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

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.

Asialoglycoprotein receptor 1 is a novel PCSK9-independent ligand of liver LDLR cleaved by furin.

The hepatic carbohydrate-recognizing asialoglycoprotein receptor (ASGR1) mediates the endocytosis/lysosomal degradation of desialylated glycoproteins following binding to terminal galactose/N-acetylgalactosamine. Human heterozygote carriers of ASGR1 deletions exhibit ∼34% lower risk of coronary artery disease and ∼10% to 14% reduction of non-HDL cholesterol. Since the proprotein convertase PCSK9 is a major degrader of the low-density lipoprotein receptor (LDLR), we investigated the degradation and functionality of LDLR and/or PCSK9 by endogenous/overexpressed ASGR1 using Western blot and immunofluorescence in HepG2-naïve and HepG2-PCSK9-knockout cells. ASGR1, like PCSK9, targets LDLR, and both independently interact with/enhance the degradation of the receptor. This lack of cooperativity between PCSK9 and ASGR1 was confirmed in livers of wildtype (WT) and Pcsk9-/- mice. ASGR1 knockdown in HepG2-naïve cells significantly increased total (∼1.2-fold) and cell-surface (∼4-fold) LDLR protein. In HepG2-PCSK9-knockout cells, ASGR1 silencing led to ∼2-fold higher levels of LDLR protein and DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate)-LDL uptake associated with ∼9-fold increased cell-surface LDLR. Overexpression of WT-ASGR1/2 primarily reduced levels of immature non-O-glycosylated LDLR (∼110 kDa), whereas the triple Ala-mutant of Gln240/Trp244/Glu253 (characterized by loss of carbohydrate binding) reduced expression of the mature form of LDLR (∼150 kDa), suggesting that ASGR1 binds the LDLR in both a sugar-dependent and -independent fashion. The protease furin cleaves ASGR1 at the RKMK103↓ motif into a secreted form, likely resulting in a loss of function on LDLR. Altogether, we demonstrate that LDLR is the first example of a liver-receptor ligand of ASGR1. We conclude that silencing of ASGR1 and PCSK9 may lead to higher LDL uptake by hepatocytes, thereby providing a novel approach to further reduce LDL cholesterol levels.

Small molecules targeting GRP78 mitigate anti-GRP78 autoantibody-mediated tissue factor procoagulant activity in cultured endothelial cells.

BACKGROUND: The 78-kDa glucose-regulated protein (GRP78) expressed on the cell surface (csGRP78) has been reported to regulate tissue factor (TF) procoagulant activity (PCA) in lesion-resident endothelial cells (ECs), which is further enhanced by circulating anti-GRP78 autoantibodies that bind to the Leu98-Leu115 epitope in GRP78. OBJECTIVES: Determine the effects of the engagement of the anti-GRP78 autoantibody to csGRP78 on ECs and the underlying mechanisms that impact TF PCA. METHODS: Immunofluorescent staining was used to determine the presence of csGRP78 in tumor necrosis factor α-treated ECs. An established TF PCA assay was used to evaluate human ECs following treatment with anti-GRP78 autoantibodies. The Fura 2-AM assay (Abcam) was used to quantify changes in intracellular Ca2+ levels. Small molecules predicted to bind GRP78 were identified using artificial intelligence. Enzyme-linked immunosorbent assays were used to assess the ability of these GRP78 binders to mitigate TF activity and interfere with the autoantibody/csGRP78 complex. RESULTS: In tumor necrosis factor α-treated ECs, anti-GRP78 autoantibodies increased TF PCA. This observation was further enhanced by endoplasmic reticulum stress-induced elevation of csGRP78 levels. Anti-GRP78 autoantibody treatment increased intracellular Ca2+ levels. Sequestering the anti-GRP78 autoantibody with a conformational peptide or blocking with heparin attenuated anti-GRP78 autoantibody-induced TF PCA. We identified B07∗, as a GRP78 binder that diminished anti-GRP78 autoantibody-induced TF PCA on ECs. CONCLUSION: These findings show how anti-GRP78 autoantibodies enhance TF PCA that contributes to thrombosis and identify novel GRP78 binders that represent a potential novel therapeutic strategy for treating and managing atherothrombotic disease.

Intraductal papillary mucinous neoplasm of the pancreas: clinical characteristics and treatment outcomes of 118 consecutive patients from a single center.

BACKGROUND/PURPOSE: Appropriate surgical treatment strategies based on clinicopathological findings are unavailable for intraductal papillary mucinous neoplasm (IPMN) of the pancreas. We investigated the clinical features of pancreatic IPMN in a single-center database in order to design an optimal surgical strategy. METHODS: The medical records of 118 consecutive patients who had undergone surgical resection between August 1994 and December 2004, in whom IPMN was histologically confirmed, were reviewed retrospectively for radiological and pathological findings. RESULTS: Most of the invasive carcinomas in these patients were detected as the main-duct type (88.5%). The type of tumor (main-duct type vs branched-duct type), the tumor size, and the dilated duct size were significant predictive factors associated with malignancy. The relative risk of malignancy was greatest at 13-mm or more ductal dilation in the main-duct type (Odds ratio, 4.1), at 35-mm or more tumor size (Odds ratio, 7.6), and for main-duct type (Odds ratio, 3.9). Major pancreatic resections such as total pancreatectomy and pancreatoduodenectomy were performed in 14.5% and 69% of the patients, respectively. There was a 19.5% rate of incomplete resection, with these patients having a positive resection margin. However, significant recurrence did not occur in patients with a benign IPMN lesion which remained at the resection margin. The overall postoperative survival rate at 5 years was 98.2% for benign IPMN and 65.3% for malignant IPMN. CONCLUSIONS: Function-preserving strategies, based on the clinical status of the patient, are necessary in order to avoid possible severe metabolic complications following extended pancreatectomy in patients with benign IPMN because of the low recurrence rate and good prognosis of this entity, irrespective of margin status.