Journal: The FASEB Journal

Article Title: Intracellular Sphingosine‐1‐Phosphate Induces Lipolysis Through Direct Activation of Protein Kinase C Zeta

doi: 10.1096/fj.202403272R

Figure Lengend Snippet: S1P induces lipolysis through a PKCzeta/MAPK/HSL pathway in differentiated 3T3‐L1 cells. (A) Glycerol release per mg protein after 16 h treatment of differentiated 3T3‐L1 cells with vehicle (0.02% BSA), 10 μM S1P (in 0.02% BSA) in the presence or absence of 10 μM PD98059 or 25 μM small molecular HSL inhibitor (smHSLi). ( n = 4 per group). (B) Representative Western blots for p‐p44/42 MAPK and p‐HSL Ser660 in differentiated T3‐L1 adipocytes treated with 10 μM S1P or vehicle (0.02% BSA) in the presence or absence of 3 μM bisindolylmaleimid I (Bis) for 15 min ( n = 4–5 per condition). Quantification of p‐p44/42 MAPK and p‐HSL Ser660 normalized to GAPDH. (C) Glycerol release per mg protein after 16 h treatment of differentiated 3T3‐L1 cells with vehicle or 1 μM S1P lyase inhibitor VD‐78. ( n = 5 per group). (D) Glycerol release per mg protein after 16 h treatment of primary mouse adipocytes with vehicle or 1 μM S1P lyase inhibitor VD‐78 ( n = 6 per group). Data are presented as mean ± SEM. A one‐way ANOVA with Tukey's multiple‐comparisons test was used for statistical analysis. (E) Gylcerol release per mg protein after 16h treatment of differentiated 3T3‐L1 cells with vehicle (0.02% BSA) vs 10 µM S1P or vehicle (0.1% DMSO) versus 1 µM Isoproterenol in the presence or absence of 100 µM of the adenylate cyclase inhibitor SQ22536. ( n = 4 per group). (F) Glycerol release per mg protein after 16 h treatment of differentiated 3T3‐L1 cells with 10 µM S1P or vehicle control (0.02% BSA) in presence or absence of the 100 nM PI3K Inhibitor Wortmannin. ( n = 3–8 per group). (G) Relative kinase activity of recombinant PKC zeta treated with 10 µM S1P or vehicle (MeOH) for 10 min as determined by in vitro phosphorylation assay. ( n = 3 per group). (H) Glycerol release per mg protein after 16 h treatment of differentiated 3T3‐L1 cells with 10 µM S1P or vehicle control (0.02% BSA) in presence or absence of the 3 µM PKC inhibitor bisindolylmaleimid I (Bis). ( n = 3–8 per group). (I) Glycerol release per mg protein after 16 h treatment of differentiated 3T3‐L1 cells with 10 µM S1P or vehicle (0.02% BSA) in the presence or absence of 10 µM PKC zeta pseudo‐substrate inhibitor (PSI). ( n = 4 per group). (J) Representative Western blots for T410 phosphorylated PKC zeta, total PKC zeta and GAPDH in differentiated 3T3‐L1 adipocytes treated with 10 µM S1P or vehicle (0.02% BSA) for 15 min. ( n = 4 per condition). Quantification of phospho‐PKC zeta T410 normalized GAPDH. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Glycerol release from differentiated 3T3‐L1 cells and primary adipocytes was measured 16 h after induction of lipolysis in the presence or absence of 10 μM S1P, 10 μM AUY954 (Cayman Chemical), 10 μM CYM5520 (Cayman Chemical), 10 μM CYM5541 (Cayman Chemical), 1 μM VD‐78 (Novartis) or 1 μM isoproterenol (Merck) with or without pre‐incubation with 3 μM Bisindolylmaleimide I, 10 μM PKC zeta pseudo‐substrate inhibitor no. sc‐3098 (Santa Cruz), 25 μM small molecular HSL inhibitor no. NNC0076‐0079 (Novo Nordisk), 10 μM PD98059 (SelleckChemicals), 100 nM Wortmannin (SelleckChemicals), 100 μM SQ22536 (SelleckChemicals) or 25 μM Fumonisin B1 (Cayman Chemical) for 1 h. After incubation, cell culture supernatants were collected.

