Restoration of the ER stress response protein TDAG51 in hepatocytes mitigates NAFLD in mice

Abstract

Endoplasmic reticulum stress is associated with insulin resistance and the development of nonalcoholic fatty liver disease. Deficiency of the endoplasmic reticulum stress response T-cell death-associated gene 51 (TDAG51) (TDAG51^-/-) in mice promotes the development of high-fat diet (HFD)-induced obesity, fatty liver, and hepatic insulin resistance. However, whether this effect is due specifically to hepatic TDAG51 deficiency is unknown. Here, we report that hepatic TDAG51 protein levels are consistently reduced in multiple mouse models of liver steatosis and injury as well as in liver biopsies from patients with liver disease compared to normal controls. Delivery of a liver-specific adeno-associated virus (AAV) increased hepatic expression of a TDAG51-GFP fusion protein in WT, TDAG51^-/-, and leptin-deficient (ob/ob) mice. Restoration of hepatic TDAG51 protein was sufficient to increase insulin sensitivity while reducing body weight and fatty liver in HFD fed TDAG51^-/- mice and in ob/ob mice. TDAG51^-/- mice expressing ectopic TDAG51 display improved Akt (Ser473) phosphorylation, post-insulin stimulation. HFD-fed TDAG51^-/- mice treated with AAV-TDAG51-GFP displayed reduced lipogenic gene expression, increased beta-oxidation and lowered hepatic and serum triglycerides, findings consistent with reduced liver weight. Further, AAV-TDAG51-GFP-treated TDAG51^-/- mice exhibited reduced hepatic precursor and cleaved sterol regulatory-element binding proteins (SREBP-1 and SREBP-2). In vitro studies confirmed the lipid-lowering effect of TDAG51 overexpression in oleic acid-treated Huh7 cells. These studies suggest that maintaining hepatic TDAG51 protein levels represents a viable therapeutic approach for the treatment of obesity and insulin resistance associated with nonalcoholic fatty liver disease.

Keywords: hepatocyte; insulin resistance; lipid metabolism; liver; obesity; triglyceride.

Figure 1

Hepatic TDAG51 protein expression is significantly reduced inhuman NASH andmultiple mouse models of liver injury or steatosis. Representative immunoblot and densitometry of TDAG51 protein levels relative to GAPDH protein in the livers of A, WT mice on an 18-day normal chow or methionine-choline–deficient (MCD) diet (n = 8), B, WT, human-only cystathionine-beta-synthase (HO), and cystathionine-beta-synthase-null also known as Maeda KO (MKO) mice (n = 8), (C) HO mice treated with acetaminophen (APAP) compared to saline-treated controls (n = 8), (D) WT mice treated with APAP compared to saline-treated controls (n = 8). E, H&E-stained sections of human liver. Histologic assessment of the livers was determined using light microscopy at 20× magnification. Sections for normal, NASH with steatosis (i, ii), and NASH without steatosis (iii) correspond to the same liver lysates immunoblotted in Figure 1F. F, representative immunoblots and densitometry of human liver lysates from normal and NASH patients probed for PHLDA1 (human homolog of mouse TDAG51), and NASH markers, MRP2 and MRP4, relative to ERK2 as a loading control. Statistical comparisons were assessed with an independent two-tailed Student’s t tests for TDAG51/PHLDA1 densitometry and increased levels of MRP2 and MRP4 were assessed with independent one-tailed Student’s t tests. Data are represented as means with error bars representing SD. A, ∗∗∗∗p = 0.00001 versus control. B, ∗∗p = 0.0026 versus WT. C, ∗p = 0.028 versus HO. D, ∗p = 0.048 versus WT. F, ∗∗p < 0.007 versus normal, ∗∗p < 0.009 versus normal, and ∗p = 0.028 versus normal. Scale bar represents 100 μm. AAV, adeno-associated virus; ERK2, extracellular signal-regulated kinase 2; MRP, multidrug resistance–associated protein; NASH, nonalcoholic steatohepatitis; PHLDA, Pleckstrin homology–like domain A-1; TDAG51, T-cell death–associated gene 51.

