Journal: Science translational medicine

Article Title: Overproduction of inter-α-trypsin inhibitor heavy chain 1 after loss of Gα 13 in liver exacerbates systemic insulin resistance in mice.

doi: 10.1126/scitranslmed.aan4735

Figure Lengend Snippet: Fig. 3. Identification of ITIH1 as a hepatocyte-secreted protein enhanced by loss of Ga13. (A) Immunoblots for phosphorylated-Akt (p-Akt) and total Akt expres- sion in protein lysates of 3T3-L1 and C2C12 cells incubated with conditioned media (CM) that were collected from primary hepatocytes isolated from WT or G13 LKO mice fed a high-fat diet (HFD) for 5 weeks. (B) The numbers of proteins in primary hepatocyte conditioned media from WT and G13 LKO mice in (A) that were differentially secreted (n = 3). (C) List of top differentially secreted proteins from (B) detected by semi-quantitative secretome analysis. (D) Immunoblots for ITIH1 expression in protein lysates from the livers of WT or G13 LKO mice fed an HFD for 9 weeks. Quantification of densitometry analysis for bands is shown next to the immunoblot (n = 7 per group). b-Actin was the loading control. (E) Measurements for serum ITIH1 concentrations for WT or G13 LKO mice fed an HFD for 12 weeks. Albumin was used as the loading control. (F) Quantitative analysis of ELISA for serum ITIH1 for the same mice as in (E) (n = 7 per group). Values are expressed as means ± SEM. **P < 0.01 for G13 LKO versus WT mice (D and F). Data were analyzed by two-tailed Student’s t test (D and F). N.S., not significant.

Article Snippet: ITIH1-enriched sample preparation for proteomics of O-GlcNAcylation HEK293A cells overexpressed with human ITIH1 (Origene, Rockville, MD, USA) were subjected to SDS-PAGE.

Techniques: Western Blot, Incubation, Isolation, Expressing, Control, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Science translational medicine

Article Title: Overproduction of inter-α-trypsin inhibitor heavy chain 1 after loss of Gα 13 in liver exacerbates systemic insulin resistance in mice.

doi: 10.1126/scitranslmed.aan4735

Figure Lengend Snippet: Fig. 5. Increased ITIH1 deposition onto hyaluronan surrounding peripheral tissues in G13 LKO mice. (A) Quantitative analyses of ELISA for ITIH1 in homogenates of epididymal fat tissue (n = 8 or 10 per group) and gastrocnemius muscle (n = 12 or 13 per group) from wild-type (WT) or G13 LKO mice fed a high-fat diet (HFD) for 9 weeks. (B) Quantitative analyses of ELISA for hyaluronan (HA) in the homogenates of epididymal fat tissue and gastrocnemius muscle of the same animals as in (A) (n = 8 per group). (C) Representative images of epididymal fat tissue and gastrocnemius muscle sections from mice as described in (A) stained for ITIH1 (brown) (n = 4 per group). The slideswere pretreatedwith hyaluronidase (20U/ml)orvehicle for2 hoursat 37°C. Scale bars, 100mm. (D)Representative images ofepididymalfattissue andgastrocnemiusmuscle sectionsfrom wild-type (WT) C57BL/6 mice injected via the tail vein with controllentiviruses (Lv-Con)or lentiviruses expressing mouse Ga13 (Lv-Ga13) (1.1 ×107 transduction units). The injected mice were subsequently fed a high-fat diet (HFD)for 11 weeks (n= 4 per group). Scale bars, 100 mm. (E)Immunoblot analyses for ITIH1 expression in fractionsenriched for extracellular matrix (ECM) prepared from epididymal fat tissue or tibialis anterior muscle of the same mice as in (A). ECM proteins were normalized to tissue weight and were stained with Coomassie blue stain after SDS-PAGE separation. (F) Representative immunofluorescence images of epididymal fat tissue, gastrocnemius muscle, and liver sections from the same mice as in (C) stained for ITIH1 (red) and hyaluronan binding protein (HABP) (green) (n = 3 per group). For liver tissue, blue color in merged images represents DAPI staining for nuclei. Arrowheads indicate merged color (yellow/orange). Scale bars, 25 mm. For (A) and (B), values are expressed as means ± SEM. *P < 0.05 for G13 LKO versus WT mice (A). Data were analyzed by two-tailed Student’s t test. N.S., not significant.