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

Journal: The FASEB Journal

Article Title: Intracellular Sphingosine‐1‐Phosphate Induces Lipolysis Through Direct Activation of Protein Kinase C Zeta

doi: 10.1096/fj.202403272R

Figure Lengend Snippet: S1P lyase inhibition increases free fatty acids and reduces gWAT mass in HFD‐fed mice due to PKC zeta/HSL activation. (A) Absolute S1P concentrations and relative S1P increase in plasma and gWAT of male C57BL/6J mice after 16 weeks of HFD versus 10 weeks HFD + 6 weeks HFD/DOP treatment. ( n = 5–11 mice per group). (B) Body weight (left) and gWAT weight (right) of male C57BL/6J mice after 16 weeks of HFD versus 10 weeks HFD + 6 weeks HFD/DOP treatment. ( n = 11–13 mice per group). (C) Representative H&E staining of perigonadal WAT from male C57BL/6J mice after 16 weeks of HFD versus 10 weeks HFD + 6 weeks HFD/DOP treatment (left). Quantification of adipocyte diameter and area (right). ( n = 11–13 mice per group). (D) Representative H&E staining of inguinal WAT (iWAT) from male C57BL/6J mice after 16 weeks of HFD versus 10 weeks HFD + 6 weeks HFD/DOP treatment (left) and quantification of adipocyte diameter and area (right). ( n = 4–5 mice per group). (E) Plasma free fatty acids (FFA) of male C57BL/6J mice after 16 weeks of HFD versus 10 weeks HFD + 6 weeks HFD/DOP treatment. ( n = 10–12 mice per group). (F) FFA in gWAT of male C57BL/6J mice after 12 weeks of HFD versus 6 weeks HFD + 6 weeks HFD/DOP treatment. ( n = 5–11 mice per group). (G) Representative Western blots of p‐PKC zeta T410 in gWAT of male C57BL/6J mice after 16 weeks of HFD versus 10 weeks HFD + 6 weeks HFD/DOP treatment. Quantification of p‐PKC zeta T410 to GAPDH. ( n = 4–5 mice per group). (H) PKC zeta kinase activity as measured by in vitro phosphorylation assay with immunoprecipitated PKC zeta in gWAT of male C57BL/6J mice after 16 weeks of HFD versus 10 weeks HFD + 6 weeks HFD/DOP treatment. ( n = 6–7 mice per group). (I) Representative Western blots of p‐HSL Ser660 in gWAT of male C57BL/6J mice after 12 weeks of HFD versus 6 weeks HFD + 6 weeks HFD/DOP treatment. Quantification of p‐HSL Ser660 to total beta‐actin. ( n = 5 mice per group). Data are presented as mean ± SEM. A two‐tailed t ‐test was used for statistical analysis. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Glycerol release from differentiated 3T3‐L1 cells and primary adipocytes was measured 16 h after induction of lipolysis in the presence or absence of 10 μM S1P, 10 μM AUY954 (Cayman Chemical), 10 μM CYM5520 (Cayman Chemical), 10 μM CYM5541 (Cayman Chemical), 1 μM VD‐78 (Novartis) or 1 μM isoproterenol (Merck) with or without pre‐incubation with 3 μM Bisindolylmaleimide I, 10 μM PKC zeta pseudo‐substrate inhibitor no. sc‐3098 (Santa Cruz), 25 μM small molecular HSL inhibitor no. NNC0076‐0079 (Novo Nordisk), 10 μM PD98059 (SelleckChemicals), 100 nM Wortmannin (SelleckChemicals), 100 μM SQ22536 (SelleckChemicals) or 25 μM Fumonisin B1 (Cayman Chemical) for 1 h. After incubation, cell culture supernatants were collected.

