This application claims the benefit under 35 U.S.C. 119 of Great Britain application number GB 0012229.1, filed May 19, 2000.
The invention relates to the use of microscopic nematodes such as C. elegans in functional high throughput in vivo assays suitable for the detection of inhibitors or activators of intestinal lipid uptake. Compounds identified as modulators of intestinal lipid uptake using the assays of the invention may provide lead compounds for the development of pharmaceutical agents useful in the treatment of diseases of the human or animal (e.g. mammal) body, and in particular diseases and disorders of human and/or animal metabolism, fat handling and/or fat storage, such as obesity, impaired fat metabolism and other related diseases such as diabetes type II and cardiovascular diseases.
When fat reaches the intestines in vertebrates, the pancreatic lipase hydrolyses the triglycerides into smaller components designated free fatty acids and monoglycerides (mainly 2-monoacylglycerols). Fatty acids are long-chain hydrocarbon molecules containing a carboxylic moiety at one end. The numbering of carbons in fatty acids begins with the carbon of the carboxylate group.
Metabolically, fatty acids are important energy substrates because of their high calorific content. In a typical diet of Western developed countries, approximately 30-40% of the dietary calories are derived from lipids, mainly in the form of di- and triglycerides. The linkage between excessive dietary lipid consumption and several common pathophysiologic disorders, including heart disease, obesity and diabetes and cancer, has been widely documented (Watts, et al., Am J Clin Nutr 64, 202-9 (1996); Storlien et al., Science 237, 885-8 (1987)).
There are three major roles in the body for the free fatty acids:
1) as the components of more complex membrane lipids.
2) as the major components of stored fat in the form of triacylglycerols.
3) Metabolism of fatty acids by xcex2-oxidation is also the major source of ATP as energy for most organisms, especially for mammalian cardiac muscle.
Until recently it was considered that the adsorption of fatty acids into the body during digestion was through passive diffusion rather than the active transport process, as was known for carbohydrates and amino acids. Presently at least five plasma membrane proteins have been identified and proposed as candidates for fatty acid transporters thus far. These include, but are not restricted to:
Plasma Membrane Fatty Acid Binding Protein (FABPpm),
Fatty Acid Translocase (FAT)
Caveolin, a 22-kDa fatty acid binding protein
Renal 56-kb FABP
Fatty Acid Transport Protein (FATP)
An overview of these membrane proteins has been published by Yuen Hui and David A. Bernlohr, Bioscience 2, 222-231 (1997).
The expression of FATP is regulated by certain transcription factors, such as the xe2x80x9cPPARxe2x80x9d (peroxysome proliferator activated receptor)-transcription factors the xe2x80x9cRXRxe2x80x9d (Retinoid X receptor)-transcription factors, and similar factors. Therefore, activators of these receptors, respectively fibrates or antidiabetic thiazolidinedione and retinoic acid, can increase FATP expression. One of the six known human FATPs, FATP4, has recently been shown to possess the functional characteristics (presence and absence is correlated with increase or decrease in fatty acid uptake) and cellular location (highly expressed in the microvilli of intestinal enterocytes) that would be required in a major intestinal fatty acid transport protein (Stahl A, et al. Mol Cell Biol 4,299-308 (1999)). It is highly probable that the expression and activity of the other transporter proteins is regulated too.
Nucleotide sequences encoding for and protein sequences of these fatty acid transporter proteins, both the human proteins and their C. elegans homologues, can be found in publicly accessible sequence databases, such as GenBank (accessible at the National Center for Biotechnology Information website, http://www.ncbi.nlm.nih.gov/PubMed/) and the C. elegans database of the Sanger Centre, UK (accessible at the Sanger Centre website, http://wormbase.sanger.ac.uk/). Some examples of sequences and designation numbers are:
Although less well documented, proteins involved in the uptake of other lipids have been described in literature, and sequences of these proteins and genes have been published. For example, it has been shown that ABC transporters and more particularly the ABC transporter ABCB1 (ABC8) plays an important role in the regulation of Cholesterol and phospholipid transport (Klucken et al., Proc. Natl. Acad. Sci. USA, 2000, 97:817-822). Delivery of lipids, and more particularly sterols and cholesterol is done by the scavenger receptor-BI (Stangl et al., J. Biol. Chem., 1999, 274:32692-32698). Izzat et al. describe other putative targets to reduce the intestinal cholesterol uptake in the Journal of Pharmacology and Experimental Therapeutics 2000, 293:315-320. Such targets include bile acid transporters and HMG-CoA reductase. Cholesterol is taken up by the gut membrane without the involvement of a transporter. Compounds that interact stoichiometrically with the cholesterol in the intestinal lumen would also reduce cholesterol uptake. Other transporter proteins can be found in literature.
Controlling the uptake of lipids in the intestines would allow the treatment of obesity as well as the treatment of some related diseases such as diabetes mellitus, cardiovascular diseases such as arteriosclerosis, hypertension, stroke and certain forms of cancer. Enhancing lipid uptake in other tissues may also have specific therapeutic applications. For example, enhanced uptake of fatty acids by the skin will result in improved cosmetics.
The present inventors describe herein a functional assay to measure the uptake of lipids in vivo in a real intestine environment using small microscopic nematodes, such as Caenorhabditis elegans, as an animal model. Specific applications of this method are also described.
