Patent Application
20140309137
1. Field of the Invention
The invention relates generally to nutritional or pharmaceutical modulation of body composition and particularly to gene expression profiles associated with improved or maintained lean body mass or reduced body fat and the use of such profiles for the identification of pharmaceutical, nutraceutical, or dietary substances that modulate or contribute to desired phenotypes in animals.
2. Description of the Related Art
Body weight is primarily a function of lean body mass and fat mass in an individual. Lean body mass is the weight of bones, muscles, organs, body water, and all other non-fat constituents of the body. Fat mass is the weight of the body's storage lipids. Disproportionate or excessive fat mass is a hallmark for an individual being overweight or obese.
Excess fat mass and obesity are recognized as a worldwide health problem among humans. The World Health Organization estimates that there are over 1 billion overweight adults, with just under one third of them classified as obese. In addition, obesity is also increasingly recognized as a problem for animals, particularly for companion animals such as dogs and cats. According to the Centers for Disease Control (CDC), obesity is closely associated with at least a risk of other health problems, including hypertension, dyslipidemia, Type II diabetes, heart disease, stroke, sleep apnea, and certain cancers such as breast, endometrial, and colon cancers. Risk factors for an individual becoming obese include genetics, emotions/stress, overeating, and a sedentary lifestyle.
Enhancing lean body mass can enhance the body's basal metabolic rate. Enhancing metabolic rate can facilitate the loss of excess fat mass when dietary caloric intake is insufficient to meet the body's energy needs or can reduce the accumulation of fat mass when dietary caloric intake exceeds the body's maintenance energy requirement. Various approaches to increase lean body mass have been described. One such approach is dietary supplementation with conjugated linoleic acid (CLA). CLA is a term used to describe isomers of octadecadienoic acid that are found in many foods such as dairy products (Terpstra A H M (2004) Am. J. Clin. Nutr. 79:352-61). CLA has been shown to reduce fat mass in mice and humans and has been implicated in an increase in lean body mass (Bhattacharya A et al. (2005) J. Nutr. 135:1124-30; Gaullier J-M et al. (2004) Am. J. Clin. Nutr. 79:1118-25; Blankson H et al. (2000) J. Nutr. 130:2943-8; and, Park Y et al. (1997) Lipids 32:853-8). Another approach to increase lean body mass is consumption of a high protein diet. Studies suggest that diets with higher protein content, coupled with reduced carbohydrate consumption and/or regular exercise, can enhance the loss of fat mass and reduce the loss of lean body mass (Layman D K et al. (2005) J. Nutr. 135:1903-10; Layman D K et al. (2004) J. Nutr. 134:968 S-73S; Marsset-Baglieri A et al. (2004) J. Nutr. 134:2646-52; and, Due A et al. (2004) Int. J. Obes. Relat. Metab. Disord. 28:1283-90). A third approach to increase lean body mass and decrease fat mass is regular exercise (Bhattacharya A et al. (2005) J. Nutr. 135:1124-30; Layman D K et al. (2005) J. Nutr. 135:1903-10; and, Tsai A C et al. (2003) J. Nutr. Biochem. 14:541-9).
Various studies have evaluated different aspects of the genetics of lean body mass and obesity. Association studies have revealed links between bodyweight, overweight, and obesity and polymorphisms in various genes (Chagnon Y C et al. (2003) Obesity Res. 11:313-67). Similarly, association studies have identified genes relating to body fat mass, percentage of body fat, and skin folds, body fat distribution (waist-to-hip ratio, waist circumference, etc.), resting energy expenditure, and adipocyte lipolysis (Chagnon Y C et al. (2003) Obesity Res. 11:313-67). In addition, studies have evaluated associations between candidate genes and changes in body weight, including genes associated with spontaneous weight gain over time, genes associated with endurance training-induced changes in fat mass, and genes associated with weight loss during a three year lifestyle change (Chagnon Y C et al. (2003) Obesity Res. 11:313-67). A listing of all such genes can be found in the prior art (Chagnon Y C et al. (2003) Obesity Res. 11:313-67). Studies have also analyzed the genetic aspects of lean body mass. Lean body mass studies have linked polymorphisms in Type 1 Deiodinase with higher lean body mass and muscle strength (Peeters R P et al. (2005) J. Clin. Endocrinol. 90:256-63), and polymorphisms in the glucocorticoid receptor with higher muscle mass and muscle strength (van Rossum E F C et al. (2004) J. Clin. Endocrinol. 89:4004-9).