Figure 2

Liver-specific expression of GFP or TDAG51-GFP fusion protein in chow-fed mice. WT C57BL/6J mice 8 weeks of age were injected with AAV encoding either GFP or TDAG51-GFP. After 4 weeks post-injection, tissues were immunoblotted for GFP or TDAG51-GFP. A, representative immunoblots for GFP (27-kDa) or TDAG51-GFP (67-kDa) using an anti-GFP antibody. Immunoblots were reprobed for β-actin as a loading control. Both GFP and TDAG51-GFP protein were exclusively expressed in liver compared to other tissues. B, GFP fluorescent images of liver sections from WT C57BL/6J mice 4 weeks postinjection with 5 × 10¹¹ AAV-GFP, 5 × 10¹¹ AAV-TDAG51-GFP genome containing particles or PBS. Green represents GFP while blue represents 4′,6-diamidino-2-phenylindole (nuclei). C, representative images of H&E-stained livers 4 weeks post-injection (bottom panel). N = 6 mice per group. The scale bar represents 10 μm. AAV, adeno-associated virus; HFD, high-fat diet; TDAG51, T-cell death–associated gene 51.

Figure 3

Expression of hepatic TDAG51 AAV in TDAG51^−/−mice.A, fifteen-week-old TDAG51^−/− mice were injected with 5 × 10¹¹ genome containing particles of AAV-GFP or AAV-TDAG51-GFP. Four weeks after AAV injection, mice were fed HFD and glucose tolerance (GTT) and insulin tolerance tests (ITT) were performed 4 weeks later (8 weeks postinjection). To assess hepatic TDAG51-GFP protein levels, mice were sacrificed at 27 weeks old, and the livers harvested, snap-frozen, and total liver lysates immunoblotted for TDAG51 or GFP. B, representative immunoblots for TDAG51 and GFP from livers of TDAG51^−/− mice injected with AAV-GFP or AAV-TDAG51-GFP compared to WT controls. The 68-kDa TDAG51-GFP fusion protein was detected by both the anti-GFP and anti-TDAG51 antibody. Hepatic expression of the TDAG51-GFP fusion protein was reduced in the livers of TDAG51^−/− mice compared to WT. C, representative GFP fluorescent images of liver sections from TDAG51^−/− mice fed HFD. Green represents GFP, and blue represents 4′,6-diamidino-2-phenylindole (nuclei). N = 3 to 4 mice per group. The scale bar represents 10 μm. AAV, adeno-associated virus; HFD, high-fat diet; TDAG51, T-cell death–associated gene 51.

Figure 4

Restoration of hepatic TDAG51 protein improves insulin sensitivity in TDAG51^−/−mice fed HFD.A, glucose tolerance test (GTT) of TDAG51^−/− mice after 4 weeks on HFD. Data represent the mean ± SD. Groups were compared by two-way ANOVA using repeated measures (time × AAV: F = 0.68, p > 0.05; time: F = 24.63, p < 0.0001; AAV: F = 0.08, p > 0.05). Area under the curve (AUC) is represented in the right panel; groups were compared by student’s independent t test (two-tailed). Data represent the mean ± SD. B, insulin tolerance test (ITT) of TDAG51^−/− mice after 4 weeks on HFD. Data represent the mean ± SD. Groups were compared by two-way ANOVA using repeated measures (time × AAV: F = 1.1, p > 0.05; time: F = 99.7, p < 0.0001; AAV: F = 5.5, p < 0.02). Sidak’s multiple comparison used post hoc (90 min, ∗∗p < 0.01). Area under the curve (AUC) is represented in the right panel; groups were compared by student’s independent t test (two-tailed). Data represent the mean ± SD (∗∗p = 0.01). C, fourteen-week-old TDAG51^−/− mice injected with AAV-GFP or AAV-TDAG51-GFP fed HFD were fasted for 6 h and livers collected 15 min after injection of insulin. Representative immunoblots for total and phospho-Akt (S473) from liver lysates. N = 4 mice per group. Data represent the mean ± SD (∗∗p = 0.01). Statistical comparisons were assessed by an independent Student’s t tests (two-tailed). AAV, adeno-associated virus; HFD, high-fat diet; TDAG51, T-cell death–associated gene 51.

