Journal: Journal of Clinical Investigation

Article Title: Adipocyte-specific deletion of Ip6k1 reduces diet-induced obesity by enhancing AMPK-mediated thermogenesis

doi: 10.1172/jci85510

Figure Lengend Snippet: Figure 6. IP6K1 reduces AMPK-mediated adipocyte browning. (A) AMPK stimulatory phosphorylation (T172) and its activity are enhanced in WT mice following acute cold exposure (n = 4–5 mice per group). (B) AMPK phosphorylation is dramatically augmented in acute-cold-exposed AdKOs (n = 3 mice per group). (C) AMPK activity is higher in 2 separate preparations (n = 6 mice per preparation) of AdKO beige adipocytes differentiated from IWAT SVF in vitro. (D) siRNA-mediated AMPKα depletion substantially reduces AMPKα protein level in LoxP and AdKO beige adipocytes. Moreover, PGC1-α and UCP1 protein levels, which are higher in AdKO adipocytes, are reduced following AMPKα depletion. Data represent results obtained from 2 independent experiments. (E) Ucp1 mRNA expression is slightly reduced by AMPKα siRNA treatment in LoxP mice. Conversely, Ucp1 expression, which is significantly higher in AdKO adipocytes compared with LoxPs, is substantially reduced following AMPKα depletion. Data represent results obtained from 5 experimental replicates. (F) AdKO beige adipocytes express higher levels of Ucp1 compared with LoxP adipocytes under basal conditions (control). TNP (5 μM, 16 hours) sub- stantially enhances Ucp1 expression in LoxP adipocytes, whereas it does not influence Ucp1 in AdKO adipocytes. The AMPK activator AICAR enhances Ucp1 expression to a higher extent in LoxP adipocytes. Data represent results obtained from 5 experimental replicates. (G) TNP (5 μM) enhances AMPK phosphorylation and activity in beige adipocytes differentiated in vitro from isolated from WT IWAT SVF. Immunoblot represents results from at least 3 independent experiments. (H and I) A single dose of TNP (20 mg/kg body weight; i.p.) enhances AMPK phosphorylation and activity on ACC in the IWAT of HFD-fed mice (n = 3 mice per group; t test). Data in all panels are expressed as mean ± SEM. *P < 0.05, **P < 0.01.

Article Snippet: LKB1 only (catalog L15-30G), unphosphorylated/inactive AMPK (α2/β1/γ1) (catalog P48-14H), active CaMKKβ (catalog C18-10G), and phosphorylated/active AMPK (catalog P48-10H) were from SignalChem.

Techniques: Phospho-proteomics, Activity Assay, In Vitro, Expressing, Control, Isolation, Western Blot

Journal: Journal of Clinical Investigation

Article Title: Adipocyte-specific deletion of Ip6k1 reduces diet-induced obesity by enhancing AMPK-mediated thermogenesis

doi: 10.1172/jci85510

Figure Lengend Snippet: Figure 7. IP6 and IP6K1 differentially regulate AMPK stimulatory phosphorylation. (A) Immunoblot demonstrates that overexpression of GST-IP6K1 diminishes AMPK and ACC phosphorylation levels in HEK293 cells under basal (+), glucose-deprived (–), and glucose-reintroduced (–/+) conditions. Data represent results from at least 3 independent experiments. (B) IP6, at increasing concentrations, stimulates AMPKα2/β1/γ1 (AMPKα T172) phosphorylation mediated by LKB1-only (120 ng/reaction). Data represent results from at least 3 independent experiments. (C) Quantification of B reveals that IP6 leads to 6- to 7-fold enhancements in LKB1-mediated AMPK phosphorylation in vitro. Data represent results from at least 3 independent experiments (1-way ANOVA). (D) Among inositol phosphates, IP6 most potently stimulates LKB1-only–mediated AMPKα2/β1/γ1 phosphorylation (IP6 > IP5 > IP4 = IP5* > IP3 > 5-IP7). IP5 displays partial stimulatory effect. IP6K1 products IP5* and 5-IP7 are ineffective. Data represent results from duplicate experiments. (E) IP6K1 inhibits IP6-mediated AMPKα2/β11 phosphorylation by LKB1-only, whereas the inactive IP6K1 mutant is much less efficient. Data represent results from 3 independent experiments. (F) 5-IP7 reverses IP6-mediated AMPKα2/β1/γ1 phosphorylation by LKB1-only. Data represent results obtained from 3 indepen- dent experiments. (G) IP6 also enhances LKB1-complex–mediated AMPKα2/β11 phosphorylation. Other inositol polyphosphates are ineffective. Data rep- resent results obtained from duplicate experiments. (H) IP6 enhances CaMKKβ-mediated AMPKα21/γ1 phosphorylation. Data represent results obtained from duplicate experiments. (I) IP6 does not enhance LKB1-mediated phosphorylation of heat-denatured AMPKα2/β1/γ1. Data represent results obtained from 3 independent experiments. (J) IP6-mediated enhancement of AMPK phosphorylation is largely similar when AMPKα is used alone or in a complex with AMPKα2/β1/γ1. Data represent results obtained from 3 independent experiments. (K) AMP stimulates LKB1-mediated AMPKα2/β11 phosphorylation at 50- to 100-μM concentrations. Data represent results obtained from duplicate experiments. (L) IP6 or 5-IP7 does not influence active AMPK’s activity on its target SAMStide (n = 3; 1-way ANOVA). Data are expressed as mean ± SEM. §P < 0.0001.