Although lean body mass and fat mass are directly related, few studies have attempted to explore the genetic mechanisms that mediate a higher proportion of one type relative to the other. A detailed knowledge of these mechanisms would provide a better understanding of the conditions that favor a high level of lean body mass and/or reduced body fat and would provide a better understanding of how to promote a lean phenotype in an animal. Because a higher proportion of lean body mass, especially relative to fat mass, has positive implications for improved health and decreased risk for obesity-related ailments, it is desirable for an individual to increase the ratio of lean body mass to fat mass, either by increasing lean body mass and/or by reducing body fat.
It is, therefore, an object of the invention to provide one or more genes or gene segments that are differentially expressed in animals exhibiting a lean phenotype resulting from one or more lean phenotype-promoting treatments comprising (1) administration of CLA, (2) consumption of a high protein diet, and (3) increased exercise.
It is another object of the invention to provide a combination comprising a plurality of polynucleotides that are differentially expressed in animals exhibiting a lean phenotype resulting from one or more lean phenotype-promoting treatments comprising (1) administration of CLA, (2) consumption of a high protein diet, and (3) increased exercise.
It is another object of the invention to provide compositions of two or more polynucleotide or polypeptide probes suitable for detecting the expression of genes differentially expressed in animals exhibiting a lean phenotype resulting from one or more lean phenotype-promoting treatments comprising (1) administration of CLA, (2) consumption of a high protein diet, and (3) increased exercise, and devices such as substrate arrays containing the probes.
It is a further object of the invention to provide methods for detecting differential expression of one or more genes differentially expressed in animals exhibiting a lean phenotype resulting from one or more lean phenotype-promoting treatments comprising (1) administration of CLA, (2) consumption of a high protein diet, and (3) increased exercise, as compared with normal or untreated animals.
It is another object of the invention to provide a method for measuring the effect of a test substance (e.g., lean body mass promoting nutrients or bioactives) on the expression profile of one or more genes differentially expressed in animals exhibiting a lean phenotype resulting from one or more lean phenotype-promoting treatments comprising (1) administration of CLA, (2) consumption of a high protein diet, and (3) increased exercise, as compared with normal or untreated animals.
One or more of these other objects are achieved using novel combinations of polynucleotides or polypeptides representing genes and gene segments that are differentially expressed in animals exhibiting a lean phenotype resulting from one or more lean phenotype-promoting treatments comprising (1) administration of CLA, (2) consumption of a high protein diet, and (3) increased exercise. The polynucleotides are used to produce compositions, probes, devices based on the probes, and methods for determining the status of polynucleotides differentially expressed in animals exhibiting a lean phenotype as compared to normal or untreated animals, which are useful for achieving the above-identified objects, e.g., prognosing and diagnosing conditions relating to the phenotype and for screening substances to determine if they are likely to be useful for promoting the phenotype. Such substances, once identified, may be used to promote the phenotype. Various kits comprising combinations of probes, devices utilizing the probes, and substances are also provided, as are various computer programs for manipulating information, and communication media for communicating information pertaining to the differentially expressed genes and methods of their use.
Other and further objects, features, and advantages of the invention will be readily apparent to those skilled in the art.
All percentages expressed herein are by weight of the composition on a dry matter basis unless specifically stated otherwise. The skilled artisan will appreciate that the term “dry matter basis” means that an ingredient's concentration in a composition is measured after any free moisture in the composition is removed.
As used throughout, ranges are used herein as shorthand, so as to avoid having to set out at length and describe each and every value within the range. Any appropriate value within the range can be selected, where appropriate, as the upper value, lower value, or the terminus of the range. It is understood that any and all whole or partial integers between any ranges or intervals set forth herein are included herein.
As used herein and in the appended claims, the singular form of a word includes the plural, and vice versa, unless the context clearly dictates otherwise. Thus, the references “a,” “an,” and “the” are generally inclusive of the plurals of the respective terms. For example, reference to “an animal”, “a method”, or “a substance” includes a plurality of such “animals”, “methods”, or “substances”. Similarly, the words “comprise”, “comprises”, and “comprising” are to be interpreted inclusively rather than exclusively.
