Yi Jen Liao

Glycine N-methyltransferase deficiency affects Niemann-Pick type C2 protein stability and regulates hepatic cholesterol homeostasis.

Nonalcoholic fatty liver disease (NAFLD) is associated with the development of metabolic syndromes and hepatocellular carcinoma (HCC). Cholesterol accumulation is related to NAFLD, whereas its detailed mechanism is not fully understood. Previously, we reported that glycine N-methyltransferase (GNMT) knockout (Gnmt-/-) mice develop chronic hepatitis and HCC. In this study, we showed that Gnmt-/- mice had hyperlipidemia and steatohepatitis. Single photon emission computed tomography images of mice injected with 131I-labeled 6β-iodocholesterol demonstrated that Gnmt-/- mice had slower hepatic cholesterol uptake and excretion rates than wild-type mice. In addition, genes related to cholesterol uptake (scavenger receptor class B type 1 [SR-B1] and ATP-binding cassette A1 (ABCA1)), intracellular trafficking (Niemann-Pick type C1 protein [NPC1] and Niemann-Pick type C2 protein [NPC2]) and excretion (ATP-binding cassette G1 [ABCG1]) were downregulated in Gnmt-/- mice. Yeast two-hybrid screenings and coimmunoprecipitation assays elucidated that the C conserved region (81–105 amino acids) of NPC2 interacts with the carboxyl-terminal fragment (171–295 amino acids) of GNMT. Confocal microscopy demonstrated that when cells were treated with low-density lipoprotein, NPC2 was released from lysosomes and interacts with GNMT in the cytosol. Overexpression of GNMT doubled the half-lives of both NPC2 isoforms and reduced cholesterol accumulation in cells. Furthermore, GNMT was downregulated in the liver tissues from patients suffering with NAFLD as well as from mice fed a high-fat diet, high-cholesterol diet or methionine/choline-deficient diet. In conclusion, our study demonstrated that GNMT regulates the homeostasis of cholesterol metabolism, and hepatic cholesterol accumulation may result from downregulation of GNMT and instability of its interactive protein NPC2. Novel therapeutics for steatohepatitis and HCC may be developed by using this concept

Deficiency of Glycine N-methyltransferase aggravates atherosclerosis in apolipoprotein E-null mice

The mechanism underlying the dysregulation of cholesterol metabolism and inflammation in atherogenesis is not understood fully. Glycine N-methyltransferase (GNMT) has been implicated in hepatic lipid metabolism and the pathogenesis of liver diseases. However, little is known about the significance of GNMT in atherosclerosis. We showed the predominant expression of GNMT in foamy macrophages of mouse atherosclerotic aortas. Genetic deletion of GNMT exacerbated the hyperlipidemia, inflammation and development of atherosclerosis in apolipoprotein E-deficient mice. In addition, ablation of GNMT in macrophages aggravated oxidized low-density lipoprotein-mediated cholesterol accumulation in macrophage foam cells by downregulating the expression of reverse cholesterol transporters including ATP-binding cassette transporters-A1 and G1 and scavenger receptor BI. Furthermore, tumor necrosis factor-α-induced inflammatory response was promoted in GNMT-null macrophages. Collectively, our data suggest that GNMT is a crucial regulator in cholesterol metabolism and in inflammation, and contributes to the pathogenesis of atherosclerosis. This finding may reveal a potential therapeutic target for atherosclerosis.

Higher susceptibility to aflatoxin B1‐related hepatocellular carcinoma in glycine N‐methyltransferase knockout mice