Article Snippet: ITIH1-enriched sample preparation for proteomics of O-GlcNAcylation HEK293A cells overexpressed with human ITIH1 (Origene, Rockville, MD, USA) were subjected to SDS-PAGE.

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Injection, Expressing, Transduction, Western Blot, SDS Page, Immunofluorescence, Binding Assay, Two Tailed Test

Journal: Science translational medicine

Article Title: Overproduction of inter-α-trypsin inhibitor heavy chain 1 after loss of Gα 13 in liver exacerbates systemic insulin resistance in mice.

doi: 10.1126/scitranslmed.aan4735

Figure Lengend Snippet: Fig. 6. Increase in O-GlcNAc transferase–mediated O-GlcNAcylation ofproteins afterloss of Ga13. (A) Immunoblot analyses for O-GlcNAc proteins (CTD110.6 or RL2 clones) or ITIH1 expression in protein lysates of livers from WT or G13 LKO mice fed a high-fat diet (HFD) for 5 weeks. (B) Immunoblot analyses for O-GlcNAc proteins (CTD110.6 or RL2 clones) or ITIH1 expression in protein lysates of primary hepatocytes isolated from WT or G13 LKO mice fed an HFD for 5 weeks. Primary hepatocytes were incubated with high glucose (25 mM) for 24 hours. (C and D) Immunoblot analyses for ITIH1 or O-GlcNAc transferase (OGT) expression in protein lysates of the liver (C) or in sera (D) from WT or G13 LKO mice. Glucose (2 g/kg body weight) was orally administered to mice, and the liver tissues were harvested after 6 hours. The relative band intensities were quantified by densi- tometry analyses for the immunoblots (n = 3 per group). b-Actin and albumin were the loading controls for liver and serum samples, respectively. (E) Immunoblot analyses for OGT in the protein lysates of liver or hepatocytes isolated from WT or G13 LKO mice fed an HFD for 9 or 5 weeks, respectively. (F) Immunoblot analysis for OGT and ITIH1 in protein lysates of liver from C57BL/6 mice injected via the tail vein with control lentiviruses (Lv-Con) or lentiviruses expressing mouse Ga13 (Lv-Ga13) (1.1 × 107 transduction units). Injected mice were subsequently fed an HFD for 10 weeks. Immunoblots were quantified by densitometry (n = 3 per group). (G) Immunoblot analysis for OGT and ITIH1 expression in protein lysates of primary hepatocytes infected with adenoviruses carrying an active mutant of Ga13 (Ad-G13QL) or green fluorescent protein (Ad-GFP) as a control. (H) Immu- noblot analysis for ITIH1 expression in conditioned media (CM) from the same primary hepatocytes as in (G). Albumin was the loading control for conditioned media samples. (I) Immunoblot analyses for O-GlcNAc proteins (CTD110.6 clone) or O-GlcNAc transferase (OGT) in ITIH1 immunoprecipitates from the liver homogenates or sera of mice fed a high-fat diet (HFD) for 9 weeks. Values are expressed as means ± SEM. *P < 0.05, **P < 0.01 for Lv-Ga13 versus Lv-Con (F). Data were analyzed by one-way ANOVA, followed by least significant difference (LSD) post hoc test (C) or two-tailed Student’s t test (F).

Article Snippet: ITIH1-enriched sample preparation for proteomics of O-GlcNAcylation HEK293A cells overexpressed with human ITIH1 (Origene, Rockville, MD, USA) were subjected to SDS-PAGE.