The term “animal” means a human or other animal, including avian, bovine, canine, equine, feline, hicrine, murine, ovine, and porcine animals, that has adipose tissue. When the term is used in the context of comparing test subjects, the animals that are compared are animals of the same species and possibly of the same race or breed. A “companion animal” is any domesticated animal, and includes, without limitation, cats, dogs, rabbits, guinea pigs, ferrets, hamsters, mice, gerbils, horses, cows, goats, sheep, donkeys, pigs, and the like. Preferably, the animal is a human or a companion animal such as a canine or feline.
The term “antibody” means any immunoglobulin that binds to a specific antigen, including IgG, IgM, IgA, IgD, and IgE antibodies. The term includes polyclonal, monoclonal, monovalent, humanized, heteroconjugate, antibody compositions with polyepitopic specificity, chimeric, bispecific antibodies, diabodies, single-chain antibodies, and antibody fragments such as Fab, Fab′, F(ab′)2, and Fv, or other antigen-binding fragments.
The term “array” means an ordered arrangement of at least two probes on a substrate. At least one of the probes is a control or standard and at least one of the probes is a diagnostic probe. The arrangement of from about two to about 40,000 probes on a substrate assures that the size and signal intensity of each labeled complex formed between a probe and a sample polynucleotide or polypeptide is individually distinguishable.
The term “binding complex” refers to a complex formed when a polypeptide in a sample specifically binds (as defined herein) to a binding partner, such as an antibody or functional fragment thereof.
The term “dietary supplement” means a product that is intended to be ingested in addition to the normal diet of an animal. Dietary supplements may be in any form—e.g., solid, lid, gel, tablets, capsules, powder, and the like. Preferably they are provided in convenient dosage forms. In some embodiments they are provided in bulk consumer packages such as bulk powders or liquids. In other embodiments, supplements are provided in bulk quantities to be included in other food items such as snacks, treats, supplement bars, beverages and the like.
The term “differential expression” or “differentially expressed” means increased or unregulated gene expression or means decreased or downregulated gene expression as detected by the absence, presence, or at least statistically significant in the amount of transcribed messenger RNA or translated protein in a sample.
The term “food” or “food composition” means a composition that is intended for consumption by an animal, including a human, and provides nutrition thereto. A “food product formulated for human consumption” is any composition specifically intended for ingestion by a human being. “Pet foods” are compositions intended for consumption by pets, preferably by companion animals. A “complete and nutritionally balanced pet food,” is one that contains all known required nutrients for the intended recipient or consumer of the food, in appropriate amounts and proportions, based for example on recommendations of recognized authorities in the field of companion animal nutrition. Such foods are therefore capable of serving as a sole source of dietary intake to maintain life or promote production, without the addition of supplemental nutritional sources. Nutritionally balanced pet food compositions are widely known and widely used in the art.
The term “fragment” means (1) an oligonucleotide or polynucleotide sequence that is a portion of a complete sequence and that has the same or similar activity for a particular use as the complete polynucleotide sequence or (2) a peptide or polypeptide sequence that is a portion of a complete sequence and that has the same or similar activity for a particular use as the complete polypeptide sequence. Such fragments can comprise any number of nucleotides or amino acids deemed suitable for a particular use. Generally, oligonucleotide or polynucleotide fragments contain at least about 10, 50, 100, or 1000 nucleotides and polypeptide fragments contain at least about 4, 10, 20, or 50 consecutive amino acids from the complete sequence. The term encompasses polynucleotides and polypeptides variants of the fragments.
The term “gene” or “genes” means a complete or partial segment of DNA involved in producing a polypeptide, including regions preceding and following the coding region (leader and trailer) and intervening sequences (introns) between individual coding segments (exons). The term encompasses any DNA sequence that hybridizes to the complement of gene coding sequences.
The term “gene product” means the product of transcription of a gene, such as mRNA or derivatives thereof (e.g., cDNA), or translation of a gene transcript. The term “gene product” generally refers to the translation product, which is a protein. The term “gene product” may be used interchangeably with the term “protein” herein.
The term “high protein diet” refers to a diet comprising foods or dietary supplements that result in an animal's intake of protein on a regular basis being at least about 10% higher than a comparable control animal. In certain embodiments, the animal's protein intake may be 20, 30, 40, 50, 60, 70, 80, 90 or 100% (i.e., two-fold in the latter case) higher than that of a comparable control animal. In other embodiments, the animal's protein intake may be three- or four-fold or more higher than that of a comparable control animal. Typically, a high protein diet is formulated to comprise the same calorie intake as a regular diet. Often, but not always, this is accomplished by lowering the carbohydrate content of the diet. Alternatively or in addition, the fat content of the diet may be lowered. In particular embodiments, the protein content of a high protein diet may comprise at least about 25% of the total calories as protein. In other embodiments, the protein content of a high protein diet may comprise at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or 80% of the total calories as protein.
