The present invention relates to a method of using Glycine N-methyltransferase (GNMT) to treat or prevent fatty liver related diseases including nonalcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC). In particular, the use of GNMT for treating or preventing fatty liver diseases is achieved by enhancing GNMT-NPC2 interaction, and thus decreasing or preventing the accumulation of lipid and cholesterol in hepatic cells or tissues.
Glycine N-methyltransferase (GNMT) regulates the ratio of S-adenosylmethionine(SAM) to S-adenosylhomocysteine (SAH) by catalyzing sarcosine synthesis from glycine (Kerr S J. J Biol Chem 1972; 247: 4248-4252). In addition to folate, GNMT binds polyaromatic hydrocarbons (PAHs) and aflatoxins (Yeo E J, et al. Proc Natl Acad Sci U S A 1994; 91: 210-214). We previously described diminished GNMT expression levels in both human HCC cell lines and tumor tissues (Liu H H, et al. J Biomed Sci 2003; 10: 87-97). Results from genotypic analyses of several human GNMT gene polymorphisms revealed a loss of heterozygosity in 36-47% of tumor tissues collected from HCC patients.
Fatty liver, also known as fatty liver disease (FLD), is a reversible condition where large vacuoles of triglyceride fat accumulate in liver cells via the process of steatosis (i.e. abnormal retention of lipids within a cell). Despite having multiple causes, fatty liver can be considered a single disease that occurs worldwide in those with excessive alcohol intake and those who are obese (with or without effects of insulin resistance). The condition is also associated with other diseases that influence fat metabolism (Reddy J K, Rao M S, Am. J. Physiol. Gastrointest. Liver Physiol. 2006; 290 (5): G852-8). Morphologically, it is difficult to distinguish alcoholic FLD from nonalcoholic FLD, and both Show microvesicular and macrovesicular fatty changes at different stages.
Non-alcoholic fatty liver diseases (NAFLD) include illnesses ranging from hepatic steatosis to intermediate lesions, non-alcoholic steatohepatitis (NASH), and cirrhosis. Epidemiologists estimate that between 20% and 30% of all adults living in the United States and other developed countries have some form of NAFLD. NAFLD is considered the hepatic event in the metabolic syndrome and is associated with hepatocellular carcinoma (HCC) development. The liver plays a critical role in whole-body lipid metabolism. Deterioration in lipid uptake, transport, excretion, synthesis, and catabolic mechanisms serves as the basis for NAFLD development. It is important to note that cholesterol content is increased in human fatty liver tissues, suggesting that cholesterol metabolism is deregulated in NAFLD. However, the reason for the cholesterol accumulation is still unclear.
Niemann-Pick Type C2 (NPC2) protein is a small soluble glycoprotein and mainly expresses in liver, kidney and testis. NPC2 plays an important role in regulating intracellular cholesterol trafficking and homeostasis through direct binding with free cholesterol. Deficiency of NPC2 in mice results in cholesterol accumulation in the liver tissues. Glycine N-methyltransferase (GNMT) is abundantly expressed in the liver cytosol, but is down-regulated in human HCC tissues. Previously, we generated a GNMT knockout (Gnmt−/−) mice and reported on their tendencies toward chronic hepatitis, glycogen storage (U.S. Pat. No. 7,759,542).
Based on the demonstration 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 fatty liver related diseases, such as Non-alcoholic fatty liver diseases, steatohepatitis, and HCC may be developed by using this concept.
This invention is based on the unexpected discovery that Gnmt−/− mice impaired cholesterol metabolism and developed steatohepatitis. In this invention, we used full-length human GNMT as bait in a yeast two-hybrid screen system and identified NPC2 as a GNMT-interactive protein. Accordingly, this study aimed to investigate the role of GNMT-NPC2 interaction in the regulation of hepatic cholesterol homeostasis.
Therefore, in one aspect of the present invention, it is provided a pharmaceutical composition for in treating or preventing fatty liver related disease, comprising Glycine N-methyltransferase (GNMT) protein as active ingredient, and a pharmaceutically acceptable carrier, diluent or excipient.
In one embodiment of this invention, the GNMT protein is used to enhance the activity and stability of NPC2 protein in liver cells. In certain embodiments, the pharmaceutical composition of this invention is used to improve GNMT-NPC2 interaction, and further to decrease or prevent cholesterol accumulation in liver cells.
