Lipid uptake assays

Details Lipid uptake assays Lipid uptake assays

2001-05-21

2004-03-09

Gitomer, Ralph (Department: 1651)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving hydrolase

C435S023000, C435S375000

Reexamination Certificate

active

06703214

ABSTRACT:

RELATED APPLICATIONS
This application claims the benefit under 35 U.S.C. 119 of Great Britain application number GB 0012229.1, filed May 19, 2000.
FIELD OF THE INVENTION
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.
BACKGROUND OF THE INVENTION
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 &bgr;-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 “PPAR” (peroxysome proliferator activated receptor)-transcription factors the “RXR” (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:
FATP:
C. elegans
F25D1.9 and D1009.1
H. sapiens
AF055899,
Caveolin:
C. elegans
C56A3.7 and T13F2.8
H. sapiens
NM_001233, NM_001753, and
NM_001234
FAT:
C. elegans
Y49E10.20 and Y76A2B.6
H. sapiens
P16671
FABP:
C. elegans
W02D3.7, W02D36.5, T22G5.2 and
F40F4.2
H. sapiens
NM_001445, NM_001443, and M10617
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.
SUMMARY OF THE INVENTION
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

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