WDR76 mediates obesity and hepatic steatosis via HRas destabilization.
ABSTRACT: Ras/MAPK (mitogen active protein kinase) signaling plays contradictory roles in adipocyte differentiation and is tightly regulated during adipogenesis. However, mechanisms regulating adipocyte differentiation involving Ras protein stability regulation are unknown. Here, we show that WD40 repeat protein 76 (WDR76), a novel Ras regulating E3 linker protein, controls 3T3-L1 adipocyte differentiation through HRas stability regulation. The roles of WDR76 in obesity and metabolic regulation were characterized using a high-fat diet (HFD)-induced obesity model using Wdr76-/- mice and liver-specific Wdr76 transgenic mice (Wdr76Li-TG). Wdr76-/- mice are resistant to HFD-induced obesity, insulin resistance and hyperlipidemia with an increment of HRas levels. In contrast, Wdr76Li-TG mice showed increased HFD-induced obesity, insulin resistance with reduced HRas levels. Our findings suggest that WDR76 controls HFD-induced obesity and hepatic steatosis via HRas destabilization. These data provide insights into the links between WDR76, HRas, and obesity.
Project description:Stability regulation of RAS that can affect its activity, in addition to the oncogenic mutations, occurs in human cancer. However, the mechanisms for stability regulation of RAS involved in their activity and its roles in tumorigenesis are poorly explored. Here, we identify WD40-repeat protein 76 (WDR76) as one of the HRAS binding proteins using proteomic analyses of hepatocellular carcinomas (HCC) tissue. WDR76 plays a role as an E3 linker protein and mediates the polyubiquitination-dependent degradation of RAS. WDR76-mediated RAS destabilization results in the inhibition of proliferation, transformation, and invasion of liver cancer cells. WDR76-/- mice are more susceptible to diethylnitrosamine-induced liver carcinogenesis. Liver-specific WDR76 induction destabilizes Ras and markedly reduces tumorigenesis in HRasG12V mouse livers. The clinical relevance of RAS regulation by WDR76 is indicated by the inverse correlation of their expressions in HCC tissues. Our study demonstrates that WDR76 functions as a tumor suppressor via RAS degradation.
Project description:BACKGROUND:Stabilization of RAS is a key event for the hyper-activation of Wnt/?-catenin signaling and activation of cancer stem cell (CSC) in colorectal cancer (CRC). WD Repeat protein 76 (WDR76) mediates the polyubiquitination-dependent degradation of RAS in hepatocellular carcinoma (HCC). We investigated whether WDR76 destabilizes RAS and acts as a tumor suppressor inhibiting CSC activation in CRC. METHODS:We generated mice with deletion of Wdr76 (Wdr76-/-) and crosses of Wdr76-/- with ApcMin/+ (Wdr76-/-; ApcMin/+) and compared them with wildtype mice (Wdr76+/+) and ApcMin/+ mice (Wdr76+/+; ApcMin/+), respectively. Intestinal crypt lengthening, tumorigenesis and CSC activation were analyzed by histology, immunohistochemistry, and immunoblotting. CRC cell line was engineered to stably express or knockdown WDR76 or control vector and was analyzed after spheroid culture. RESULTS:Wdr76-/- mice, with increased Ras level, displayed crypt elongation and hyper-proliferation. Wdr76-/-; ApcMin/+ mice developed more tumors with bigger sizes than ApcMin/+ mice and their tumors showed increased proliferation and CSC activation with elevated RAS and ?-catenin levels. In CRC cells, overexpression or knockdown of WDR76 decreased or increased the numbers and sizes of CRC spheroids with inhibition or activation of CSC markers, respectively. In human CRC, lower level of WDR76 was associated with poor patient survival. CONCLUSIONS:In analyses of mice with deletion of Wdr76 and CRC spheroids, we found that RAS stability plays important roles in tumorigenesis by affecting proliferation and CSC activation. Our results suggest that destabilization of RAS by WDR76 is a potential strategy for targeting malignant CRC involving CSC activation.
Project description:Numerous studies have suggested that beige adipocyte abundance is correlated with improved metabolic performance, but direct evidence showing that beige adipocyte expansion protects animals from the development of obesity is missing. Previously, we have described that the liver kinase b1 (LKB1) regulates beige adipocyte renaissance in subcutaneous inguinal white adipose tissue (iWAT) through a class IIa histone deacetylase 4 (HDAC4)-dependent mechanism. This study investigates the physiological impact of persistent beige adipocyte renaissance in energy homeostasis in mice. Here we present that the transgenic mice H4-TG, overexpressing constitutively active HDAC4 in adipocytes, showed beige adipocyte expansion in iWAT at room temperature. H4-TG mice exhibited increased energy expenditure due to beige adipocyte expansion. They also exhibited reduced adiposity under both normal chow and high-fat diet (HFD) feeding conditions. Specific ablation of beige adipocytes reversed the protection against HFD-induced obesity in H4-TG mice. Taken together, our results directly demonstrate that beige adipocyte expansion regulates adiposity in mice and targeting beige adipocyte renaissance may present a novel strategy to tackle obesity in humans.
