Journal: Diabetes & metabolism journal

Article Title: Serpina3c Mitigates Adipose Tissue Inflammation by Inhibiting the HIF1α-Mediated Endoplasmic Reticulum Overoxidation in Adipocytes.

doi: 10.4093/dmj.2024.0441

Figure Lengend Snippet: Fig. 1. Serine (or cysteine) peptidase inhibitor, clade A, member 3C (Serpina3c) knockdown (3cKD) in adipocytes enhanced en doplasmic reticulum overoxidation and endoplasmic reticulum stress (ERS), and promoted the expression of pro-inflammatory cytokines and adipocyte apoptosis under lipotoxicity injury. (A) Western blotting of Serpina3c protein level in 3cKD 3T3-L1 adi pocytes and its control LV3 group, as well as Serpina3c overexpression (3cOV) 3T3-L1 adipocytes and its control LV5 group. (B) Cell counting kit-8 (CCK8) assay measured the cell viability (%) of LV3 group and 3cKD group after 500 μM palmitic acid (PA)- treated for 24 or 48 hours. (C-J) In these experiments, LV3, 3cKD, LV5, and 3cOV groups were treated by 500 μM PA for 48 hours. (C, D) The relative mRNA levels of indicated genes in 3T3-L1 adipocytes. (E) The protein levels of indicated genes in 3T3- L1 adipocytes and quantification of the relative protein band density (n=3 for each group). (F) Hydrogen peroxide (H2O2) level in 3T3-L1 adipocytes was determined. (G) The protein levels of ERS makers in 3T3-L1 adipocytes and quantification of the relative protein band density (n=3 for each group). (H) Reactive oxygen species (ROS) level in 3T3-L1 adipocytes was detected. (I) The protein levels of mitogen-activated protein kinase signaling pathway in 3T3-L1 adipocytes and quantification of the relative pro tein band density (n=3 for each group). (J) Caspase-3 activity in 3T3-L1 adipocytes was determined. Data were presented as mean±standard error of the mean (n=5 for each group unless otherwise mentioned). NS, no significance; IL-6, interleukin-6; CCL2 or CCL5, C-C motif chemokine ligand 2 or 5; CXLC1 or CXCL10, C-X-C motif chemokine ligand 1 or 10; HIF1α, hypox ia-inducible factor 1α; Ero1α, endoplasmic reticulum oxidoreductase 1α; PDIA3 or PDIA4, protein disulfide isomerase family A member 3 or 4; GRP78, glucose regulated protein 78; CHOP, C/EBP homologous protein; p-eIF2α, phosphorylated eukaryotic initiation factor 2α; c-ATF6, cleaved activating transcription factor 6; XBP1S, spliced X-box binding protein 1; p-JNK, phosphor ylated c-Jun N-terminal kinase; p-ERK, phosphorylated extracellular signal-regulated kinase. aP<0.05, bP<0.01, cP<0.001, dP<0.0001. (Continued to the next page)

Article Snippet: The following primary antibodies were used in this study: Serpina3c (50375- RP01, Sino Biological), HIF1α (BF8002, Affinity Biosciences, Cincinnati, OH, USA), Ero1α (67416-1-Ig, Proteintech, Rosemont, IL, USA), protein disulfide isomerase family A member 3 (PDIA3; DF6230, Affinity Biosciences), PDIA4 (DF12114, Affinity Biosciences), glucose regulated protein 78 (GRP78; AF5366, Affinity Biosciences), C/EBP homologous protein (CHOP; AF6277, Affinity Biosciences), phosphorylated eukaryotic initiation factor 2α (p-eIF2α; AF3087, Affinity Biosciences), eIF2α (AF6087, Affinity Biosciences), activating transcription factor 6 (ATF6; DF6009, Affinity Biosciences), spliced X-box binding protein 1 (XBP1S; 24868-1-AP, Proteintech), phosphorylated c-Jun N-terminal kinase (p-JNK; AF3318, Affinity Biosciences), JNK (AF6318, Affinity Biosciences), phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2; AF1015, Affinity Biosciences), ERK (AF0155, Proteintech), p-P38 (28796-1-AP, Proteintech), P38 (14064-1-AP, Proteintech), α-tubulin (66031-1-Ig, Proteintech).