The term “prophylactic” or “therapeutic” treatment is art-recognized and refers to administration of a drug to a host. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) then the treatment is prophylactic, i.e., it protects the host against developing the unwanted condition, whereas if administered after manifestation of the unwanted condition, the treatment is therapeutic (i.e., it is intended to diminish, ameliorate or maintain the existing unwanted condition or side effects therefrom).
GNMT protein can be formulated for a variety of modes of administration, including systemic and topical or localized administration. Techniques and formulations generally may be found in Remington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa. For parenteral administration, injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, the compounds can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution. In addition, the compounds may be formulated in solid form and re-dissolved or suspended immediately prior to use. Lyophilized forms are also included.
For oral administration, the compositions can be formulated by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compositions of the invention to be formulated as tablets, pills, lozenges, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient. Pharmaceutical preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding other suitable auxiliaries if desired, to obtain tablets or dragee cores. Useful excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol, cellulose preparations such as, for example, maize starch, wheat starch, rice starch and potato starch and other materials such as gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinyl-pyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid. A salt such as a sodium alginate may also be used.
In another aspect, this invention features a method for diagnosing the occurrence of fatty liver disease in a tested animal, comprising collecting a blood or liver sample from the animal; and detecting the content or expression level of GNMT protein by using a detecting agent.
In certain embodiments, the detecting agent for GNMT protein comprises an anti-GNMT antibody. The term “antibody” herein is used in the broadest sense and specifically includes full-length monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired biological activity.
Other features or advantages of the present invention will be apparent from the following drawings and detailed description of several embodiments, and also from the appending claims.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office a upon request and payment of the necessary fee.
The term “agent” is used herein to denote a chemical compound, a mixture of chemical compounds, a biological macromolecule (such as a nucleic acid, an antibody, a protein or portion thereof, e.g., a peptide), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. The activity of such agents may render it suitable as a “therapeutic agent” which is a biologically, physiologically, or pharmacologically active substance (or substances) that acts locally or systemically in a subject.
In certain embodiments, the method of using Glycine N-methyltransferase (GNMT) to treat or prevent fatty liver related diseases may comprises administrating GNMT protein to an animal having need of the treatment. The animal may include human and other mammals.
The specific examples below are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. All publications cited herein are hereby incorporated by reference in their entirety. Further, any mechanism proposed below does not in any way restrict the scope of the claimed invention.
Firstly, we used a GNMT knock-out mouse which showed abnormal liver function and suffered from glycogen storage disease (Gnmt−/− mouse model, disclosed in U.S. Pat. No. 7,759,542) to investigate the role of GNMT in lipid metabolism, specifically comparing hepatic steatosis and serum lipid levels between WT and Gnmt−/− mice.
As shown in
To delineate the molecular mechanisms responsible for hepatic cholesterol accumulation in Gnmt−/− mice, we analyzed protein and mRNA expression levels for the following genes during different stages of cholesterol metabolism: (a) hepatic cholesterol uptake: ABCA1 and SR-B1; (b) cholesterol synthesis: HMGCR and SREBP2; (c) cholesterol trafficking: NPC1, NPC2 and StAR; and (d) hepatic cholesterol efflux: ABCG1. As shown in
To further evaluate and compare cholesterol uptake and excretion between WT and Gnmt mice, we injected 131I-labeled 6β-iodocholesterol into mouse penis and normalized liver single photon emission computed tomography (SPECT) images to heart SPECT images at different time points (
Identification of NPC2 as a GNMT interacting protein
To identify proteins that interact with GNMT, we used full-length human GNMT as bait in a yeast two-hybrid screen system for a human liver cDNA library. After three rounds of screening, we selected a cDNA clone containing a nucleotide sequence encoding amino acid residues 43-151 of the NPC2 protein for further study. Specific GNMT-NPC2 interaction and colocalization were analyzed by coimmunoprecipitation, confocal microscopic examination and a combination of lysosomal/cytosol fractionation and Western blot analysis.