In both humans and rodents, males are known to be more susceptible than females to hepatocarcinogenesis. We have previously reported that glycine N‐methyltransferase (GNMT) interacts with aflatoxin B1 (AFB1) and reduces both AFB1‐DNA adduct formation and hepatocellular carcinoma (HCC) in mice. We also reported that 50% of the males and 100% of the females in a small group of Gnmt null (Gnmt−/−) mice developed HCC, with first dysplastic hepatocellular nodules detected at mean ages of 17 and 16.5 months, respectively. In our study, we tested our hypothesis that male and female Gnmt−/− mice are susceptible to AFB1 carcinogenesis, and that the absence of Gnmt expression may accelerate AFB1‐induced liver tumorigenesis. We inoculated Gnmt−/− and wild‐type mice intraperitoneally with AFB1 at 7 days and 9 weeks of age and periodically examined them using ultrasound. Dysplastic hepatocellular nodules were detected in six of eight males and five of five females at 12.7 and 12 months of ages, respectively. Dysplastic hepatocellular nodules from 5/8 (62.5%) male and 4/5 (80%) female Gnmt−/− mice were diagnosed as having HCC, ∼6 months earlier than AFB1‐treated wild‐type mice. Results from microarray and real‐time PCR analyses indicate that five detoxification pathway‐related genes were downregulated in AFB1‐treated Gnmt−/− mice: Cyp1a2, Cyp3a44, Cyp2d22, Gsta4 and Abca8a. In summary, we observed overall higher susceptibility to AFB1‐related HCC in Gnmt−/− mice, further evidence that GNMT overexpression is an important contributing factor to liver cancer resistance.

Characterization of Niemann-Pick Type C2 Protein Expression in Multiple Cancers Using a Novel NPC2 Monoclonal Antibody

Niemann-Pick Type C2 (NPC2) plays an important role in the regulation of intracellular cholesterol homeostasis via direct binding with free cholesterol. However, little is known about the significance of NPC2 in cancer. In this study, we have pinpointed the impact of various different cancers on NPC2 expression. A series of anti-NPC2 monoclonal antibodies (mAbs) with the IgG2a isotype were generated and peptide screening demonstrated that the reactive epitope were amino acid residues 31-40 of the human NPC2 protein. The specificity of these mAbs was confirmed by Western blotting using shRNA mediated knock-down of NPC2 in human SK-Hep1 cells. By immunohistochemical staining, NPC2 is expressed in normal kidney, liver, breast, colon, lung, esophageal, uterine cervical, pancreatic and stomach tissue. Strong expression of NPC2 was found in the distal and proximal convoluted tubule of kidney and the hepatocytes of liver. Normal esophageal, uterine cervical, pancreatic, stomach, breast, colon and lung tissue stained moderately to weakly. When compared to their normal tissue equivalents, NPC2 overexpression was observed in cancers of the breast, colon and lung. Regarding to breast cancer, NPC2 up-regulation is associated with estrogen receptor (-), progesterone receptor (-) and human epidermal growth factor receptor (+). On the other hand, NPC2 was found to be down-regulated in renal cell carcinoma, liver cirrhosis and hepatoma tissues. By antigen-capture enzyme immunoassay ELISA, the serum NPC2 is increased in patients with cirrhosis and liver cancer. According to western blot data, the change of glycosylated pattern of NPC2 in serum is associated with cirrhosis and liver cancer. To the best of our knowledge, this is the first comprehensive immunohistochemical and serological study investigating the expression of NPC2 in a variety of different human cancers. These novel monoclonal antibodies should help with elucidating the roles of NPC2 in tumor development, especially in liver and breast cancers.

Deficiency of glycine N-methyltransferase results in deterioration of cellular defense to stress in mouse liver

Purpose: Previously, we reported that glycine N‐methyltransferase (GNMT) interacts with benzo[a]pyrene (BaP) and inhibits BaP‐DNA adducts formation. In addition, Gnmt knockout (Gnmt−/−) mice developed chronic hepatitis and hepatocellular carcinoma (HCC). The aims of this study were to understand the gene expression profile of Gnmt−/− mice and to study the interaction between BaP and GNMT deficiency in vivo.