Figure 5

Hepatic TDAG51 AAV reduces body weight,in addition toliverand adipose tissue weight and results in higher serum adiponectin levels in TDAG51^−/−mice fed HFD.A, total body weight of AAV-injected TDAG51^−/− mice fed HFD over time and at endpoint. Groups were compared by two-way ANOVA using repeated measures (Time × AAV: F = 5.99, p < 0.0001; time: F = 138.6, p < 0.0001; AAV: F = 0.06, p = 0.057). Sidak’s multiple comparison used post hoc (12 weeks, p < 0.002). Data represent the mean ± SD. At endpoint, groups were compared with an independent Student’s t tests (two-tailed) (∗∗p = 0.008). B, food intake of AAV-injected TDAG51^−/− HFD mice. C, epididymal fat pad weight at endpoint from AAV-injected TDAG51^−/− mice fed HFD (∗p = 0.04). D, total liver weight at endpoint from AAV-injected TDAG51^−/− mice fed HFD (∗∗p = 0.004). E, liver weight as a percentage of total body weight in TDAG51^−/− mice fed HFD (∗∗p = 0.01). F, plasma adiponectin levels at endpoint from AAV-injected TDAG51^−/− mice after an overnight fast (∗p = 0.04). G, plasma leptin levels at endpoint from AAV-injected TDAG51^−/− mice after an overnight fast. N = 3 to 4 mice per group. Data represent the mean ± SD. B–G, statistical comparison of two groups were compared with independent Student’s t tests (two-tailed). AAV, adeno-associated virus; HFD, high-fat diet; TDAG51, T-cell death–associated gene 51.

Figure 6

Restoration of hepatic TDAG51 protein reduces total hepatic lipid content by increasing hepatic gene expression ofbeta-oxidation markers while reducing de novo lipogenesis.A, twenty-six-week-old TDAG51^−/− mice injected with AAV-GFP or AAV-TDAG51-GFP fed HFD were fasted for 16 h and liver RNA and total protein lysates were collected and subjected to RT-PCR and immunoblotting. Fold change in FAS, Scd-1, DGAT-2, SREBP-2, LDLR, and PCSK9 mRNA expression in AAV-injected TDAG51^−/− mice fed HFD. B, representative immunoblots for precursor (p) and cleaved (n) SREBP1 and SREBP2 from liver lysates. N = 4 mice per group. C, Fold change in ADRP, SREBP-1, PPAR-alpha, CPT1, and CPT2 normalized to 18S housekeeping gene and relative to AAV-GFP controls. D, plasma (mg/dl) and E, hepatic (mg/g tissue) triglyceride content measured by a colorimetric assay. F, representative images of H&E-stained livers from TDAG51^−/− mice (bottom panels). The scale bar represents 10 μm. Table represents pathologist scoring of percent steatosis per 20× liver tissue. Statistical comparison of two groups were compared with independent Student’s t tests (two-tailed), ∗∗p < 0.01 versus GFP and ∗p < 0.05 versus GFP. A–E, data represent the mean ± SD. AAV, adeno-associated virus; DGAT-2, diacylglycerol O-acyltransferase-2; FAS, fatty acid synthase; HFD, high-fat diet; Scd-1, stearoyl-CoA desaturase-1; SREBP, sterol regulatory–element binding protein; TDAG51, T-cell death–associated gene 51.

Figure 7

Overexpression of hepatic TDAG51 protein reduces total hepatic lipid content in vitro by decreasing cleaved SREBP-1 protein expression.A, Huh7 cells transfected with GFP or TDAG51-GFP plasmid then stained and quantified for Oil Red O. B, representative immunoblots for SREBP-1, GFP, and GFP-TDAG51 from Huh7 cells transfected with GFP or GFP-TDAG51. C, Huh7 cells transfected with GFP or TDAG51-GFP plasmid were treated with oleic acid then stained and quantified for Oil Red O. D, total triglyceride content measured in Huh7 cell lysate transfected with GFP or GFP-TDAG51 and treated with oleic acid. Data represent the mean ± SD. Statistical comparison of two groups were compared with independent Student’s t tests (two-tailed). ∗p < 0.05 versus GFP. The scale bar represents 100 μm. AAV, adeno-associated virus; SREBP, sterol regulatory–element binding protein; TDAG51, T-cell death–associated gene 51.