Techniques: Knockdown, Expressing, Western Blot, Control, Over Expression, Cell Counting, CCK-8 Assay, Activity Assay, Binding Assay

Journal: Diabetes & metabolism journal

Article Title: Serpina3c Mitigates Adipose Tissue Inflammation by Inhibiting the HIF1α-Mediated Endoplasmic Reticulum Overoxidation in Adipocytes.

doi: 10.4093/dmj.2024.0441

Figure Lengend Snippet: Fig. 3. Serine (or cysteine) peptidase inhibitor, clade A, member 3C (Serpina3c) ablation exacerbated endoplasmic reticulum (ER) overoxidation, inflammation and apoptosis of epididymal white adipose tissue (eWAT) in the high-fat diet (HFD)-fed mice. These mice were the same batch of 12 week-HFD-fed wild type (WT) and Serpina3c global knockout (Serpina3c–/–) mice as shown in Fig. 2. (A) The relative mRNA levels of indicated genes in eWAT. (B) The protein levels of indicated genes in eWAT and quantification of the relative protein band density (n=3 for each group). (C) Hydrogen peroxide (H2O2) level in eWAT was mea sured. (D) The protein levels of mitogen-activated protein kinase signaling pathway in eWAT and quantification of the relative protein band density (n=3 for each group). (E) Caspase-3 activity of eWAT was determined. (F) Representative images of termi nal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) stained eWAT sections. Scale bar: 100 µm. (G) Representative photographs of ER in ultrathin eWAT sections observed by transmission electron microscopy (TEM) at 50,000 magnification (scale bar: 200 nm; red arrow: ER). Data were presented as mean±standard error of the mean (n=5 for each group). TNF-α, tu mor necrosis factor-alpha; IL-6, interleukin-6; CCL2 or CCL5, C-C motif chemokine ligand 2 or 5; CXLC1 or CXCL10, C-X-C motif chemokine ligand 1 or 10; HIF1α, hypoxia-inducible factor 1α; Ero1α, endoplasmic reticulum oxidoreductase 1α; PDIA3 or PDIA4, protein disulfide isomerase family A member 3 or 4; GRP78, glucose regulated protein 78; CHOP, C/EBP homologous protein; p-JNK, phosphorylated c-Jun N-terminal kinase; p-ERK, phosphorylated extracellular signal-regulated kinase; DAPI, 4’,6-diamidino-2-phenylindole. aP<0.05, bP<0.01, cP<0.001.

Article Snippet: The following primary antibodies were used in this study: Serpina3c (50375- RP01, Sino Biological), HIF1α (BF8002, Affinity Biosciences, Cincinnati, OH, USA), Ero1α (67416-1-Ig, Proteintech, Rosemont, IL, USA), protein disulfide isomerase family A member 3 (PDIA3; DF6230, Affinity Biosciences), PDIA4 (DF12114, Affinity Biosciences), glucose regulated protein 78 (GRP78; AF5366, Affinity Biosciences), C/EBP homologous protein (CHOP; AF6277, Affinity Biosciences), phosphorylated eukaryotic initiation factor 2α (p-eIF2α; AF3087, Affinity Biosciences), eIF2α (AF6087, Affinity Biosciences), activating transcription factor 6 (ATF6; DF6009, Affinity Biosciences), spliced X-box binding protein 1 (XBP1S; 24868-1-AP, Proteintech), phosphorylated c-Jun N-terminal kinase (p-JNK; AF3318, Affinity Biosciences), JNK (AF6318, Affinity Biosciences), phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2; AF1015, Affinity Biosciences), ERK (AF0155, Proteintech), p-P38 (28796-1-AP, Proteintech), P38 (14064-1-AP, Proteintech), α-tubulin (66031-1-Ig, Proteintech).

