oncology Letters

Article Title: Serum pyruvate dehydrogenase kinase as a prognostic marker for cholangiocarcinoma

doi: 10.3892/ol.2019.10185

Figure Lengend Snippet: Candidate mitochondrial proteins involved in the glucose metabolism of cholangiocarcinoma.

Article Snippet: Then, the endogenous peroxidase activity of the sections was blocked with 3% H 2 O 2 in methanol for 1 h in the dark and non-specific background binding was blocked by incubation with 20% fetal bovine serum (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 2 h. The sections were then incubated with 150 µl rabbit polyclonal antibody against human PDK1, PDK2, PDK3 and PDK4 (Biorbyt, Cambridge, UK; cat. nos. of the anti-PDK1, -PDK2, -PDK3 and -PDK4 antibodies are orb14422, orb137873, orb312719 and orb136059, respectively) at a dilution of 1:100 at 4°C overnight.

Techniques:

Journal: Oncology Letters

Article Title: Serum pyruvate dehydrogenase kinase as a prognostic marker for cholangiocarcinoma

doi: 10.3892/ol.2019.10185

Figure Lengend Snippet: (A) Representative PDK staining in cancerous and non-cancerous tissues of the surgical specimens from patients with CCA (magnification, ×400; scale bar, 50 µm). Arrows in the ‘Adjacent’ column indicate cholangiocytes, and arrows in the ‘CCA’ column indicate CCA cells. (B) PDK1, PDK2 and PDK3, but not PDK4, were significantly overexpressed in CCA tissues compared with adjacent non-cancerous tissues (P<0.0001). PDK, pyruvate dehydrogenase kinase; CCA, cholangiocarcinoma.

Article Snippet: Then, the endogenous peroxidase activity of the sections was blocked with 3% H 2 O 2 in methanol for 1 h in the dark and non-specific background binding was blocked by incubation with 20% fetal bovine serum (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 2 h. The sections were then incubated with 150 µl rabbit polyclonal antibody against human PDK1, PDK2, PDK3 and PDK4 (Biorbyt, Cambridge, UK; cat. nos. of the anti-PDK1, -PDK2, -PDK3 and -PDK4 antibodies are orb14422, orb137873, orb312719 and orb136059, respectively) at a dilution of 1:100 at 4°C overnight.

Techniques: Staining

Journal: Oncology Letters

Article Title: Serum pyruvate dehydrogenase kinase as a prognostic marker for cholangiocarcinoma

doi: 10.3892/ol.2019.10185

Figure Lengend Snippet: PDK1, PDK2 and PDK3 levels in the sera of CCA, BBD and healthy control groups. While PDK1 and PDK2 were rarely detected in all three groups, the PDK3 level in the CCA group was significantly higher when compared with the other two groups. Slightly less than a half of the patients with CCA exhibited high levels of PDK3 in the sera. PDK, pyruvate dehydrogenase kinase; CCA, cholangiocarcinoma; BBD, benign biliary disease.

Article Snippet: Then, the endogenous peroxidase activity of the sections was blocked with 3% H 2 O 2 in methanol for 1 h in the dark and non-specific background binding was blocked by incubation with 20% fetal bovine serum (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 2 h. The sections were then incubated with 150 µl rabbit polyclonal antibody against human PDK1, PDK2, PDK3 and PDK4 (Biorbyt, Cambridge, UK; cat. nos. of the anti-PDK1, -PDK2, -PDK3 and -PDK4 antibodies are orb14422, orb137873, orb312719 and orb136059, respectively) at a dilution of 1:100 at 4°C overnight.

Techniques:

Journal: Experimental & molecular medicine

Article Title: Increased oxidative phosphorylation through pyruvate dehydrogenase kinase 2 deficiency ameliorates cartilage degradation in mice with surgically induced osteoarthritis.

doi: 10.1038/s12276-025-01400-9

Figure Lengend Snippet: Fig. 2 PDK2 deficiency reduces the severity of cartilage degradation, oxidative stress-related DNA damage and pain-related behaviors in DMM-induced OA mice. a Safranin-O staining and immunostaining for MMP13 and 8-oxo-dG were performed on WT and Pdk2 KO mice 8 weeks after DMM and sham surgery, and representative images are displayed. Scale bars, 100 μm. b OARSI grade, subchondral bone plate thickness and osteophyte size were quantified and are expressed as mean ± s.e.m. *P < 0.05, Mann–Whitney U test. n = 7. c Quantification of the percentage of MMP13- and 8-oxo-dG-positive chondrocytes above the tidemark. ***P < 0.001; mean ± s.e.m.; Mann–Whitney U test; n = 7. d Pain behavior tests were conducted once a week on DMM and sham-operated mice. Weight bearing on the hind paw was assessed by an incapacitance test, which represents the ratio of weight bearing between the ipsilateral and contralateral hind paws; thus, any percentage less than 100% indicates hind limb unweighting. Thermal and mechanical pain sensations were assessed with the Hargreaves test via a hot plate and the von Frey test, respectively. *P < 0.05, compared between WT and Pdk2 KO mice in the DMM group, Mann–Whitney U test. n = 7.

