Ube4A maintains metabolic homeostasis and facilitates insulin signaling in vivo.

OBJECTIVE: Defining the regulators of cell metabolism and signaling is essential to design new therapeutic strategies in obesity and NAFLD/NASH. E3 ubiquitin ligases control diverse cellular functions by ubiquitination-mediated regulation of protein targets, and thus their functional aberration is associated with many diseases. The E3 ligase Ube4A has been implicated in human obesity, inflammation, and cancer. However, its in vivo function is unknown, and no animal models are available to study this novel protein. METHODS: A whole-body Ube4A knockout (UKO) mouse model was generated, and various metabolic parameters were compared in chow- and high fat diet (HFD)-fed WT and UKO mice, and in their liver, adipose tissue, and serum. Lipidomics and RNA-Seq studies were performed in the liver samples of HFD-fed WT and UKO mice. Proteomic studies were conducted to identify Ube4A's targets in metabolism. Furthermore, a mechanism by which Ube4A regulates metabolism was identified. RESULTS: Although the body weight and composition of young, chow-fed WT and UKO mice are similar, the knockouts exhibit mild hyperinsulinemia and insulin resistance. HFD feeding substantially augments obesity, hyperinsulinemia, and insulin resistance in both sexes of UKO mice. HFD-fed white and brown adipose tissue depots of UKO mice have increased insulin resistance and inflammation and reduced energy metabolism. Moreover, Ube4A deletion exacerbates hepatic steatosis, inflammation, and liver injury in HFD-fed mice with increased lipid uptake and lipogenesis in hepatocytes. Acute insulin treatment resulted in impaired activation of the insulin effector protein kinase Akt in liver and adipose tissue of chow-fed UKO mice. We identified the Akt activator protein APPL1 as a Ube4A interactor. The K63-linked ubiquitination (K63-Ub) of Akt and APPL1, known to facilitate insulin-induced Akt activation, is impaired in UKO mice. Furthermore, Ube4A K63-ubiquitinates Akt in vitro. CONCLUSION: Ube4A is a novel regulator of obesity, insulin resistance, adipose tissue dysfunction and NAFLD, and preventing its downregulation may ameliorate these diseases.

Enhancing Hepatic MBOAT7 Expression in Mice With Nonalcoholic Steatohepatitis.

BACKGROUND AND AIMS: Polymorphisms near the membrane bound O-acyltransferase domain containing 7 (MBOAT7) genes are associated with worsened nonalcoholic fatty liver (NASH), and nonalcoholic fatty liver disease (NAFLD)/NASH may decrease MBOAT7 expression independent of these polymorphisms. We hypothesized that enhancing MBOAT7 function would improve NASH. METHODS: Genomic and lipidomic databases were mined for MBOAT7 expression and hepatic phosphatidylinositol (PI) abundance in human NAFLD/NASH. Male C57BL6/J mice were fed either choline-deficient high-fat diet or Gubra Amylin NASH diet and subsequently infected with adeno-associated virus expressing MBOAT7 or control virus. NASH histological scoring and lipidomic analyses were performed to assess MBOAT7 activity, hepatic PI, and lysophosphatidylinositol (LPI) abundance. RESULTS: Human NAFLD/NASH decreases MBOAT7 expression and hepatic abundance of arachidonate-containing PI. Murine NASH models display subtle changes in MBOAT7 expression, but significantly decreased activity. After MBOAT7 overexpression, liver weights, triglycerides, and plasma alanine and aspartate transaminase were modestly improved by MBOAT7 overexpression, but NASH histology was not improved. Despite confirmation of increased activity with MBOAT7 overexpression, content of the main arachidonoylated PI species was not rescued by MBOAT7 although the abundance of many PI species was increased. Free arachidonic acid was elevated but the MBOAT7 substrate arachidonoyl-CoA was decreased in NASH livers compared to low-fat controls, likely due to the decreased expression of long-chain acyl-CoA synthetases. CONCLUSION: Results suggest decreased MBOAT7 activity plays a role in NASH, but MBOAT7 overexpression fails to measurably improve NASH pathology potentially due to the insufficient abundance of its arachidonoyl-CoA substrate.