Techniques: Inhibition, Activation Assay, Clinical Proteomics, Staining, Western Blot, Activity Assay, In Vitro, Phospho-proteomics, Immunoprecipitation, Two Tailed Test

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: PKMζ, a Brain-specific PKCζ Isoform, is Required for Glycolysis and Myofibroblastic Activation of Hepatic Stellate Cells

doi: 10.1016/j.jcmgh.2024.101429

Figure Lengend Snippet: HSCs express brain-specific PKMζ isoform, but not full-length PKCζ. ( A ) Upper , Primary human HSCs were collected for RNA extraction and RT-PCR for transcript variants of PKCζ. Lower , A PDX of brain cancer was subjected to RT-PCR. Transcript variants 2 and 5 (V2 and V5) were detected from both samples. Data are representative of 3 repeats with similar results. ( B ) Upper, the RT-PCR products of HSCs were subjected to Sanger sequencing, and the sequences obtained were aligned to V2 of PKCζ by the Sequencher software. The translation initiation codon is highlighted. Lower , Sequence alignment confirmed that the variant detected is V2 encoding PKMζ. ( C ) Upper , HSCs transduced with lentiviruses encoding NT shRNA or PKMζ shRNA were collected for WB. A PKMζ band about 50 kilodalton (Kd) was detected with its density reduced upon shRNA-mediated knockdown. Lower, a PDX of brain cancer was subjected to WB, revealing a PKM band about 50 Kd. Data are representative of 3 repeats with similar results. ( D ) Upper , Primary HSCs were subjected to RT-PCR for transcript variants V1 to V3 of murine PKCζ. V2 encoding PKMζ was detected from day 5 culture of murine HSCs. Lower , Murine cerebral cortex tissues were subjected to RT-PCR and V2 of murine PKCζ was detected. Data are representative of 3 repeats with similar results. ( E ) The RT-PCR product of murine HSCs was subjected to Sanger sequencing. The sequence obtained were aligned to the V2 of murine PKCζ. The translation initiation codon is highlighted in green . ( F ) Left , Murine HSCs were collected for WB, revealing a time-dependent increase of the expression of PKMζ and αSMA from day 1 to day 5 culture of HSCs. Right, a predominant band of PKMζ about 50 Kd was detected from murine cerebral cortex tissues. Data are representative of 3 repeats with similar results. ( G ) IF was performed on murine HSCs, and confocal fluorescence images were obtained by a Zeiss microscope (Zeiss LSM 900 with Airyscan 2) with a 40× lens and the Zen 2.3 lite software. IF confirmed a time-dependent increase of PKMζ expression in primary murine HSCs. ∗∗ P < .01 by ANOVA; n = 10 cells per group. Bar: 20 μm.

Article Snippet: PKCζ pseudosubstrate inhibitor (PKCζ PS) was purchased from Santa Cruz Biotechnology (sc-3098 Lot# E2819).

Techniques: RNA Extraction, Reverse Transcription Polymerase Chain Reaction, Sequencing, Software, Variant Assay, Transduction, shRNA, Knockdown, Expressing, Fluorescence, Microscopy

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: PKMζ, a Brain-specific PKCζ Isoform, is Required for Glycolysis and Myofibroblastic Activation of Hepatic Stellate Cells