Techniques: Knock-Out, Activity Assay, End Labeling, TUNEL Assay, Staining, Transmission Assay, Electron Microscopy

Journal: Diabetes & metabolism journal

Article Title: Serpina3c Mitigates Adipose Tissue Inflammation by Inhibiting the HIF1α-Mediated Endoplasmic Reticulum Overoxidation in Adipocytes.

doi: 10.4093/dmj.2024.0441

Figure Lengend Snippet: Fig. 5. Serine (or cysteine) peptidase inhibitor, clade A, member 3C (Serpina3c) inhibited endoplasmic reticulum overoxidation and its downstream in adipocytes at least partially by suppressing hypoxia-inducible factor 1α (HIF1α)/endoplasmic reticulum oxidoreductase 1α (Ero1α)-protein disulfide isomerase (PDI) signaling. (A) The effect of transcription regulatory factor HIF1α on the promoter activity of protein disulfide isomerase family A member 3 (PDIA3) and PDIA4 in 293T cells was detected by Dual-luciferase reporter assay. (B-H) Serpina3c knockdown (3cKD) 3T3-L1 adipocytes were pretreated with 100 ng/mL recom binant mouse Serpina3c protein (rSerpina3c) or 5 μM HIF1α inhibitor acriflavine (ACF) for 2 hours before being stimulated with 500 μM palmitic acid (PA) for 48 hours. (B) The mRNA levels of indicated genes in 3cKD 3T3-L1 adipocytes. (C, D) The protein levels of indicated genes in 3cKD 3T3-L1 adipocytes and quantification of the relative protein band density (n=3 for each group). (E) Hydrogen peroxide (H2O2) level in 3cKD 3T3-L1 adipocytes was determined. (F, G) The protein levels of mitogen-activated protein kinase (MAPK) signaling pathway in 3cKD 3T3-L1 adipocytes and quantification of the relative protein band density (n=3 for each group). (H) Caspase-3 activity in 3cKD 3T3-L1 adipocytes was determined. (I-L) 3cKD 3T3-L1 adipocytes were pretreated with 10 μM Ero1α inhibitor EN460 or 0.4 μM PDI inhibitor propynoic acid carbamoyl methyl amide 31 (PACMA31) alone or in combination for 2 hours before being stimulated with 500 μM PA for 48 hours. (I) The mRNA levels of indicated genes in 3T3-L1 adipocytes. (J) H2O2 level in 3cKD 3T3-L1 adipocytes was measured. (K) The protein levels of MAPK signaling path way in 3cKD 3T3-L1 adipocytes and quantification of the relative protein band density (n=3 for each group). (L) Caspase-3 activ ity in 3cKD 3T3-L1 adipocytes was determined. Data were presented as mean±standard error of the mean. MCS, multiple clon ing site; 6xHis, hexahistidine; IL-6, interleukin-6; CCL2 or CCL5, C-C motif chemokine ligand 2 or 5; CXLC1 or CXCL10, C-X- C motif chemokine ligand 1 or 10; GRP78, glucose regulated protein 78; CHOP, C/EBP homologous protein; p-JNK, phosphory lated c-Jun N-terminal kinase; p-ERK, phosphorylated extracellular signal-regulated kinase; NS, no significance. aP<0.05, bP<0.01, cP<0.001, dP<0.0001. (Continued to the next page)

Article Snippet: The following primary antibodies were used in this study: Serpina3c (50375- RP01, Sino Biological), HIF1α (BF8002, Affinity Biosciences, Cincinnati, OH, USA), Ero1α (67416-1-Ig, Proteintech, Rosemont, IL, USA), protein disulfide isomerase family A member 3 (PDIA3; DF6230, Affinity Biosciences), PDIA4 (DF12114, Affinity Biosciences), glucose regulated protein 78 (GRP78; AF5366, Affinity Biosciences), C/EBP homologous protein (CHOP; AF6277, Affinity Biosciences), phosphorylated eukaryotic initiation factor 2α (p-eIF2α; AF3087, Affinity Biosciences), eIF2α (AF6087, Affinity Biosciences), activating transcription factor 6 (ATF6; DF6009, Affinity Biosciences), spliced X-box binding protein 1 (XBP1S; 24868-1-AP, Proteintech), phosphorylated c-Jun N-terminal kinase (p-JNK; AF3318, Affinity Biosciences), JNK (AF6318, Affinity Biosciences), phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2; AF1015, Affinity Biosciences), ERK (AF0155, Proteintech), p-P38 (28796-1-AP, Proteintech), P38 (14064-1-AP, Proteintech), α-tubulin (66031-1-Ig, Proteintech).