293T cells were cotransfected with GNMT-Flag and NPC2-HA for 24 h and harvested for commmunoprecipitation. GNMT was precipitated by anti-Flag beads and immunoblotted with anti-HA to detect NPC2 expression (arrowhead). NPC2 was precipitated by anti-HA beads and immunoblotted with anti-Flag to detect GNMT expression (arrowhead). As shown in
To prove GNMT colocalization and interaction with NPC2 in the cytosol, we isolated various lysosomal and cytosolic fractions from WT mouse liver for Western blot and communoprecipitation analyses. As a result, approximately 80% of the NPC2 was expressed in lysosomes (indicated by the lysosomal markers LAMP 1 and cathepsin D), and 20% was expressed in the cytosol. In contrast, GNMT was expressed in the cytosol (
Liver cytosol fractions from WT mice were precipitated with anti-GNMT antibodies and immunoblotted with anti-NPC2 antibodies to detect endogenous NPC2 expression, and to confirm GNMT-NPC2 interaction within the cytosol (
To map GNMT-NPC2 interactive domains, we constructed nine plasmids containing different regions of either GNMT or NPC2. The results indicate that the amino-terminal half of GNMT was not capable of pulling down NPC2, but the carboxyl-terminal half (containing amino acid residues 171-295) of GNMT was capable. On the other hand, the C region of NPC2 (containing amino acid residues 81-105) was the primary domain for GNMT interaction.
GNMT Enhances NPC2 Protein Stability
Because the protein level of NPC2 decreased significantly in Gnmt−/− mice liver tissues while mRNA level remained unchanged (
GNMT-NPC2 Influences Intracellular Cholesterol Homeostasis
To monitor dynamic distribution and colocalization between GNMT and NPC2, we used immunofluorescent staining, confocal microscopy and Western blot to detect NPC2-GNMT colocalization in cells treated with LDL and progesterone. Progesterone inhibits cholesteryl ester synthesis and blocks cholesterol trafficking pathways. When CHO cells were transfected with NPC2-DsRed and treated with LDL and progesterone for 24 h, NPC2 was accumulated in lysosomes (
When CHO cells overexpressed GNMT-GFP and NPC2-DsRed, colocalization signals between GNMT and NPC2 became prominent in the cytosol between 6 and 18 h after the removal of progesterone (FIG. 5B-5-7). According to cytosol-lysosome isolation results, endogenous NPC2 was predominantly expressed in the lysosomal fractions of SK-Hep1 cells before LDL and progesterone treatment (
To evaluate whether GNMT coordinates with NPC2 in regulating intracellular homeostasis of cholesterol, SK-Hep1. cells were infected with Ad-GNMT for 24 h before treatment with LDL and progesterone. Compared to cells infected with a vector control, the cholesterol levels in GNMT-overexpressed SK-Hep1 cells decreased significantly 6-18 h after the removal of LDL and progesterone (
Expression Pattern Of NPC2 Is Dysregulated In Fatty Liver Tissues
In mice, dietary models (such as MCD, high-cholesterol and high-fat diets) and genetic models (such as ob/ob mice) both mimic NAFLD in humans. We therefore fed WT mice a high-cholesterol diet, high-fat diet or MCD diet to prove the downregulation of GNMT in fatty liver tissues. As expected, we found that GNMT was downregulated in mice fed a high-cholesterol diet, high-fat diet or MCD diet but not in ob/ob mice (
No appreciable differences in NgBR expression levels were observed in Gnmt−/− mice. In addition, co-immunoprecipitate GNMT did not observe the expression of NgBR, suggesting that the interaction between GNMT and NPC2 is independent of NgBR. Because NPC2 cholesterol binding occurs at both acidic and neutral pH levels, GNMT may physiologically interact with NPC2 and maintain NPC2 function in the cytosol before NPC2-NgBR interaction. In addition to supporting the idea of GNMT as an NPC2-interacting protein, these findings suggest avenues for enhancing NPC2 protein stability and preventing intracellular cholesterol accumulation.
According to the two-hit hypothesis, lipid accumulation and subsequent inflammation and oxidative stress may trigger liver damage and HCC. In example 1, it is showed that Gnmt−/− mice had signs of hepatic steatosis with increased cholesterol deposition in liver tissues, as well as inflammatory infiltration before HCC formation (
This invention firstly discloses the demonstration that GNMT plays an important role in regulating cholesterol homeostasis via interaction with NPC2. GNMT deficiency attenuates NPC2 protein stability and triggers cholesterol accumulation in the liver tissues. Because NASH is one of the key factors involved in cirrhosis and HCC development, data presented in the examples have both physiological and pathological significance. These novel findings may provide important implications regarding the development of diagnostic or therapeutic strategies for fatty liver patients, and possibly for HCC as well.
All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.
From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.
Number | Date | Country | Kind |
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101141570 | Nov 2012 | TW | national |