doi: 10.1016/j.jcmgh.2024.101429

Figure Lengend Snippet: PKMζ is required for myofibroblastic activation of HSCs. ( A and B ) HSCs expressing NT shRNA or PKMζ shRNA by lentiviral transduction were stimulated with TGFβ1 (5 ng/mL) and collected 24 hours later for WB ( A ) and αSMA IF ( B ). TGFβ1-stimulated HSC activation was suppressed by PKMζ knockdown. Note that the basal αSMA level was also reduced by PKMζ knockdown. ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001 by ANOVA. For WB, n = 3 repeats (data were compiled from 3 independent experiments); for IF, n = 10 randomly picked microscopic fields with each containing more than 100 cells. Bar, 50 μm. IF images were captured under a Zeiss Axio observer with a 20× lens and the Zen 2.3 lite software. ( C and D ) HSCs expressing PKMζ-HA or LacZ (control) by retroviral transduction were stimulated with TGFβ1 and collected for WB and IF. TGFβ1-induced HSC activation was potentiated by overexpression of PKMζ. Note that the basal αSMA level was also enhanced by overexpression of PKMζ. ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001 by ANOVA. For WB, n = 3 repeats (data were compiled from 3 independent experiments); for αSMA IF, n = 10 randomly picked microscopic fields with each containing more than 100 cells. Bar, 50 μm. IF images were captured under a Zeiss Axio observer with a 20× lens and the Zen 2.3 lite software. ( E ) HSCs pre-incubated with PKCζ pseudo-substrate peptide (PKCζ PS; PKCζ/PKMζ inhibitor) were stimulated with TGFβ1 and collected for WB. TGFβ1-stimulated HSC activation was suppressed by PKCζ PS (1 μM). ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001 by ANOVA, n = 3 repeats (data were compiled from 3 independent experiments). ( F ) Murine HSCs incubated without or with PKCζ PS (1 μM) were collected at day 5 for WB ( left ) and αSMA IF ( right ). HSCs at day 1 were used as a control for WB. Targeting PKMζ suppressed spontaneous myofibroblastic activation of murine HSCs in vitro . For WB, ∗∗ P < .01; ∗∗∗ P < .001 by ANOVA, n = 3 repeats (data were compiled from 3 independent experiments). For IF, ∗∗∗∗ P < .0001 by t -test, n = 9, 10 randomly picked microscopic fields with each containing more than 50 cells. Bar, 50 μm. IF images were captured under a Zeiss Axio observer with a 20× lens and the Zen 2.3 lite software.

Article Snippet: PKCζ pseudosubstrate inhibitor (PKCζ PS) was purchased from Santa Cruz Biotechnology (sc-3098 Lot# E2819).

Techniques: Activation Assay, Expressing, shRNA, Transduction, Knockdown, Software, Control, Retroviral, Over Expression, Incubation, In Vitro

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: PKMζ, a Brain-specific PKCζ Isoform, is Required for Glycolysis and Myofibroblastic Activation of Hepatic Stellate Cells

doi: 10.1016/j.jcmgh.2024.101429

Figure Lengend Snippet: PKMζ complexes with VASP and Glut1 and induces VASP phosphorylation. ( A ) Left , Triple IF was performed on HSCs and confocal microscopy was done with a Zeiss microscope (Zeiss LSM 900 with Airyscan 2) with a 63× lens and the Zen 2.3 lite software. IF demonstrated PKMζ/VASP/Glut1 co-localization at the PM of HSCs ( arrowheads ). Bar, 20 μm. Right , HSCs without or with overexpression of PKM-HA and FLAG-Glut1 were collected for co-IP with anti-FLAG antibody. PKMζ bound to VASP and Glut1 in HSCs and the binding was increased by TGFβ1. ∗ P < .05 by t -test, n = 3 repeats (data were compiled from 3 independent experiments). ( B ) Biotinylation assay showed that VASP knockdown abolished PM Glut1 induced by TGFβ1. ∗ P < .05; ∗∗ P < .01 by ANOVA, n = 3 repeats (data were compiled from 3 independent experiments). ( C ) Control and PKMζ knockdown HSCs were stimulated with TGFβ1 for 30 minutes and collected for WB to detect VASP phosphorylation at Serines 157 (P-VASPS157), 239 (P-VASPS239), and Threonine 278 (P-VASPT278). TGFβ1 promoted P-VASPS157 and P-VASPS239 level in a PKMζ-dependent manner. ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001 by ANOVA, n = 3 repeats (data were compiled from 3 independent experiments). ( D ) Double IF was performed on HSCs, and confocal microscopy was done with a Zeiss microscope (Zeiss LSM 900 with Airyscan 2) with a 63× lens and the Zen 2.3 lite software. IF demonstrated Glut1/P-VASPS157 co-localization ( arrows ) and Glut1/P-VASPS239 co-localization ( arrowheads ) at the peripheral PM of HSCs. Bar, 20 μm. ( E ) Left, GST-VASP fusion protein was expressed by E. Coli and purified with Glutathione Sepharose 4B beads. Its quality and concentration were determined by Coomassie staining. Middle and right , in vitro phosphorylation assay revealed that VASP was phosphorylated by PKCζ/PKMζ. ∗ P < .05 by ANOVA, n = 3 repeats (data were compiled from 3 independent experiments).