Techniques: Activity Assay, Luciferase, Reporter Assay, Knockdown

Journal: Diabetes & metabolism journal

Article Title: Serpina3c Mitigates Adipose Tissue Inflammation by Inhibiting the HIF1α-Mediated Endoplasmic Reticulum Overoxidation in Adipocytes.

doi: 10.4093/dmj.2024.0441

Figure Lengend Snippet: Fig. 6. Adeno-associated virus (AAV) mediated adipocyte-specific overexpression of serine (or cysteine) peptidase inhibitor, clade A, member 3C (Serpina3c) or knockdown of hypoxia-inducible factor 1α (HIF1α) in epididymal white adipose tissue (eWAT) alleviated endoplasmic reticulum (ER) overoxidation, fibrosis and macrophage infiltration in eWAT of high-fat diet (HFD)-fed Serpina3c global knockout (Serpina3c–/–) mice. (A) Scheme of Serpina3c overexpression or HIF1α knockdown in the eWAT of Serpina3c–/– mice. (B) Western blotting of Serpina3c protein expression in eWAT of mice. (C) Body weight change of mice. (D) Body weight of mice at 12-week-HFD feeding. (E) Representative photographs of mice. (F) Representative pictures of eWAT. (G) Representative images of hematoxylin-eosin (H&E) staining, Masson’s trichrome staining and F4/80 immunohisto chemistry (IHC) staining of eWAT sections (scale bar: 100, 100, 50 µm, respectively). (H) Representative photographs of ER in ultrathin eWAT sections observed by transmission electron microscopy (TEM) at 50,000 magnification (scale bar: 200 nm; red arrow: ER). (I, J) The relative mRNA levels of indicated genes in eWAT. (K) Hydrogen peroxide (H2O2) level in eWAT was mea sured. (L) Caspase-3 activity in eWAT was determined. Data were presented as mean±standard error of the mean (n=5 for each group). Adipoq-AAV8, adiponectin-promoter-driven adeno-associated virus serotype 8; NS, no significance; TNF-α, tumor ne crosis factor-alpha; IL-6, interleukin-6; CCL2 or CCL5, C-C motif chemokine ligand 2 or 5; CXLC1 or CXCL10, C-X-C motif chemokine ligand 1 or 10; Ero1α, endoplasmic reticulum oxidoreductase 1α; PDIA3 or PDIA4, protein disulfide isomerase family A member 3 or 4; GRP78, glucose regulated protein 78; CHOP, C/EBP homologous protein. aP<0.05, bP<0.01, cP<0.001. (Continued to the next page)

Article Snippet: The following primary antibodies were used in this study: Serpina3c (50375- RP01, Sino Biological), HIF1α (BF8002, Affinity Biosciences, Cincinnati, OH, USA), Ero1α (67416-1-Ig, Proteintech, Rosemont, IL, USA), protein disulfide isomerase family A member 3 (PDIA3; DF6230, Affinity Biosciences), PDIA4 (DF12114, Affinity Biosciences), glucose regulated protein 78 (GRP78; AF5366, Affinity Biosciences), C/EBP homologous protein (CHOP; AF6277, Affinity Biosciences), phosphorylated eukaryotic initiation factor 2α (p-eIF2α; AF3087, Affinity Biosciences), eIF2α (AF6087, Affinity Biosciences), activating transcription factor 6 (ATF6; DF6009, Affinity Biosciences), spliced X-box binding protein 1 (XBP1S; 24868-1-AP, Proteintech), phosphorylated c-Jun N-terminal kinase (p-JNK; AF3318, Affinity Biosciences), JNK (AF6318, Affinity Biosciences), phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2; AF1015, Affinity Biosciences), ERK (AF0155, Proteintech), p-P38 (28796-1-AP, Proteintech), P38 (14064-1-AP, Proteintech), α-tubulin (66031-1-Ig, Proteintech).

Techniques: Virus, Over Expression, Knockdown, Knock-Out, Western Blot, Expressing, Staining, Immunohistochemistry, Transmission Assay, Electron Microscopy, Activity Assay