Research ArticleMetabolism

Overproduction of inter-α-trypsin inhibitor heavy chain 1 after loss of Gα13 in liver exacerbates systemic insulin resistance in mice

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Science Translational Medicine  09 Oct 2019:
Vol. 11, Issue 513, eaan4735
DOI: 10.1126/scitranslmed.aan4735
  • Fig. 1 Decrease in hepatic Gα13 in obese mice and patients with diabetes.

    (A) Immunoblot analyses for Gα13 expression of protein lysates of liver, adipose tissue (epididymal fat), and skeletal muscle (gastrocnemius) collected from C57BL/6 mice fed a normal diet (ND) or a high-fat diet (HFD) for 9 weeks. The relative band intensities of the immunoblots were quantified by densitometry and expressed as relative intensity normalized to β-actin (n = 4 per group). (B) Representative images of liver sections from C57BL/6 mice fed a normal diet (ND) or a high-fat diet (HFD) for 9 weeks [as in (A)] immunostained for Gα13 (brown) using a polyclonal antibody against Gα13 (n = 3 to 4 per group). (C) Immunoblot analysis for Gα13 expression in protein lysates of primary hepatocytes isolated from C57BL/6 mice fed either an ND or HFD for 12 weeks. The relative band intensities of the immunoblots were quantified by densitometry and expressed as relative intensity normalized to β-actin (n = 4 per group). (D) Immunoblot analyses for Gα13 expression in protein lysates of liver collected from wild-type (WT), obese ob/ob (top), and diabetic db/db (bottom) mice fed normal chow. Quantification of densitometry analyses for immunoblots is shown below the representative gels (n = 5 per group). The relative band intensities of the immunoblots were quantified by densitometry and expressed as intensity relative to β-actin. (E) Correlation analyses between hepatic Gα13 expression and fasting blood glucose concentrations in C57BL/6 mice fed a normal diet (ND) or high-fat diet (HFD) for 9 weeks (left, n = 8 or 13 per group) and wild-type (WT), obese ob/ob, or diabetic db/db mice (middle and right, n = 5 per group) fed a normal diet. An identical correlation was reanalyzed (far right box) using combined data for the same obese ob/ob and diabetic db/db mice (n = 23). (F) Immunoblot analyses for Gα13 expression in protein lysates of liver specimens from individuals with normal glucose tolerance (NGT), impaired glucose tolerance (IGT), and overt diabetes (T2DM) (NAFLD cohort #1). Quantification of densitometry analyses for immunoblots relative to β-actin is shown below the representative gels (n = 8 to 10 per group). (G) Representative images of liver sections obtained from the same individuals described in (F) stained for Gα13 expression (brown) using a polyclonal antibody against Gα13 (left, n = 3 to 4 per group) or stained with hematoxylin and eosin (H&E) (right, n = 1 to 2 per group). Scale bars, 100 μm. (H) Correlation analyses between hepatic Gα13 expression and diabetic indices such as the homeostatic model assessment of insulin resistance (HOMA-IR) test, insulin, fasting glucose, HbA1c, adipose tissue insulin resistance index (adipose tissue IR), and C-peptide in subjects with non-alcoholic fatty liver disease (NAFLD) or normal controls (n = 27, total number of subjects including normal controls and NAFLD patients). Values are expressed as means ± SEM. (**P < 0.01 versus ND or WT). Data were analyzed by two-tailed Student’s t test (A and D) or Pearson correlations (E and H). N.S., not significant.

  • Fig. 2 Impaired glucose homeostasis and insulin resistance in G13 LKO mice fed a high-fat diet.