The term “homolog” means (1) a polynucleotide, including polynucleotides from the same or different animal species, having greater than 30%, 50%, 70%, or 90% sequence similarity to a reference polynucleotide, and having the same or substantially the same properties and performing the same or substantially the same function as the reference polynucleotide, or having the capability of specifically hybridizing to a reference polynucleotide under stringent conditions or (2) a polypeptide, including polypeptides from the same or different animal species, having greater than 30%, 50%, 70%, or 90% sequence similarity to a reference polypeptide and having the same or substantially the same properties and performing the same or substantially the same function as the reference polypeptide, or having the capability of specifically binding to a reference polypeptide. When referring to fragments of full length coding sequences, the function of those fragments may simply be to encode a selected portion of a polypeptide of a certain sequence, or to be of suitably similar sequence to hybridize to another polynucleotide fragment encoding that polypeptide. When referring to fragments of polypeptides, the function of those fragments may simply be to form an epitope suitable for generation of an antibody. Sequence similarity of two polypeptide sequences or of two polynucleotide sequences is determined using methods known to skilled artisans, e.g., the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990)). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (J. Mol. Biol. 215:403-410 (1990)). To obtain gapped alignments for comparison purposes, Gapped Blast can be utilized as described in Altschul et al. (Nucl. Acids Res. 25: 3389-3402 (1997)). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) are used. See http://ww.ncbi.nlm.nih.gov.
The term “hybridization complex” means a complex that is formed between sample polynucleotides when the purines of one polynucleotide hydrogen bond with the pyrimidines of the complementary polynucleotide, e.g., 5′-A-G-T-C-3′ base pairs with 3′-T-C-A-G-5′. The degree of complementarily and the use of nucleotide analogs affect the efficiency and stringency of hybridization reactions.
The term “increased exercise” refers to an increase in physical activity of least about 10% higher than that of a comparable control animal in the same period of time. In certain embodiments, the animal's physical activity may be 20, 30, 40, 50, 60, 70, 80, 90 or 100% (i.e., two-fold in the latter case) higher than that of a comparable control animal. In other embodiments, the animal's activity may be three- or four-fold or more higher than that of a comparable control animal. An animal's physical activity may be measured by a variety of techniques well known to the person of skill in the art.
The term “individual” when referring to an animal means an individual animal of any species or kind.
The term “lean phenotype” refers to any molecular, biochemical, physiologic, cellular, systemic, and physical effects observed in an animal resulting from the differential expression of genes that occurs when the animal exercises, consumes a specialized diet regimen such as a high protein diet, and/or is administered a compound, composition, or dietary supplement to modulate the expression of genes associated with increasing or maintaining lean body mass, and/or reducing body fat. An exemplary compound of this type is CLA. The term “lean phenotype” also includes the “transition to lean phenotype,” which refers to any molecular, biochemical, physiologic, cellular, systemic, and physical effects observed in an animal resulting from the differential expression of genes that occurs when the animal is undergoing the change from normal (as defined below) to lean phenotype.
The term “administration” means to administer a substance, a diet, or a test treatment (such as increased physical exercise) to an animal. Administration periods include terms consistent with the particular substance, diet, or treatment and the animal. “Long term administration” generally refers to periods in excess of one week. Periods of longer than two or three weeks, or one, two, three, or four months are contemplated. Also included are more extended periods that include longer than 5, 6, 7, 8, 9, or 10 months. Periods in excess of 11 months or 1 year are also included. Long term use extending over 1, 2, 3 years or more is also contemplated herein. In the case of certain animals, it is envisioned that the animal would be administered substances or treatment regimens identified by the present methods on a regular basis. “Regular basis” refers to at least monthly administration. More frequent administration, such as weekly or two or three times weekly is included. Also included are regimens that comprise at least once, twice, three times or more daily administration. Any dosing frequency, regardless of whether expressly exemplified herein, is considered useful. The skilled artisan will appreciate that dosing frequency will be a function of the substance that is being administered, and some compositions may require more or less frequent administration to maintain a desired biochemical, physiological or gene expression effects, namely effects including one or more of food intake, satiety, lipid metabolism, and fat utilization, and the gene expression profile associated therewith. The term “extended regular basis” refers to long term administration of a substance on a regular basis.