Therefore, in accordance with a first aspect of the invention there is provided a method of assaying lipid uptake in microscopic nematode worms which comprises:
incubating the said microscopic nematode worms in the presence of a probe molecule comprising a lipid moiety linked to a signal generating label; and
determining the amount of probe molecule taken up by the said microscopic nematode worms by detecting a signal generated by the label part of the probe molecule.
In the assays of the invention, the nematode worms are incubated to a medium containing the probe molecule. Upon such incubation, the probe is taken up into the gastrointestinal tract of the nematode (e.g. by pharynx pumping), and in particular into the gut (lumen) of the nematode.
From the gut lumen, the probe may then pass from the gut lumen through the wall of the gastrointestinal tract (i.e. of the gut) into the body of the nematode, where it may concentrate in specific cells or tissues, such as the gut granules and/or other cells and tissues that store or handle lipids.
According to the invention, this passing of the probe molecule(s) from the gut lumen of the nematode into the body of the nematode is used as an in vivo model for lipid transport across biological membranes or barriers, not just in nematodes, but also in higher multicellular organisms, such as vertebrates, mammals and even humans. (In this respect, it should also be noted that the passing of the probe molecule(s) from the gut lumen of the nematode into the body of the nematode may not just be used as a model for lipid transport across the wall of the gastrointestinal tract, but generally as a model for lipid transport across any biological membrane and/or barrier, including epithelial cells/cell layersxe2x80x94such as the walls of blood vesselsxe2x80x94and cell membranes.)
The invention is not particularly limited as to the mechanism or pathway via which said lipid transport may take place (i.e. both in nematode used in the assay of the invention as well as in the higher organism for which the nematode serves as an in vivo model) and may for instance include mechanisms such as active transport (which is preferred) and (passive) diffusion, or a combination thereof. In one embodiment, the invention may even be used to determine if, under the conditions of the assay (i.e. as used as a model), a probe or lipid is transported by an active transport mechanism or by a passive transport mechanism (e.g. by diffusion).
This may also depend on the specific type of lipid or probe involved (i.e. on the type of lipid moiety present in the probe), andxe2x80x94for the assay of the inventionxe2x80x94on the specific worm strain used, on genetic factors influencing the worm strain used (such as gene suppression, e.g. as induced by RNAi methodology) and/or on the induction or suppression of specific pathways in the nematode(s) used, e.g. as a result of exposure of the worms to compounds that induce or suppress (the expression of) certain pathways involved in lipid transport and/or of specific genes and/or enzymes that are involved in such pathways (as further described below.)
As will be appreciated, to measure (active or passive) transport of a probe molecule from the gut lumen into the body of the nematode in vivo, it would in principle be necessary to measure, over a relevant period of time, either the amount and/or concentration of probe in the gut lumen (and/or any changes therein), the amount and/or concentration of probe in the body of the nematode (and/or any changes therein), or both. However, this would be very elaborate, would require examination of individual wormsxe2x80x94e.g. using microscopy techniquesxe2x80x94and even so would probably still not afford quantitative and/or statistically relevant results. Also, any such measurement would not be suitable for automation and/or for screening at medium to high throughput.
Thus, it is one of the general objects of the present invention to provide an assay for measuring lipid uptake/transport in vivo, which assay can serve as an in vivo model for lipid uptake/transport across biological membranes or barriers in (higher) multicellular animals, and which assay can be susceptible toxe2x80x94i.e. can be configured forxe2x80x94automation and/or to screening at medium to high throughput.
More specifically, it is an object of the invention to provide such an assay using nematode worms as a model organism.
One particular purpose of the invention is to provide such an assay which can be used for determining the influence on such lipid uptake/transport of small molecules, of induction or suppression of pathways involved in lipid uptake/transport (e.g. by prolonged exposure to inducing or suppressing factors), and/or of genetic factors such as mutations or RNAi-induced gene suppression.
In this way, the assay of the invention could be used to screen libraries of chemical compounds for small molecules and/or for (other) factors that can influence lipid uptake/transport across biological membranes/barrier, which small molecules or factors could then be used (as a starting point) in the development of compounds for use in compositions for increasing or decreasing lipid uptake/transport across biological membranes/barriers in multicellular organisms, including vertebrates such as mammals and humans, and more generally for influencing or altering fat and/or lipid handling or storage by such multicellular organisms, which can have relevance for a number of disease areas in animals and humans, including but not limited to obesity, diabetes and cardiovascular diseases (and in particular those relating to cholesterol handing and metabolism).
In addition to screening chemical libraries, the assay of the invention could also be used in drug development, e.g. in hits-to-leads chemistry or lead development, but also in genetic screens, gene discovery techniques, target validation techniques and/or other (functional) genomics techniques.
In a specific embodiment, the assays of the invention may also be used to measure/determine the uptake of specific components of food or food compositions (e.g. for human or animal use), such as fatty acids, fats, oils cholesterol-like compounds and/or other lipids (e.g. as defined below), as well as of compounds that are intended to replace fats, oils and/or lipids (for instance in food compositions for human consumption), and/or for additivesxe2x80x94e.g. for food compositionsxe2x80x94that are intended to lower cholesterol in the animal or person that consumes said composition. In this specific aspect of the invention, the assay may be used as a model for the uptake of such compounds from the gastrointestinal tract of a higher animal, such as an animal or human, e.g. to determine components which show increased or reduced uptake from the g.i. tract (i.e. compared to a reference). Thus, the assays of the invention may also be used in the development of components/additives for food, such as dietary compositions for human consumption, (specialized) nutritional compositions, and/or infant formula, or components for use therein.
The invention achieves the objects referred to above by providing the assay techniques described herein.