Techniques: Western Blot, Clone Assay, Expressing, Isolation, Incubation, Injection, Control, Transduction, Infection, Mutagenesis, Two Tailed Test

Journal: Science translational medicine

Article Title: Overproduction of inter-α-trypsin inhibitor heavy chain 1 after loss of Gα 13 in liver exacerbates systemic insulin resistance in mice.

doi: 10.1126/scitranslmed.aan4735

Figure Lengend Snippet: Fig. 7. Overproduction and O-GlcNAcylation of ITIH1 induced by O-GlcNAc transferase. (A) Immunoblot analysis for O-GlcNAc proteins (CTD110.6) or ITIH1 expression in protein lysates of livers from WT or G13 LKO mice injected with ST045849, an inhibitor of O-GlcNAc transferase (OGT; 20 mg/kg body weight). Mice were subjected to vehicle (left, n = 2 per group) orglucose gavage (right, n = 3 to4 per group). Glucose (2 g/kg body weight) was orally administered to mice, and the liver tissues were harvested after6 hours. (B) Immunoblot analysis for ITIH1 in serum samples from the same mice as in (A). (C) Analysis of the glucose tolerance test (2 g/kg body weight) in WT and G13 LKO mice injected with ST045849 (OGT inhibitor, 20 mg/kg body weight) (n = 6 per group). Inset represents area under the curve (AUC) for the glucose tolerance test (GTT). (D) Immunoblot analysis for ITIH1 expression in protein lysates of liver or in sera from 8-week-old C57BL/6 mice injected with lentiviral vector carrying the human OGT gene with an albumin promoter (Lv-OGTalb) or empty control vector (Lv-Con) via the tail vein (2 × 107 transduction units each). Albumin was the loading control for serum samples. (E and F) The effect of hepatic Ga13 overexpression on glucose metabolism was determined using a glucose test (E, n = 10 per group) or insulin tolerance test (F, n = 9 to 10 per group) in the same mice as in (D). Insets show the area under the curve (AUC) for the glucose tolerance test (GTT) or insulin tolerance test (ITT). (G) Immunoblot analyses for ITIH1 and O-GlcNAc ITIH1 in primary hepatocytes isolated from 8-week-old C57BL/6 mice injected via tail vein with lentiviral vector carrying the human OGT gene with an albumin promoter (Lv-OGTalb) or empty control vector (Lv-Con) (2 × 107 transduction units). Immunoblotting for O-GlcNAc proteins (CTD110.6 clone) was performed on immunoprecipitates of ITIH1 in primary hepatocyte cell lysates. (H) Immunoblot analyses for OGT in protein lysates of liver, epididymal fat tissue, and gastrocnemius muscle of 8-week-old C57BL/6 mice injected via tail vein with lentiviral vector carrying the human OGT gene with an albumin promoter (Lv-OGTalb) or empty vector control (Lv-Con) (2 × 107 transduction units) (n = 3 per group). The relative band intensities were quantified by densitometry analyses for the immunoblots. Values were expressed as means ± SEM. *P < 0.05 for G13 LKO + vehicle versus WT + ve- hicle; ##P < 0.01 for G13 LKO + ST045849 versus G13 LKO + vehicle (C); and *P < 0.05, **P < 0.01 for Lv-OGTalb versus Lv-Con (E, F, and H). Data were analyzed by one-way ANOVA, followed by least significant difference (LSD) (A), Bonferroni post hoc tests (C), or two-tailed Student’s t test (E, F, and H). N.S., not significant.

Article Snippet: ITIH1-enriched sample preparation for proteomics of O-GlcNAcylation HEK293A cells overexpressed with human ITIH1 (Origene, Rockville, MD, USA) were subjected to SDS-PAGE.

Techniques: Western Blot, Expressing, Injection, Plasmid Preparation, Control, Transduction, Over Expression, Isolation, Two Tailed Test