    (A) Measurement of fasting blood glucose concentrations in wild-type (WT) mice or mice with liver-specific ablation of Gna13 (G13 LKO) fed a high-fat diet (HFD) for 9 weeks (n = 5 per group). (B) Results of the glucose tolerance test (glucose gavage; 2 g/kg body weight) in WT and G13 LKO mice fed an HFD for 10 weeks (n = 6 per group). (C) Results of the insulin tolerance test (insulin injection; 1.5 insulin units/kg body weight) in WT and G13 LKO mice fed an HFD for 13 weeks (n = 12 per group). (D) Measurements of serum insulin concentrations during the glucose tolerance test in WT and G13 LKO mice fed an HFD for 10 weeks (n = 6 per group). (E) Measurements of fasting blood glucose concentrations in WT or G13 LKO mice fed an HFD for 5 weeks (n = 4 or 5 per group). Mice were subjected to fasting and then refeeding (fasted for 16 hours and then refed for 4 hours). (F) Measurements of serum insulin and C-peptide concentrations in the same mice as in (E) (n = 6 per group). (G) Representative images of pancreas sections from WT and G13 LKO mice fed an HFD for 9 weeks and stained for hematoxylin and eosin (n = 3 to 4 per group). (H) Immunoblots for phosphorylated-Akt (p-Akt) and total Akt expression in protein lysates of the livers from WT or G13 LKO mice fed an HFD for 5 weeks and injected with a single dose of insulin (2 insulin units/kg body weight; i.p., 15 min) or from WT or G13 LKO mice subjected to fasting and then refeeding (fasted for 16 hours and then refed for 4 hours). (I) Immunoblots for phosphorylated-Akt (p-Akt) and total Akt expression in protein lysates of primary hepatocytes isolated from WT or G13 LKO mice fed an HFD for 5 weeks and treated with a single dose of insulin (100 nM, 15 min). (J) Measurement of glucose production rate in hepatocytes from WT or G13 LKO mice fed an HFD for 5 weeks (n = 3 per group, experiments performed in triplicate). (K) Immunoblots for phosphorylated-Akt (p-Akt) and total Akt expression in protein lysates of epididymal fat tissue or soleus muscle from the same mice as in (E). Quantification of densitometry analyses for immunoblots relative to β-actin is presented below each blot (n = 3 per group). (L) Measurement of 2-deoxyglucose uptake rate in epididymal fat tissue or soleus muscle from WT or G13 LKO mice fed an HFD for 5 weeks (n = 4 or 6 per group). (M and N) The effect of hepatic Gα13 overexpression on glucose metabolism was determined by analyzing glucose (M) or insulin tolerance (N) in C57BL/6 mice injected via the tail vein with control lentiviruses (Lv-Con) or lentiviruses expressing mouse Gα13 (Lv-Gα13) (1.1 × 107 transduction units). Injected mice were subsequently subjected to HFD feeding for 8 or 9 weeks (n = 6 or 7 per group). For (B) to (D) and (M) and (N), insets represent area under the curve (AUC). Values are expressed as means ± SEM. *P < 0.05, **P < 0.01 for G13 LKO versus WT (A to C and K) or Lv-Gα13 versus Lv-Con (M and N). Data were analyzed by two-tailed Student’s t test (A to D, K, M, and N) or one-way ANOVA followed by Bonferroni post hoc tests (E, F, J, and L). N.S., not significant.

  • Fig. 3 Identification of ITIH1 as a hepatocyte-secreted protein enhanced by loss of Gα13.

    (A) Immunoblots for phosphorylated-Akt (p-Akt) and total Akt expression 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). β-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.

  • Fig. 4 Increased hepatic and serum ITIH1 concentrations in NAFLD subjects with diabetes.

    (A) Immunoblot analyses for ITIH1 and ITIH2 expression in protein lysates of liver specimens from subjects with normal glucose tolerance (NGT), impaired glucose tolerance (NGT), or type 2 diabetes (T2DM) (NAFLD cohort #1). (B) Representative immunofluorescence images of liver sections from NAFLD patients with diabetes stained for ITIH1 (red) with DAPI counterstain (blue) (n = 3 to 4 per group, 60× magnification). (C) Quantitative analysis of ELISA for serum ITIH1 from the same subjects as in (A) (n = 7 per group). (D and E) Correlation analyses between serum ITIH1 concentrations and hepatic Gα13 expression (D, n = 17) and serum ITIH1 concentrations and insulin resistance–related indices including HOMA-IR, insulin, glucose, HbA1c, adipose tissue IR, and C-peptide (E, n = 21) in subjects with NAFLD or normal controls. (F) Changes in diabetic indices in relation to serum ITIH1 concentrations for human subjects categorized into two subgroups by the median value of serum ITIH1 (n = 10 per group). Data are shown as box and whisker plots. Box, interquartile range (IQR); whiskers, 5 to 95 percentiles; horizontal line within box, median. Values are expressed as means ± SEM. Data were analyzed by one-way ANOVA followed by Bonferroni post hoc tests (C), Pearson correlations (D and E), or Mann-Whitney tests (F).