Article Snippet: LKB1 only (catalog L15-30G), unphosphorylated/inactive AMPK (α2/β1/γ1) (catalog P48-14H), active CaMKKβ (catalog C18-10G), and phosphorylated/active AMPK (catalog P48-10H) were from SignalChem.

Techniques: Phospho-proteomics, Western Blot, Over Expression, In Vitro, Mutagenesis, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation of p62 by AMP-activated protein kinase mediates autophagic cell death in adult hippocampal neural stem cells

doi: 10.1074/jbc.M117.780874

Figure Lengend Snippet: AMPK activation is required for ACD in I(−) HCN cells. A, time-course analysis of AMPK activation and autophagy induction following insulin withdrawal. Blots shown are representative of three independent experiments, which yielded similar results. B, pharmacological inhibition of AMPK with CC (0.5 μm) decreased cell death (n = 5). C, administration of CC attenuated autophagy. The levels of LC3-II and p62 were measured 5 h after insulin withdrawal. D, quantification of LC3-II and p62 after normalization to β-actin (n = 5). E, HCN cells were transfected with the GFP-LC3 plasmid, and LC3 puncta were visualized after insulin withdrawal for 5 h. F, analysis of E. Quantification of the puncta from three independent experiments (n = 40) is shown. G, AMPK α2 knock-out decreased cell death following insulin withdrawal. Cell death of AMPK α2 KO (Sg-AMPK α2) and control (Sg-con) HCN cells was measured at the indicated time points following insulin withdrawal (n = 4). H, autophagy flux following insulin withdrawal (5 h) was abrogated in AMPK α2 KO HCN cells. BafA1 (20 nm) was added 1 h before sampling. I, quantification of LC3-II and p62 after normalization to β-actin (n = 4). Error bars represent ±S.D. from independent assays. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Scale bar, 10 μm. p-AMPK, phospho-AMPK; t-AMPK, total AMPK.

Article Snippet: In addition, recombinant human AMPK protein (combination of α2/β1/γ1 subunits; SignalChem, P48-10H) expressed in Sf9 cells was used for the in vitro kinase assay.

Techniques: Activation Assay, Inhibition, Transfection, Plasmid Preparation, Knock-Out, Control, Sampling

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation of p62 by AMP-activated protein kinase mediates autophagic cell death in adult hippocampal neural stem cells

doi: 10.1074/jbc.M117.780874

Figure Lengend Snippet: Genetic activation of AMPK enhances ACD in I(−) HCN cells. A, overexpression of AMPK-CA increased the level of LC3-II and decreased that of p62 but did not activate caspase-3 following insulin withdrawal for 5 h. STS (0.5 μm for 8 h) was used as a positive control for apoptosis. C-cas3, cleaved caspase-3. B, quantification of LC3-II and p62 after normalization to β-actin (n = 4). C, AMPK-CA increased cell death more than did I(−) alone. Z-VAD (20 μm) failed to reduce cell death induced by AMPK-CA but efficiently reduced STS-induced apoptosis (n = 5). D, AMPK-CA increased autophagic flux in I(−) HCN cells. Insulin was withdrawn for 5 h, and BafA1 (20 nm) was added 1 h before sampling. E, quantification of LC3-II and p62 after normalization to β-actin (n = 5). F, AMPK-CA (CA) increased autophagic flux. Autophagic flux was measured using tandem mRFP-GFP-LC3 after 5-h insulin withdrawal. G, analysis of F. The red and yellow puncta were counted from three independent experiments (n = 29). H, Atg7 knockdown abrogated AMPK-CA-triggered cell death. Cell death was measured 24 h after insulin withdrawal (n = 6). I, Atg7 knockdown abrogated AMPK-CA-triggered autophagic flux. AMPK-CA failed to affect LC3-II or p62 in Sh-Atg7 HCN cells analyzed 5 h after insulin withdrawal. J, quantification of LC3-II and p62 after normalization to β-actin (n = 9). Error bars represent ±S.D. from independent assays. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant. Scale bar, 10 μm. EV, empty vector; con, control.