Project description:C-type natriuretic peptide (CNP) is expressed in diverse tissues, including adipose and endothelium, and exerts its effects by binding to and activating its receptor, guanylyl cyclase B. Natriuretic peptides regulate intracellular cGMP and phosphorylated vasodilator-stimulated phosphoprotein (VASP). We recently revealed that overexpression of CNP in endothelial cells protects against high-fat diet (HFD)-induced obesity in mice. Given that endothelial CNP affects adipose tissue during obesity, CNP in adipocytes might directly regulate adipocyte function during obesity. Therefore, to elucidate the effect of CNP in adipocytes, we assessed 3T3-L1 adipocytes and transgenic (Tg) mice that overexpressed CNP specifically in adipocytes (A-CNP). We found that CNP activates the cGMP-VASP pathway in 3T3-L1 adipocytes. Compared with Wt mice, A-CNP Tg mice showed decreases in fat weight and adipocyte hypertrophy and increases in fatty acid ?-oxidation, lipolysis-related gene expression, and energy expenditure during HFD-induced obesity. These effects led to decreased levels of the macrophage marker F4/80 in the mesenteric fat pad and reduced inflammation. Furthermore, A-CNP Tg mice showed improved glucose tolerance and insulin sensitivity, which were associated with enhanced insulin-stimulated Akt phosphorylation. Our results suggest that CNP overexpression in adipocytes protects against adipocyte hypertrophy, excess lipid metabolism, inflammation, and decreased insulin sensitivity during HFD-induced obesity.
Project description:Adipose triglyceride lipase (ATGL) initiates intracellular triglyceride (TG) catabolism. In humans, ATGL deficiency causes neutral lipid storage disease with myopathy (NLSDM) characterized by a systemic TG accumulation. Mice with a genetic deletion of ATGL (AKO) also accumulate TG in many tissues. However, neither NLSDM patients nor AKO mice are exceedingly obese. This phenotype is unexpected considering the importance of the enzyme for TG catabolism in white adipose tissue (WAT). In this study, we identified the counteracting mechanisms that prevent excessive obesity in the absence of ATGL. We used "healthy" AKO mice expressing ATGL exclusively in cardiomyocytes (AKO/cTg) to circumvent the cardiomyopathy and premature lethality observed in AKO mice. AKO/cTg mice were protected from high-fat diet (HFD)-induced obesity despite complete ATGL deficiency in WAT and normal adipocyte differentiation. AKO/cTg mice were highly insulin sensitive under hyperinsulinemic-euglycemic clamp conditions, eliminating insulin insensitivity as a possible protective mechanism. Instead, reduced food intake and altered signaling by peroxisome proliferator-activated receptor-gamma (PPAR-?) and sterol regulatory element binding protein-1c in WAT accounted for the phenotype. These adaptations led to reduced lipid synthesis and storage in WAT of HFD-fed AKO/cTg mice. Treatment with the PPAR-? agonist rosiglitazone reversed the phenotype. These results argue for the existence of an adaptive interdependence between lipolysis and lipid synthesis. Pharmacological inhibition of ATGL may prove useful to prevent HFD-induced obesity and insulin resistance.
Project description:A vast body of literature has established GRK2 as a key player in the development and progression of heart failure. Inhibition of GRK2 improves cardiac function post injury in numerous animal models. In recent years, discovery of several non-canonical GRK2 targets has expanded our view of this kinase. Here, we describe the novel and exciting finding that cardiac GRK2 activity can regulate whole body metabolism. Transgenic mice with cardiac-specific expression of a peptide inhibitor of GRK2 (Tg?ARKct) display an enhanced obesogenic phenotype when fed a high fat diet (HFD). In contrast, mice with cardiac-specific overexpression of GRK2 (TgGRK2) show resistance to HFD induced obesity. White adipose tissue (WAT) mass was significantly enhanced in HFD fed Tg?ARKct mice. Furthermore, regulators of adipose differentiation were differentially regulated in WAT from mice with gain or loss of GRK2 function. Using complex metabolomics we found that cardiac GRK2 signaling altered myocardial BCAA and endocannabinoid metabolism and modulated circulating BCAA and endocannabinoid metabolite profiles on a HFD, and one of the BCAA metabolites identified here enhances adipocyte differentiation in vitro. Taken together, these results suggest that metabolic changes in the heart due to GRK2 signaling on a HFD control whole body metabolism.