“Normal” as used in relation to animals manifesting a lean phenotype, refers to the absence of molecular, biochemical, physiologic, cellular, systemic, and physical effects resulting from the differential expression of genes associated with a lean phenotype.
The term “oral administration” means that an animal ingests, or a human is directed to feed, or does feed, the animal one or more of the substances described herein. The term “ingestion” is used herein interchangeably with the term “oral administration.” The term “consumption” is also used herein to refer to ingestion of a substance, particularly a food composition, on an extended regular basis. When a human is directed to orally administer or feed the substance, such direction may be that which instructs and/or informs the human that use of the substance may and/or will provide the referenced benefit. Such direction may be oral direction (e.g., through oral instruction from, for example, a physician, veterinarian, or other health professional, or radio or television media (i.e., advertisement), or written direction (e.g., through written direction from, for example, a physician, veterinarian, or other health professional (e.g., prescriptions), sales professional or organization (e.g., through, for example, marketing brochures, pamphlets, or other instructive paraphernalia), written media (e.g., internet, electronic mail, or other computer-related media), and/or packaging associated with the substance.
The term “polynucleotide” or “oligonucleotide” means a polymer of nucleotides. The term encompasses DNA and RNA (including cDNA and mRNA) molecules, either single or double stranded and, if single stranded, its complementary sequence in either linear or circular form. The term also encompasses fragments, variants, homologs, and alleles, as appropriate for the sequences, which have the same or substantially the same properties and perform the same or substantially the same function as the original sequence. In particular, the term encompasses homologs from different species, e.g., a mouse and a dog or cat. The sequences may be fully complementary (no mismatches) when aligned or may have up to about a 30% sequence mismatch. Preferably, for polynucleotides, the chain contains from about 50 to 10,000 nucleotides, more preferably from about 150 to 3,500 nucleotides. Preferably, for oligonucleotides, the chain contains from about 2 to 100 nucleotides, more preferably from about 6 to 30 nucleotides. The exact size of a polynucleotide or oligonucleotide will depend on various factors and on the particular application and use of the polynucleotide or oligonucleotide. The term includes nucleotide polymers that are synthesized and that are isolated and purified from natural sources. The term “polynucleotide” is inclusive of “oligonucleotide.”
The term “polypeptide,” “peptide,” or “protein” means a polymer of amino acids. The term encompasses naturally occurring and non-naturally occurring (synthetic) polymers and polymers in which artificial chemical mimetics are substituted for one or more amino acids. The term also encompasses fragments, variants, and homologs that have the same or substantially the same properties and perform the same or substantially the same function as the original sequence. The term encompass polymers of any length, preferably polymers containing from about 2 to 1000 amino acids, more preferably from about 5 to 500 amino acids. The term includes amino acid polymers that are synthesized and that are isolated and purified from natural sources.
The term “probe” means (1) an oligonucleotide or polynucleotide, either RNA or DNA, whether occurring naturally as in a purified restriction enzyme digest or produced synthetically, that is capable of annealing with or specifically hybridizing to a polynucleotide with sequences complementary to the probe or (2) a compound or substance, including a peptide or polypeptide, capable of specifically binding a particular protein or protein fragment to the substantial exclusion of other proteins or protein fragments. An oligonucleotide or polynucleotide probe may be either single or double stranded. The exact length of the probe will depend upon many factors, including temperature, source, and use. For example, for diagnostic applications, depending on the complexity of the target sequence, an oligonucleotide probe typically contains about 10 to 100, 15 to 50, or 15 to 25 nucleotides. In certain diagnostic applications, a polynucleotide probe contains about 100-1000, 300-600, nucleotides, preferably about 300 nucleotides. The probes herein are selected to be “substantially” complementary to different strands of a particular target sequence. This means that the probes must be sufficiently complementary to specifically hybridize or anneal with their respective target sequences under a set of predetermined conditions. Therefore, the probe sequence need not reflect the exact complementary sequence of the target. For example, a noncomplementary nucleotide fragment may be attached to the 5′ or 3′ end of the probe, with the remainder of the probe sequence being complementary to the target sequence. Alternatively, noncomplementary bases or longer sequences can be interspersed into the probe provided that the probe sequence has sufficient complementarity with the sequence of the target polynucleotide to specifically anneal to the target polynucleotide. A peptide or polypeptide probe may be any molecule to which the protein or peptide specifically binds, including DNA (for DNA binding proteins), antibodies, cell membrane receptors, peptides, cofactors, lectins, sugars, polysaccharides, cells, cell membranes, organelles and organellar membranes.