Article Snippet: In addition, recombinant human AMPK protein (combination of α2/β1/γ1 subunits; SignalChem, P48-10H) expressed in Sf9 cells was used for the in vitro kinase assay.

Techniques: Activation Assay, Over Expression, Positive Control, Sampling, Knockdown, Plasmid Preparation, Control

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation of p62 by AMP-activated protein kinase mediates autophagic cell death in adult hippocampal neural stem cells

doi: 10.1074/jbc.M117.780874

Figure Lengend Snippet: p62 is required for ACD in I(−) HCN cells. A, schematic representation of the functional domains and predicted AMPK α2 phosphorylation sites of p62. B, knockdown of p62 abrogated an increase in LC3-II following insulin withdrawal. C, quantification of LC3-II after normalization to β-actin (n = 3). D, p62 knockdown attenuated the AMPK-CA-induced increase in cell death I(−) HCN cells (n = 6). E, p62 knockdown attenuated the AMPK-CA-induced increase in LC3-II in I(−) HCN cells. F, quantification of LC3-II after normalization to β-actin (n = 6). Error bars represent ±S.D. from independent assays. ***, p < 0.001. con, control; ZZ, ZZ-type zinc finger domain; LIR, LC3-interacting region; UBA, ubiquitin-associated domain; KIR, Keap1-interacting region.

Article Snippet: In addition, recombinant human AMPK protein (combination of α2/β1/γ1 subunits; SignalChem, P48-10H) expressed in Sf9 cells was used for the in vitro kinase assay.

Techniques: Functional Assay, Phospho-proteomics, Knockdown, Control, Ubiquitin Proteomics

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation of p62 by AMP-activated protein kinase mediates autophagic cell death in adult hippocampal neural stem cells

doi: 10.1074/jbc.M117.780874

Figure Lengend Snippet: Serine phosphorylation at a novel site in p62 is mediated by AMPK. A, endogenous p62 was phosphorylated in an AMPK-dependent manner in HCN cells. Insulin withdrawal for 5 h induced phosphorylation of p62 Ser-293, which was detected by using a phosphospecific antibody. CC (0.5 μm) reduced Ser-293 phosphorylation. B, quantification of p62 Ser-293 phosphorylation (p-p62) after normalization to total p62 (t-p62) following CC treatment (n = 6). C, AMPK α2 KO reduced Ser-293 phosphorylation. D, quantification of p62 Ser-293 phosphorylation (p-p62) after normalization to total p62 (t-p62) in AMPK α2 KO HCN cells (n = 6). E, hp62-WT, but not hp62-S294A mutant (SA), was phosphorylated in vitro by the enriched AMPK immune complex. Phosphorylation was detected by Western blotting with phosphospecific antibody. F, quantification of in vitro kinase assay results (n = 4). G, treatment of the AMPK immune complex with CC (1 μm) for 1 h abolished p62 phosphorylation. H, quantification of in vitro kinase assay results with CC (n = 3). Error bars represent ±S.D. from independent assays. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: In addition, recombinant human AMPK protein (combination of α2/β1/γ1 subunits; SignalChem, P48-10H) expressed in Sf9 cells was used for the in vitro kinase assay.

Techniques: Phospho-proteomics, Mutagenesis, In Vitro, Western Blot, Kinase Assay

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation of p62 by AMP-activated protein kinase mediates autophagic cell death in adult hippocampal neural stem cells