Role of phosphatase activity of soluble epoxide hydrolase in regulating simvastatin-activated endothelial nitric oxide synthase

Soluble epoxide hydrolase (sEH) has C-terminal epoxide hydrolase and N-terminal lipid phosphatase activity. Its hydrolase activity is associated with endothelial nitric oxide synthase (eNOS) dysfunction. However, little is known about the role of sEH phosphatase in regulating eNOS activity. Simvastatin, a clinical lipid-lowering drug, also has a pleiotropic effect on eNOS activation. However, whether sEH phosphatase is involved in simvastatin-activated eNOS activity remains elusive. We investigated the role of sEH phosphatase activity in simvastatin-mediated activation of eNOS in endothelial cells (ECs). Simvastain increased the phosphatase activity of sEH, which was diminished by pharmacological inhibitors of sEH phosphatase. In addition, pharmacological inhibition of sEH phosphatase or overexpressing the inactive phosphatase domain of sEH enhanced simvastatin-induced NO bioavailability, tube formation and phosphorylation of eNOS, Akt, and AMP-activated protein kinase (AMPK). In contrast, overexpressing the phosphatase domain of sEH limited the simvastatin-increased NO biosynthesis and eNOS phosphorylation at Ser1179. Simvastatin evoked epidermal growth factor receptor–c-Src–increased Tyr phosphorylation of sEH and formation of an sEH–Akt–AMPK–eNOS complex, which was abolished by the c-Src kinase inhibitor PP1 or c-Src dominant-negative mutant K298M. These findings suggest that sEH phosphatase activity negatively regulates simvastatin-activated eNOS by impeding the Akt–AMPK–eNOS signaling cascade.

Niemann-Pick type C2 protein regulates liver cancer progression via modulating ERK1/2 pathway: Clinicopathological correlations and therapeutical implications.

Primary hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide and the third leading cause of cancer‐related death. It is important to identify new targets for early diagnosis and treatment of HCC. Niemann‐Pick type C2 (NPC2) plays an important role in the regulation of intracellular cholesterol homeostasis via direct binding with free cholesterol. However, little is known about the significance of NPC2 in HCC tumorigenesis. In this study, we showed that NPC2 is abundantly expressed in normal liver, but is downregulated in human HCC tissues. The patients with NPC2 downregulation expressed much higher α‐fetoprotein, multiple tumor type, vascular invasion, later pathological stage and shorter survival rate. Knockdown NPC2 in liver cancer cell lines promote cell proliferation, migration and xenograft tumorigenesis. In contrast, NPC2 overexpression inhibits HuH7 promoted tumor growth. Furthermore, administration of hepatotropic adeno‐associated virus 8 (AAV8) delivered NPC2 decreased the inflammatory infiltration, the expression of two early HCC markers—glypican 3 and survivin and suppressed the spontaneous HCC development in mice. To identify the NPC2‐dependent mechanism, we emphasized on the status of MAPK/ERK signaling. MEK1/2 inhibitor treatment demonstrated that the expression of NPC2 affected the activation of ERK1/2 but not MEK1/2. In addition, cholesterol trafficking inhibitor treatment did not alter the cell proliferation and the activation of MEK/ERK. In conclusion, our study demonstrates that NPC2 may play an important role in negatively regulate cell proliferation and ERK1/2 activation that were independent of cholesterol accumulation. AAV‐NPC2 may thus represent a new treatment strategy for liver cancer.

A Study on the Dose Distributions in Various Materials from an Ir-192 HDR Brachytherapy Source

Dose distributions of 192Ir HDR brachytherapy in phantoms simulating water, bone, lung tissue, water-lung and bone-lung interfaces using the Monte Carlo codes EGS4, FLUKA and MCNP4C are reported. Experiments were designed to gather point dose measurements to verify the Monte Carlo results using Gafchromic film, radiophotoluminescent glass dosimeter, solid water, bone, and lung phantom. The results for radial dose functions and anisotropy functions in solid water phantom were consistent with previously reported data (Williamson and Li). The radial dose functions in bone were affected more by depth than those in water. Dose differences between homogeneous solid water phantoms and solid water-lung interfaces ranged from 0.6% to 14.4%. The range between homogeneous bone phantoms and bone-lung interfaces was 4.1% to 15.7%. These results support the understanding in dose distribution differences in water, bone, lung, and their interfaces. Our conclusion is that clinical parameters did not provide dose calculation accuracy for different materials, thus suggesting that dose calculation of HDR treatment planning systems should take into account material density to improve overall treatment quality.