Article Snippet: PKCζ pseudosubstrate inhibitor (PKCζ PS) was purchased from Santa Cruz Biotechnology (sc-3098 Lot# E2819).

Techniques: Phospho-proteomics, Confocal Microscopy, Microscopy, Software, Over Expression, Co-Immunoprecipitation Assay, Binding Assay, Cell Surface Biotinylation Assay, Knockdown, Control, Purification, Concentration Assay, Staining, In Vitro

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: PKMζ, a Brain-specific PKCζ Isoform, is Required for Glycolysis and Myofibroblastic Activation of Hepatic Stellate Cells

doi: 10.1016/j.jcmgh.2024.101429

Figure Lengend Snippet: S1P is required for VASP phosphorylation and PM Glut1 induced by TGFβ1. ( A ) Confocal microscopy was done with a Zeiss microscope (Zeiss LSM 900 with Airyscan 2) with a 63× lens and the Zen 2.3 lite software. IF demonstrated that PKMζ/VASP/Glut1 co-localization at the PM was reduced in VASP2A-YFP-expressing HSCs but enhanced in VASP2D-YFP-expressing HSCs. Quantitative data for PM Glut1 are shown on the right . ∗ P < .05; ∗∗ P < .01 by ANOVA, n >8 cells per group. Bar, 20 μm. ( B ) HSCs pre-incubated with PKCζ PS (1 μM) or a S1P inhibitor DMS (5 μM) were stimulated with TGFβ1 and collected for WB. Both PKCζ PS and DMS abolished P-VASPS157 induced by TGFβ1. ∗∗∗∗ P < .0001 by ANOVA, n = 4 repeats (results were compiled from 4 independent experiments). ( C ) HSCs incubated with deoxypyridoxine (4DP) (1 mM) or S1P (1 μM) for 30 minutes were collected for WB. Cells stimulated with TGFβ1 were used as the control. Both 4DP and S1P were able to promote P-VASPS157 level of HSCs like TGFβ. ∗ P < .05; ∗∗ P < .01 by ANOVA, n = 3 replicates per group. ( D ) HSCs pre-incubated with 4DP were stimulated without or with TGFβ1 and collected for biotinylation assay. 4DP potentiated the basal PM Glut1 level of HSCs. ∗ P < .05; ∗∗ P < .01 by ANOVA, n = 3 repeats (results were compiled from 3 independent experiments). ( E ) Biotinylation assay revealed that DMS suppressed PM accumulation of Glut1 induced by TGFβ1. ∗ P < .05 by ANOVA, n = 3 repeats (results were compiled from 3 independent experiments).

Article Snippet: PKCζ pseudosubstrate inhibitor (PKCζ PS) was purchased from Santa Cruz Biotechnology (sc-3098 Lot# E2819).

Techniques: Phospho-proteomics, Confocal Microscopy, Microscopy, Software, Expressing, Incubation, Control, Cell Surface Biotinylation Assay