Article Snippet: The membranes were blocked with 5% skim milk in PBS with 0.25% Tween-20 (PBST) and incubated with primary antibodies against PDK1 (Abcam, #ab202468), PDK2 (#ab68164), PDK3 (#ab154549), PDK4 (#ab214938), phospho-PDHE1α (Ser232, Millipore, #AP1063), PDH-E1 (Santa Cruz Biotechnology, #sc377092), Col2 (#ab34712), Sirt1 (Cell Signaling Technology, #9475), MMP13 (#ab51072), p-p38 (#4631), p38 (#9212), p-JNK (#9251), JNK (#9252), pAMPKα (#2531), AMPKα (#2532), p-FoxO3 (#9466), FoxO3a (#2497), p-mTOR (#2971), mTOR (#2972), p-p65 (#3033), p65 (#8242), p-Akt (#9271), Akt (#9272) and β-actin (Sigma-Aldrich, #A1978) overnight at 4 °C.

Techniques: Staining, Immunostaining, MANN-WHITNEY

Journal: Experimental & molecular medicine

Article Title: Increased oxidative phosphorylation through pyruvate dehydrogenase kinase 2 deficiency ameliorates cartilage degradation in mice with surgically induced osteoarthritis.

doi: 10.1038/s12276-025-01400-9

Figure Lengend Snippet: Fig. 3 PDK2 deficiency partially restores IL-1β-induced metabolic alterations, leading to increases in ATP and NAD+/NADH levels in primary chondrocytes. a,b, OCRs (a) and ECARs (b) were measured in primary chondrocytes isolated from WT and Pdk2 KO mice via an XF96 Seahorse analyzer. Basal respiration and ATP production in the OCR analysis and glycolysis and glycolytic capacity in the ECAR analysis at one time point were quantified and are presented as the mean ± s.e.m. *P < 0.05, Mann–Whitney U test. n = 4. c, d, The ATP levels (c) and NAD+/ NADH ratios (d) in the culture media were quantified via colorimetric assays at 570 nm and 450 nm, respectively; mean ± s.e.m., *P < 0.05, Mann–Whitney U test. n = 3.

Article Snippet: The membranes were blocked with 5% skim milk in PBS with 0.25% Tween-20 (PBST) and incubated with primary antibodies against PDK1 (Abcam, #ab202468), PDK2 (#ab68164), PDK3 (#ab154549), PDK4 (#ab214938), phospho-PDHE1α (Ser232, Millipore, #AP1063), PDH-E1 (Santa Cruz Biotechnology, #sc377092), Col2 (#ab34712), Sirt1 (Cell Signaling Technology, #9475), MMP13 (#ab51072), p-p38 (#4631), p38 (#9212), p-JNK (#9251), JNK (#9252), pAMPKα (#2531), AMPKα (#2532), p-FoxO3 (#9466), FoxO3a (#2497), p-mTOR (#2971), mTOR (#2972), p-p65 (#3033), p65 (#8242), p-Akt (#9271), Akt (#9272) and β-actin (Sigma-Aldrich, #A1978) overnight at 4 °C.

Techniques: Isolation, MANN-WHITNEY

Journal: Experimental & molecular medicine

Article Title: Increased oxidative phosphorylation through pyruvate dehydrogenase kinase 2 deficiency ameliorates cartilage degradation in mice with surgically induced osteoarthritis.

doi: 10.1038/s12276-025-01400-9

Figure Lengend Snippet: Fig. 5 PDK2 deficiency enhances FoxO3a signaling and prevents the downregulation of AMPK signaling, while it suppresses p38 MAPK activity under IL-1β-induced catabolic conditions. a The phosphorylation of FoxO3a, AMPKα, p38, JNK, mTOR, p65 and Akt was assessed in primary chondrocytes from WT and Pdk2 KO mice stimulated with IL-1β (20 ng/ml) for the indicated durations. The experiments were performed in triplicate, and representative blots are shown. b The western blot band quantification for phosphorylated proteins was based on normalization to the corresponding nonphosphorylated total protein. *P < 0.05. Mann–Whitney U test. n = 3.

Article Snippet: The membranes were blocked with 5% skim milk in PBS with 0.25% Tween-20 (PBST) and incubated with primary antibodies against PDK1 (Abcam, #ab202468), PDK2 (#ab68164), PDK3 (#ab154549), PDK4 (#ab214938), phospho-PDHE1α (Ser232, Millipore, #AP1063), PDH-E1 (Santa Cruz Biotechnology, #sc377092), Col2 (#ab34712), Sirt1 (Cell Signaling Technology, #9475), MMP13 (#ab51072), p-p38 (#4631), p38 (#9212), p-JNK (#9251), JNK (#9252), pAMPKα (#2531), AMPKα (#2532), p-FoxO3 (#9466), FoxO3a (#2497), p-mTOR (#2971), mTOR (#2972), p-p65 (#3033), p65 (#8242), p-Akt (#9271), Akt (#9272) and β-actin (Sigma-Aldrich, #A1978) overnight at 4 °C.