Figure 8

Expression of hepatic TDAG51 AAV in ob/ob mice.A, nine-week-old ob/ob mice were injected with 5 × 10¹¹ genome containing particles of AAV-GFP or AAV-TDAG51-GFP. At 16 and 19 weeks of age, glucose tolerance (GTT) and insulin tolerance tests (ITT) were performed, respectively. At 20 weeks of age (11 weeks post-injection), ob/ob mice were sacrificed and livers harvested. Total liver lysates immunoblotted for GFP, TDAG51, or TDAG51-GFP. Immunoblots were reprobed for β-actin as a loading control. B, representative immunoblots from livers of ob/ob mice injected with AAV-GFP or AAV-TDAG51-GFP. Densitometric analysis revealed that ectopic TDAG51-GFP protein levels were significantly reduced in chow-fed ob/ob mice compared to WT mice injected with AAV-TDAG51-GFP. C, GFP fluorescent images of liver sections from AAV-GFP– or AAV-TDAG51-GFP–infected ob/ob mice. Green represents GFP while blue represents 4′,6-diamidino-2-phenylindole (nuclei). The scale bar represents 10 μm. AAV, adeno-associated virus; TDAG51, T-cell death–associated gene 51.

Figure 9

Restoration of hepatic TDAG51 protein improves insulin sensitivity in the livers of ob/ob mice.A, glucose tolerance test (GTT) following 6 h fast of ob/ob mice, 10 weeks post-injection with AAV-GFP or AAV-TDAG51-GFP (left panel). Groups were compared by two-way ANOVA using repeated measures (Time × AAV: F = 0.47, p > 0.05; time: F = 30.4, p < 0.0005; AAV: F = 0.33, p < 0.02). Data represent the mean ± SD. Area under the curve (AUC) is represented in the right panel, groups were compared by student’s independent t test (two-tailed) (p = 0.01). Data represent the mean ± SD. B, insulin tolerance test (ITT) of ob/ob mice, 10 weeks post-injection with AAV-GFP or AAV-TDAG51-GFP (left panel). Groups were compared by two-way ANOVA using repeated measures (Time × AAV: F = 3.58, p < 0.02; time: F = 38.33, p < 0.0001; AAV: F = 6.26, p < 0.05). Sidak’s multiple comparison used post hoc (15 min, ∗∗p < 0.01; 30 min, ∗p < 0.04). Data represent the mean ± SD. AUC is represented in the right panel; groups were compared by student’s independent t test (two-tailed) (p = 0.02). Data represent the mean ± SD. AAV, adeno-associated virus; TDAG51, T-cell death–associated gene 51.

Figure 10

Restoration of hepatic TDAG51 protein reduces body weight and plasma triglycerides by increasing gene expression ofbeta-oxidation markers while reducing de novo lipogenesis in the livers of ob/ob.A, total body weight at endpoint (p = 0.03). B, food intake of AAV-injected ob/ob mice. C, total raw liver weight expressed as a percent of body weight. D, epididymal fat pad weight expressed as a percent of body weight. E, fold change of lipogenic and beta-oxidative markers normalized to 18S relative to AAV-GFP control. F, plasma (mg/dl) (p = 0.0012) and (G) hepatic (mg/g tissue) triglyceride content measured by a colorimetric assay. H, representative images of H&E-stained livers from ob/ob mice injected with AAV-GFP or AAV-TDAG51-GFP scored for percent steatosis. The scale bar represents 10 μm. A–H, data represent the mean ± SD. Statistical comparison of two groups were compared with independent Student’s t tests (two-tailed). ∗p < 0.05 and ∗∗p < 0.01. AAV, adeno-associated virus; TDAG51, T-cell death–associated gene 51.