The term “sample” means any animal tissue or fluid containing, e.g., polynucleotides, polypeptides, antibodies, metabolites, and the like, including cells and other tissue containing DNA and RNA. Examples include adipose, blood, cartilage, connective, epithelial, lymphoid, muscle, nervous, sputum, and the like. A sample may be solid or liquid and may be DNA, RNA, cDNA, bodily fluids such as blood or urine, cells, cell preparations or soluble fractions or media aliquots thereof, chromosomes, organdies, and the like.
The term “single package” means that the components of a kit are physically associated in or with one or more containers and considered a unit for manufacture, distribution, sale, or use. Containers include, but are not limited to, bags, boxes, bottles, shrink wrap packages, stapled or otherwise affixed components, or combinations thereof. A single package may be containers of individual food compositions physically associated such that they are considered a unit for manufacture, distribution, sale, or use.
The term “specifically bind” means a special and precise interaction between two molecules which is dependent upon their structure, particularly their molecular side groups. For example, the intercalation of a regulatory protein into the major groove of a DNA molecule, the hydrogen bonding along the backbone between two single stranded nucleic acids, or the binding between an epitope of a protein and an agonist, antagonist, or antibody.
The term “specifically hybridize” means an association between two single stranded polynucleotides of sufficiently complementary sequence to permit such hybridization under predetermined conditions generally used in the art (sometimes termed “substantially complementary”). For example, the term may refer to hybridization of a polynucleotide probe with a substantially complementary sequence contained within a single stranded DNA or RNA molecule according to an aspect of the invention, to the substantial exclusion of hybridization of the polynucleotide probe with single stranded polynucleotides of non-complementary sequence.
The term “standard” means (1) a control sample that contains tissue from an animal administered a control or reference substance, or no substance, as compared with a sample that contains tissue from an animal administered a test substance, for example, to determine if the test substance causes differential gene expression, as appropriate for the context of its use.
The term “stringent conditions” means (1) hybridization in 50% (vol/vol) formamide with 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42° C., (2) hybridization in 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C.; with washes at 42° C. in 0.2×SSC and 0.1% SDS or washes with 0.015 M NaCl, 0.0015 M sodium citrate, 0.1% Na2SO4 at 50° C. or similar procedures employing similar low ionic strength and high temperature washing agents and similar denaturing agents.
The term “variant” means (1) a polynucleotide sequence containing any substitution, variation, modification, replacement, deletion, or addition of one or more nucleotides from or to a polynucleotide sequence and that has the same or substantially the same properties and performs the same or substantially the same function as the original sequence and (2) a polypeptide sequence containing any substitution, variation, modification, replacement, deletion, or addition of one or more amino acids from or to a polypeptide sequence and that has the same or substantially the same properties and performs the same or substantially the same function as the original sequence. The term therefore includes single nucleotide polymorphisms (SNPs) and allelic variants and includes conservative and non-conservative amino acid substitutions in polypeptides. The term also encompasses chemical derivatization of a polynucleotide or polypeptide and substitution of nucleotides or amino acids with nucleotides or amino acids that do not occur naturally, as appropriate.
The term “virtual package” means that the components of a kit are associated by directions on one or more physical or virtual kit components instructing the user how to obtain the other components, e.g., in a bag containing one component and directions instructing the user to go to a website, contact a recorded message, view a visual message, or contact a caregiver or instructor to obtain instructions on how to use the kit.
The combinations, compositions, devices, methods, probes, and other advances disclosed herein are not limited to particular methodology, protocols, and reagents described herein because, as the skilled artisan will appreciate, they may vary. Further, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to and does not limit the scope of that which is disclosed or claimed.
Unless defined otherwise, all technical and scientific terms, terms of art, and acronyms used herein have the meanings commonly understood by one of ordinary skill in the art in the field(s) of the invention, or in the field(s) where the term is used. Although any compositions, methods, articles of manufacture, or other means or materials similar or equivalent to those described herein can be used in the practice of the invention, the preferred compositions, methods, articles of manufacture, or other means or materials are described herein.