CLIC1 null mice demonstrate a role for CLIC1 in macrophage superoxide production and tissue injury.

We generated and studied CLIC1 null (C1KO) mice to investigate the physiological role of this protein. C1KO and matched wild-type (WT) mice were studied in two models of acute toxic tissue injury. CLIC1 expression is upregulated following acute injury of WT kidney and pancreas and is absent in C1KOs. Acute tissue injury is attenuated in the C1KOs and this correlates with an absence of the rise in tissue reactive oxygen species (ROS) that is seen in WT mice. Infiltration of injured tissue by inflammatory cells was comparable between WT and C1KOs. Absence of CLIC1 increased PMA-induced superoxide production by isolated peritoneal neutrophils but dramatically decreased PMA-induced superoxide production by peritoneal macrophages. CLIC1 is expressed in both neutrophils and macrophages in a peripheral pattern consistent with either plasma membrane or the cortical cytoskeleton in resting cells and redistributes away from the periphery following PMA stimulation in both cell types. Absence of CLIC1 had no effect on redistribution or dephosphorylation of Ezrin/ERM cytoskeleton in macrophages. Plasma membrane chloride conductance is altered in the absence of CLIC1, but not in a way that would be expected to block superoxide production. NADPH oxidase redistributes from an intracellular compartment to the plasma membrane when WT macrophages are stimulated to produce superoxide and this redistribution fails to occur in C1KO macrophages. We conclude that the role of CLIC1 in macrophage superoxide production is to support redistribution of NADPH oxidase to the plasma membrane, and not through major effects on ERM cytoskeleton or by acting as a plasma membrane chloride channel.

Tissue and subcellular distribution of CLIC1.

BACKGROUND: CLIC1 is a chloride channel whose cellular role remains uncertain. The distribution of CLIC1 in normal tissues is largely unknown and conflicting data have been reported regarding the cellular membrane fraction in which CLIC1 resides. RESULTS: New antisera to CLIC1 were generated and were found to be sensitive and specific for detecting this protein. These antisera were used to investigate the distribution of CLIC1 in mouse tissue sections and three cultured cell lines. We find CLIC1 is expressed in the apical domains of several simple columnar epithelia including glandular stomach, small intestine, colon, bile ducts, pancreatic ducts, airway, and the tail of the epididymis, in addition to the previously reported renal proximal tubule. CLIC1 is expressed in a non-polarized distribution in the basal epithelial cell layer of the stratified squamous epithelium of the upper gastrointesitinal tract and the basal cells of the epididymis, and is present diffusely in skeletal muscle. Distribution of CLIC1 was examined in Panc1 cells, a relatively undifferentiated, non-polarized human cell line derived from pancreatic cancer, and T84 cells, a human colon cancer cell line which can form a polarized epithelium that is capable of regulated chloride transport. Digitonin extraction was used to distinguish membrane-inserted CLIC1 from the soluble cytoplasmic form of the protein. We find that digitonin-resistant CLIC1 is primarily present in the plasma membrane of Panc1 cells. In T84 cells, we find digitonin-resistant CLIC1 is present in an intracellular compartment which is concentrated immediately below the apical plasma membrane and the extent of apical polarization is enhanced with forskolin, which activates transepithelial chloride transport and apical membrane traffic in these cells. The sub-apical CLIC1 compartment was further characterized in a well-differentiated mouse renal proximal tubule cell line. The distribution of CLIC1 was found to overlap that of megalin and the sodium-phosphate cotransporter, NaPi-II, which are markers of the apical endocytic/recycling compartment in proximal tubule. CONCLUSION: The cell and tissue specific patterns of CLIC1 expression suggest it may play distinct roles in different cell types. In certain polarized columnar epithelia, it may play a role in apical membrane recycling.