Project description:Berries of Aronia melanocarpa (chokeberry) are known to be a rich source of biologically active polyphenols. In the present study, the effects of seven anti-adipogenic polyphenolic phytochemicals isolated from A. melanocarpa methanol extract on adipogenic transcription factors were investigated. Amygdalin and prunasin were found to inhibit 3T3-L1 adipocyte differentiation by suppressing the expressions of PPAR? (peroxisome proliferator-activated receptor ?), C/EBP? (CCAAT/enhancer binding protein ?), SREBP1c (sterol regulatory element binding protein 1c), FAS (fatty acid synthase), and aP2 (adipocyte fatty-acid?binding protein). A. melanocarpa extract-treated (100 or 200 mg/kg/day on body weight) high fat diet (HFD)-induced obese mice showed significant decreases in body weight, serum triglyceride (TG), and low-density lipoprotein cholesterol (LDLC) levels and improved insulin sensitivity as compared with HFD controls. This research shows A. melanocarpa extract is potentially beneficial for the suppression of HFD-induced obesity.
Project description:Development of brown and beige/brite adipocytes increases thermogenesis and helps to reduce obesity and metabolic syndrome. Our previous study suggests that dietary raspberry can ameliorate metabolic syndromes in diet-induced obese mice. Here, we further evaluated the effects of raspberry on energy expenditure and adaptive thermogenesis and determined whether these effects were mediated by AMP-activated protein kinase (AMPK). Mice deficient in the catalytic subunit of AMPK?1 and wild-type (WT) mice were fed a high-fat diet (HFD) or HFD supplemented with 5% raspberry (RAS) for 10 weeks. The thermogenic program and related regulatory factors in adipose tissue were assessed. RAS improved the insulin sensitivity and reduced fat mass in WT mice but not in AMPK?1-/- mice. In the absence of AMPK?1, RAS failed to increase oxygen consumption and heat production. Consistent with this, the thermogenic gene expression in brown adipose tissue and brown-like adipocyte formation in subcutaneous adipose tissue were not induced by RAS in AMPK?1-/- mice. In conclusion, AMPK?1 is indispensable for the effects of RAS on brown and beige/brite adipocyte development, and prevention of obesity and metabolic dysfunction.
Project description:Mitogen-activated protein kinase phosphatase 3 (MKP-3) is a negative regulator of extracellular signal-related kinase signaling. Our laboratory recently demonstrated that MKP-3 plays an important role in obesity-related hyperglycemia by promoting hepatic glucose output. This study shows that MKP-3 deficiency attenuates body weight gain induced by a high-fat diet (HFD) and protects mice from developing obesity-related hepatosteatosis. Triglyceride (TG) contents are dramatically decreased in the liver of MKP-3(-/-) mice fed an HFD compared with wild-type (WT) controls. The absence of MKP-3 also reduces adiposity, possibly by repressing adipocyte differentiation. In addition, MKP-3(-/-) mice display increased energy expenditure, enhanced peripheral glucose disposal, and improved systemic insulin sensitivity. We performed global phosphoproteomic studies to search for downstream mediators of MKP-3 action in liver lipid metabolism. Our results revealed that MKP-3 deficiency increases the phosphorylation of histone deacetylase (HDAC) 1 on serine 393 by 3.3-fold and HDAC2 on serine 394 by 2.33-fold. Activities of HDAC1 and 2 are increased in the livers of MKP-3(-/-) mice fed an HFD. Reduction of HDAC1/2 activities is sufficient to restore TG content of MKP-3(-/-) primary hepatocytes to a level similar to that in WT cells.
Project description:Obesity is the major risk factor for type 2 diabetes, cardiovascular disorders, and many other diseases. Adipose tissue inflammation is frequently associated with obesity and contributes to the morbidity and mortality. Dedicator of cytokinesis 2 (DOCK2) is involved in several inflammatory diseases, but its role in obesity remains unknown. To explore the function of DOCK2 in obesity and insulin resistance, WT and DOCK2-deficient (DOCK2-/-) mice were given chow or high-fat diet (HFD) for 12 weeks followed by metabolic, biochemical, and histologic analyses. DOCK2 was robustly induced in adipose tissues of WT mice given HFD. DOCK2-/- mice with HFD showed decreased body weight gain and improved metabolic homeostasis and insulin resistance compared with WT mice. DOCK2 deficiency also attenuated adipose tissue and systemic inflammation accompanied by reduced macrophage infiltration. Moreover, DOCK2-/- mice exhibited increased expression of metabolic genes in adipose tissues with greater energy expenditure. Mechanistically, DOCK2 appeared to regulate brown adipocyte differentiation because increased preadipocyte differentiation to brown adipocytes in interscapular and inguinal fat was observed in DOCK2-/- mice, as compared with WT. These data indicated that DOCK2 deficiency protects mice from HFD-induced obesity, at least in part, by stimulating brown adipocyte differentiation. Therefore, targeting DOCK2 may be a potential therapeutic strategy for treating obesity-associated diseases.