doi: 10.1074/jbc.M117.780874

Figure Lengend Snippet: AMPK induces mitophagy in I(−) HCN cells. A, transfection of HCN cells with the plasmids encoding GFP-LC3 and DsRed2-Mito. Mitophagy was assessed as the overlap of LC3 and DsRed2-Mito signals and was greatly enhanced following insulin withdrawal (5 h) and prevented by CC. Inset, a high-magnification image of the boxed area. B, analysis of A. Quantification of mitochondrial localization of GFP-LC3 (n = 8) is shown. C, fluorescence signal of mt-mKeima. Cells expressing mt-mKeima were excited (Ex.) at two wavelengths (458 and 561 nm), and the emission signal was collected at 620 nm after insulin withdrawal for 5 h. D, analysis of C. The relative intensity ratio as an index of mitophagy (n = 10) is shown. E, insulin withdrawal-induced mitophagy was abrogated in AMPK α2 KO HCN cells. F, analysis of E. The relative intensity ratio as an index of mitophagy is shown. CCCP (5 μm for 5 h) was added to I(+) HCN cells to induce mitophagy as a positive control (n = 8). ns, not significant; G, the amount of mitochondria in HCN cells following insulin withdrawal for 12 h and treatment with CC (5 μm for 12 h) was determined by FACS using MitoTracker Green (x axis). FSC, forward scatter. H, analysis of G. The relative ΔMitoTracker (%) as an index of mitophagic degradation of mitochondria is shown. CCCP (10 μm for 12 h) was used as a positive control (n = 5). Error bars represent ±S.D. from independent assays. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant. Scale bar, 10 μm.

Article Snippet: In addition, recombinant human AMPK protein (combination of α2/β1/γ1 subunits; SignalChem, P48-10H) expressed in Sf9 cells was used for the in vitro kinase assay.

Techniques: Transfection, Fluorescence, Expressing, Positive Control

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation of p62 by AMP-activated protein kinase mediates autophagic cell death in adult hippocampal neural stem cells

doi: 10.1074/jbc.M117.780874

Figure Lengend Snippet: Phosphorylated p62 translocates to mitochondria in an AMPK-dependent manner in I(−) HCN cells. A, co-transfection of Sh-rp62 HCN cells with plasmids encoding DsRed2-Mito and HA-hp62-WT, -S294A mutant, or -S294E mutant. Translocation of p62 to mitochondria was examined by immunocytochemistry 5 h after insulin withdrawal. CC (0.5 μm) was added as indicated. B, analysis of A. Quantification of mitochondrial localization of p62 (n = 9) is shown. C, live confocal imaging for monitoring mitochondrial translocation of GFP-p62 upon insulin withdrawal. Arrowheads indicate the immobile p62 puncta in I(+) HCN cells; arrows indicate the dynamic p62 puncta that overlap with the mitochondria and change their size over time in I(−) HCN cells. D, AMPK is required for p62 translocation to mitochondria. Insulin withdrawal-induced translocation of hp62-WT, but not its phosphomimicking S294E form, was prevented in AMPK α2 KO HCN cells. E, analysis of D. Quantification of mitochondrial localization of p62 (n = 10) is shown. F, detection of phosphorylated p62 in the mitochondrial fraction following insulin withdrawal for 5 h. Subcellular fractions were analyzed by Western blotting with p62 Ser-293 phosphospecific antibody. Blots shown are representative of three independent experiments, which yielded similar results. Error bars represent ±S.D. from independent assays. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Scale bar, 10 μm. Cyto, cytosolic fraction; Mito, mitochondrial fraction; COX, cytochrome c oxidase; p-p62, phosphorylated p62; t-p62, total p62.

Article Snippet: In addition, recombinant human AMPK protein (combination of α2/β1/γ1 subunits; SignalChem, P48-10H) expressed in Sf9 cells was used for the in vitro kinase assay.

Techniques: Cotransfection, Mutagenesis, Translocation Assay, Immunocytochemistry, Imaging, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation of p62 by AMP-activated protein kinase mediates autophagic cell death in adult hippocampal neural stem cells

doi: 10.1074/jbc.M117.780874

Figure Lengend Snippet: Phosphorylated p62 mediates AMPK-induced mitophagy in I(−) HCN cells. A, co-transfection of Sh-rp62 HCN cells with plasmids encoding mt-mKeima and HA-tagged hp62-WT, -S294A, or -S294E. Cells expressing hp62-WT or hp62-S294E underwent mitophagy, whereas the expression of hp62-S294A mutant prevented mitophagy. Ex, excitation. B, analysis of A. The relative intensity ratio as an index of mitophagy (n = 8) is shown. C, a schematic diagram illustrating p62 phosphorylation by AMPK and subsequent mitophagy and ACD in HCN cells following insulin withdrawal. Error bars represent ±S.D. from independent assays. *, p < 0.05; ***, p < 0.001. Scale bar, 10 μm.

Article Snippet: In addition, recombinant human AMPK protein (combination of α2/β1/γ1 subunits; SignalChem, P48-10H) expressed in Sf9 cells was used for the in vitro kinase assay.

Techniques: Cotransfection, Expressing, Mutagenesis, Phospho-proteomics