  • 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 slides were pretreated with hyaluronidase (20 U/ml) or vehicle for 2 hours at 37°C. Scale bars, 100 μm. (D) Representative images of epididymal fat tissue and gastrocnemius muscle sections from wild-type (WT) C57BL/6 mice injected via the tail vein with control lentiviruses (Lv-Con) or lentiviruses expressing mouse Gα13 (Lv-Gα13) (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 μm. (E) Immunoblot analyses for ITIH1 expression in fractions enriched 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 μm. 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.

  • Fig. 6 Increase in O-GlcNAc transferase–mediated O-GlcNAcylation of proteins after loss of Gα13.

    (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 densitometry analyses for the immunoblots (n = 3 per group). β-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 Gα13 (Lv-Gα13) (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 Gα13 (Ad-G13QL) or green fluorescent protein (Ad-GFP) as a control. (H) Immunoblot 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-Gα13 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).

  • 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) or glucose gavage (right, n = 3 to 4 per group). Glucose (2 g/kg body weight) was orally administered to mice, and the liver tissues were harvested after 6 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 Gα13 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 + vehicle; ##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.

  • Fig. 8 Recovery of impaired glucose tolerance in HFD-fed G13 LKO mice treated with anti-ITIH1 antibody.

    (A to C) Measurements for body weight gain and epididymal fat weights for 6-week-old C57BL/6 mice subjected to a high-fat diet (HFD) or normal diet (ND) for 10 weeks with daily injections of rabbit polyclonal anti-ITIH1 antibody or control pre-IgG antibody for the last 2 weeks of the HFD or ND diet (n = 7 to 8 per group). Body weight gain (A), liver–to–body weight ratio (B), and epididymal fat weight (C). (D, E, G, and H) Effects of anti-ITIH1 antibody treatment on the results of the glucose tolerance test (1.5 g/kg body weight) or insulin tolerance test (0.75 insulin units/kg body weight) in C57BL/6 mice fed an HFD or ND (D and E) and WT or G13 LKO mice fed an HFD (G and H). Mice were injected daily with purified anti-ITIH1 antibody or pre-IgG (i.p., 250 μg each) for the last 2 weeks of HFD or ND feeding. (D) Analysis of the glucose tolerance test in C57BL/6 mice fed an ND or HFD for 10 weeks (ND, n = 7; HFD, n = 8; HFD–pre-IgG, n = 7; HFD–anti-ITIH1 antibody, n = 8). (E) Analysis of the insulin tolerance test in C57BL/6 mice fed an ND or HFD for 11 weeks (ND, n = 7; HFD, n = 8; HFD–pre-IgG, n = 6; HFD–anti-ITIH1 antibody, n = 6). (F) Quantitative analysis of ELISA for ITIH1 in serum (ND, n = 7; HFD, n = 8; HFD–pre-IgG, n = 7; HFD–anti-ITIH1 antibody, n = 8). (G) Analysis of the glucose tolerance test in WT and G13 LKO mice fed an HFD for 11 weeks (n = 7 per group). (H) Analysis of the insulin tolerance test in WT and G13 LKO mice fed an HFD for 12 weeks (n = 6 per group). (I) Quantitative analysis of ELISA for serum ITIH1 in WT and G13 LKO mice fed an HFD (WT–pre-IgG or G13 LKO–pre-IgG, n = 7; G13 LKO–anti-ITIH1 antibody, n = 6). (J and K) Body weight gain (J) or fasting blood glucose concentrations (K) in G13 LKO mice fed an HFD for 13 weeks and treated with anti-ITIH1 antibody or control IgG as in (A) (n = 5 per group). Days 0 and 14 represent before and after treatments, respectively, with anti-ITIH1 antibody or pre-IgG control antibody. (L) Measurements of 2-deoxyglucose uptake in epididymal fat tissue of WT mice fed an HFD for 16 weeks. (Left) Basal 2-deoxyglucose uptake (n = 3 mice) and uptake after glucose gavage (n = 3 mice) in adipose tissue from mice treated with pre-IgG antibody control or anti ITIH1 antibody (right). Basal 2-deoxyglucose uptake (n = 3 mice) and uptake after glucose gavage (n = 3 mice) in skeletal soleus muscle of mice treated with pre-IgG antibody control or anti-ITIH1 antibody. For (D), (E), (G), and (H), insets represent area under the curve (AUC). Values are expressed as means ± SEM. *P < 0.05, **P < 0.01 for HFD versus ND (A, D, and E) or G13 LKO mice treated with pre-IgG versus WT mice treated with pre-IgG (G to I); #P < 0.05, ##P < 0.01 for mice fed an HFD and treated with anti-ITIH1 antibody versus mice fed an HFD and treated with pre-IgG antibody (D and E) or G13 LKO mice treated with anti-ITIH1 antibody versus G13 LKO mice treated with pre-IgG antibody (G to I). Data were analyzed by one-way ANOVA followed by Bonferroni (A to K) or least significant difference (LSD) (L) post hoc tests. N.S., not significant.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/11/513/eaan4735/DC1