Glycine N-methyltransferase deficiency in female mice impairs insulin signaling and promotes gluconeogenesis by modulating the PI3K/Akt pathway in the liver

BackgroundGlycine N-methyltransferase (GNMT) is abundantly expressed in the normal liver but is down-regulated in liver cancer tissues. GNMT knockout (Gnmt−/−) mice can spontaneously develop chronic hepatitis, fatty liver, and liver cancer. We previously demonstrated that hepatic GNMT is decreased in high-fat-diet-induced type 2 diabetes mellitus, but its contribution to metabolic syndrome is unclear. Here we show that GNMT modulates key aspects of metabolic syndrome in mice.MethodsEleven-week-old Gnmt−/− and wild-type (WT) mice with a C57BL/6 genetic background were used in this study. The metabolic defects of GNMT deficiency were measured by glucose and insulin tolerance tests, lipid homeostasis, gluconeogenesis, and insulin signaling.ResultsGnmt−/− mice, especially females, exhibited glucose intolerance and insulin resistance. However, their body fat and lean mass, food and water intakes, and energy expenditure did not differ from those of WT mice. In addition, glucose-stimulated insulin secretion and insulin-stimulated glucagon secretion were normal in the serum and pancreatic islets of Gnmt−/− mice. Importantly, we found that GNMT deficiency increased lipogenesis and triglycerides in the liver. The elevated triglycerides disrupted the ability of insulin to induce Akt and S6 ribosomal protein phosphorylation, and then triggered insulin resistance and gluconeogenesis in female Gnmt−/− mice.ConclusionsOur data indicate that hepatic GNMT regulates lipid and glucose homeostasis, and provide insight into the development of insulin resistance through modulating the PI3K/Akt pathway.

Niemann-Pick Type C2 Protein Mediates Hepatic Stellate Cells Activation by Regulating Free Cholesterol Accumulation

In chronic liver diseases, regardless of their etiology, the development of fibrosis is the first step toward the progression to cirrhosis, portal hypertension, and hepatocellular carcinoma. Hepatic stellate cells (HSCs) are the main profibrogenic cells that promote the pathogenesis of liver fibrosis, and so it is important to identify the molecules that regulate HSCs activation and liver fibrosis. Niemann-Pick type C2 (NPC2) protein plays an important role in the regulation of intracellular cholesterol homeostasis by directly binding with free cholesterol. However, the roles of NPC2 in HSCs activation and liver fibrosis have not been explored in detail. Since a high-cholesterol diet exacerbates liver fibrosis progression in both rodents and humans, we propose that the expression of NPC2 affects free cholesterol metabolism and regulates HSCs activation. In this study, we found that NPC2 is decreased in both thioacetamide- and carbon tetrachloride-induced liver fibrosis tissues. In addition, NPC2 is expressed in quiescent HSCs, but its activation status is down-regulated. Knockdown of NPC2 in HSC-T6 cells resulted in marked increases in transforming growth factor-β1 (TGF-β1)-induced collagen type 1 α1 (Col1a1), α-smooth muscle actin (α-SMA) expression, and Smad2 phosphorylation. In contrast, NPC2 overexpression decreased TGF-β1-induced HSCs activation. We further demonstrated that NPC2 deficiency significantly increased the accumulation of free cholesterol in HSCs, increasing Col1a1 and α-SMA expression and activating Smad2, and leading to sensitization of HSCs to TGF-β1 activation. In contrast, overexpression of NPC2 decreased U18666A-induced free cholesterol accumulation and inhibited the subsequent HSCs activation. In conclusion, our study has demonstrated that NPC2 plays an important role in HSCs activation by regulating the accumulation of free cholesterol. NPC2 overexpression may thus represent a new treatment strategy for liver fibrosis.