Techniques: Activity Assay, Phospho-proteomics, Western Blot, MANN-WHITNEY

Journal: Experimental & molecular medicine

Article Title: Increased oxidative phosphorylation through pyruvate dehydrogenase kinase 2 deficiency ameliorates cartilage degradation in mice with surgically induced osteoarthritis.

doi: 10.1038/s12276-025-01400-9

Figure Lengend Snippet: Fig. 6 PDK2 is involved in a positive feedback loop between p38 MAPK and mitochondrial ROS production. a p38 MAPK inhibition led to a decrease in ROS production and cellular senescence in WT chondrocytes but not in Pdk2-deficient chondrocytes. Chondrocytes from WT and Pdk2 KO plants were pretreated with the p38 MAPK inhibitor SB203580 and the mitochondrial ROS inhibitor Mito-TEMPO for 30 min. The cells were then treated with IL-1β (10 ng/ml) for 24 h and stained with DHE (red) for total cellular ROS, 8-oxo-dG (green) for DNA damage, and MitoSOX (red) for mitochondrial ROS, and their nuclei were counterstained with DAPI (blue). Cellular senescence was assessed via SA-β-gal staining. Scale bar, 100 μm. DHE, 8-oxo-dG and MitoSOX fluorescence-positive cells were quantified and expressed as ratios to DAPI-positive cells. The area of SA-β-gal expression was quantified via densitometry with ImageJ software. *P < 0.05, #P < 0.05. Mann–Whitney U test. n = 4. b p38 MAPK phosphorylation was assessed in the presence of a p38 inhibitor (SB203580) and a mitochondrial ROS inhibitor (Mito-TEMPO). Chondrocytes from WT and Pdk2 KO mice were stimulated with IL-1β (20 ng/ml) for the indicated times and subjected to western blot analysis to assess the phosphorylated and total protein levels of p38 MAPK. *P < 0.05 compared with the fold change in p-p38/p38 in WT PBS controls at 15 min after stimulation. #P < 0.05 compared with p-p38/p38 in Pdk2 KO PBS controls at 15 min after stimulation. Mann–Whitney U test. n = 4.

Article Snippet: The membranes were blocked with 5% skim milk in PBS with 0.25% Tween-20 (PBST) and incubated with primary antibodies against PDK1 (Abcam, #ab202468), PDK2 (#ab68164), PDK3 (#ab154549), PDK4 (#ab214938), phospho-PDHE1α (Ser232, Millipore, #AP1063), PDH-E1 (Santa Cruz Biotechnology, #sc377092), Col2 (#ab34712), Sirt1 (Cell Signaling Technology, #9475), MMP13 (#ab51072), p-p38 (#4631), p38 (#9212), p-JNK (#9251), JNK (#9252), pAMPKα (#2531), AMPKα (#2532), p-FoxO3 (#9466), FoxO3a (#2497), p-mTOR (#2971), mTOR (#2972), p-p65 (#3033), p65 (#8242), p-Akt (#9271), Akt (#9272) and β-actin (Sigma-Aldrich, #A1978) overnight at 4 °C.

Techniques: Inhibition, Staining, Expressing, Software, MANN-WHITNEY, Phospho-proteomics, Western Blot

Journal: Experimental & molecular medicine

Article Title: Increased oxidative phosphorylation through pyruvate dehydrogenase kinase 2 deficiency ameliorates cartilage degradation in mice with surgically induced osteoarthritis.

doi: 10.1038/s12276-025-01400-9

Figure Lengend Snippet: Fig. 7 Schematic diagram depicting the anabolic effects of metabolic reprogramming toward oxidative phosphorylation caused by PDK2 deficiency under catabolic conditions. Among PDK isoforms, PDK2 was expressed primarily under catabolic and in vivo OA conditions. The loss of PDK2 function enhanced OxPhos and ATP/NAD+ production, which led to a reduction in oxidative stress. Mechanistically, PDK2 plays a crucial role in the positive feedback loop between oxidative stress and p38 MAPK under catabolic conditions in chondrocytes.

Article Snippet: The membranes were blocked with 5% skim milk in PBS with 0.25% Tween-20 (PBST) and incubated with primary antibodies against PDK1 (Abcam, #ab202468), PDK2 (#ab68164), PDK3 (#ab154549), PDK4 (#ab214938), phospho-PDHE1α (Ser232, Millipore, #AP1063), PDH-E1 (Santa Cruz Biotechnology, #sc377092), Col2 (#ab34712), Sirt1 (Cell Signaling Technology, #9475), MMP13 (#ab51072), p-p38 (#4631), p38 (#9212), p-JNK (#9251), JNK (#9252), pAMPKα (#2531), AMPKα (#2532), p-FoxO3 (#9466), FoxO3a (#2497), p-mTOR (#2971), mTOR (#2972), p-p65 (#3033), p65 (#8242), p-Akt (#9271), Akt (#9272) and β-actin (Sigma-Aldrich, #A1978) overnight at 4 °C.

Techniques: Phospho-proteomics, In Vivo