    Materials and Methods

    Fig. S1. Decrease in hepatic Gα13 in patients with diabetes.

    Fig. S2. Correlations between hepatic Gα13 and lipid profiles in patients with diabetes.

    Fig. S3. A metabolic and liver function profile in G13 LKO mice fed a high-fat diet.

    Fig. S4. Impaired glucose homeostasis and insulin resistance in G13 LKO mice fed a normal diet.

    Fig. S5. Correlations between serum ITIH1 concentrations and lipid profiles in patients with NAFLD.

    Fig. S6. ITIH1 and ITIH2 concentrations in the liver of patients with diabetes or G13 LKO mice fed a high-fat diet.

    Fig. S7. Immunoblotting for extracellular matrix and cytoplasmic protein markers.

    Fig. S8. Immunofluorescence staining for ITIH1-hyaluronan complexes in vitro.

    Fig. S9. Increase in O-GlcNAC transferase–mediated O-GlcNAcylation and ITIH1 concentrations after loss of Gα13.

    Fig. S10. LC-MS/MS analysis of O-GlcNAc modification of human ITIH1.

    Fig. S11. O-GlcNAC transferase–mediated stabilization of ITIH1 through suppression of proteasomal degradation.

    Fig. S12. The effect of ITIH1 silencing on insulin sensitivity in vitro and effects of anti-ITIH1 antibody treatment of mice in vivo.

    Fig. S13. The effect of a high to low glucose transition on O-GlcNAcylation and Gα13 concentrations in mice in vivo.

    Table S1. Characteristics of subjects with NAFLD in cohort #1.

    Data file S1.

    References (4251)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Decrease in hepatic Gα13 in patients with diabetes.
    • Fig. S2. Correlations between hepatic Gα13 and lipid profiles in patients with diabetes.
    • Fig. S3. A metabolic and liver function profile in G13 LKO mice fed a high-fat diet.
    • Fig. S4. Impaired glucose homeostasis and insulin resistance in G13 LKO mice fed a normal diet.
    • Fig. S5. Correlations between serum ITIH1 concentrations and lipid profiles in patients with NAFLD.
    • Fig. S6. ITIH1 and ITIH2 concentrations in the liver of patients with diabetes or G13 LKO mice fed a high-fat diet.
    • Fig. S7. Immunoblotting for extracellular matrix and cytoplasmic protein markers.
    • Fig. S8. Immunofluorescence staining for ITIH1-hyaluronan complexes in vitro.
    • Fig. S9. Increase in O-GlcNAC transferase–mediated O-GlcNAcylation and ITIH1 concentrations after loss of Gα13.
    • Fig. S10. LC-MS/MS analysis of O-GlcNAc modification of human ITIH1.
    • Fig. S11. O-GlcNAC transferase–mediated stabilization of ITIH1 through suppression of proteasomal degradation.
    • Fig. S12. The effect of ITIH1 silencing on insulin sensitivity in vitro and effects of anti-ITIH1 antibody treatment of mice in vivo.
    • Fig. S13. The effect of a high to low glucose transition on O-GlcNAcylation and Gα13 concentrations in mice in vivo.
    • Table S1. Characteristics of subjects with NAFLD in cohort #1.
    • References (4251)

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    Other Supplementary Material for this manuscript includes the following:

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