All patents, patent applications, publications, and other references cited or referred to herein are incorporated herein by reference to the extent allowed by controlling law. The discussion of those references is intended merely to summarize the assertions made therein. No admission is made that any such patents, patent applications, publications or references, or any portion thereof, is relevant, material, or prior art. The right to challenge the accuracy and pertinence of any assertion of such patents, patent applications, publications, and other references as relevant, material, or prior art is specifically reserved.
The invention is based in part on the clear demonstration that treatments known to promote a lean phenotype as defined herein are associated with significant changes in the gene expression profiles in three different tissues of animals subjected to those treatments. The association was determined by comparing expression of the genes in normal tissue, namely muscle, liver and adipose tissue, with tissue from animals manifesting a lean phenotype (LP) as a result of one or more LP-promoting treatments, namely (1) administration of CLA, (2) consumption of a high protein diet, and/or (3) increased exercise.
In one embodiment, several hundred genes were found to be differentially expressed as a result of all three treatments (sometimes referred to herein as “three treatments”); i.e., CLA supplementation, a high protein diet, or increased exercise caused the differential expression of this subset of genes, the encoded proteins of which are listed in Table 6 (Example 3) herein. The proteins set forth in Table 6 are divided into groups based upon different criteria. First, the proteins are listed by tissue in Table 6, representing genes differentially expressed in adipose tissue (Table 6A), liver (Table 6B) and muscle (quadriceps) (Table 6C). Table 6 also lists subsets of proteins representing genes differentially expressed in two or more of the three tissue types, namely (1) adipose and liver (Table 6D), (2) adipose and muscle (Table 6E), (3) liver and muscle (Table 6F), and (4) all three tissues (Table 60). Second, the proteins are divided into groups based upon the function or physiological role of the encoded protein, as well as the tissue in which the differential expression occurs. Those groupings are set forth in Table 7 (adipose), Table 8 (liver) and Table 9 (muscle). Those functions include: (1) in adipose tissue, cholesterol biosynthetic pathway, statin pathway, adipogenesis, apoptosis, cell motility, mitochondrial fatty acid betaoxidation, fatty acid biosynthesis, fatty acid metabolism, glycolysis, regulation of cell proliferation, inflammation, immunity and stress response (including the subcategories of Rn T-cell receptor, Rn B-cell receptor, leukocyte transendothelial migration, tight junction, adherens junction, antigen processing, response to unfolded proteins, response to wounding, response to external stimulus, inflammatory response, immune response, T-cell activation and Rn IL-4), multicellular organismal development, and regulation of apoptosis; (2) in liver, PPAR signaling pathway, and fatty acid metabolism; and (3) in muscle, lipid metabolism.
In another embodiment, the high protein diet treatment, analyzed singly, was found to cause differential expression of several thousand genes. The proteins encoded by these genes are listed in Table 10 (Example 4) herein. As with the “three treatments” subset, the proteins set forth in Table 10 are divided into groups based upon different criteria. First, the proteins are listed by tissue in Table 10, representing genes differentially expressed in adipose tissue (Table 10A), liver (Table 10B) and muscle (quadriceps) (Table 10C). Table 10 also lists subsets of proteins representing genes differentially expressed in two or more of the three tissue types, namely (1) adipose and liver (Table 10D), (2) adipose and muscle (Table 10E), (3) liver and muscle (Table 10F), and (4) all three tissues (Table 100). Second, the proteins are divided into groups based upon the function or physiological role of the encoded protein, as well as the tissue in which the differential expression occurs. Those groupings are set forth in Table 11 (adipose), Table 12 (liver) and Table 13 (muscle). Those functions include: (1) in adipose tissue, immune response, inflammatory response, response to stress, chemotaxis, response to unfolded protein, defense response, cell activation, lymphocyte activation, locomotory behavior, lipid metabolic process, lipid biosynthetic process, steroid biosynthetic process, cholesterol metabolic process, steroid metabolic process, glycolysis, glucose metabolic process, organ development, muscle development, positive regulation of cell proliferation, angiogenesis, blood vessel morphogenesis, anti-apoptosis, muscle contraction, phosphate transport, protein complex assembly, calcium-mediated signaling, regulation of GTPase activity, protein amino acid glycosylation, regulation of cell shape, Rattus norvegicus (Rn) B cell receptor NetPath 12 (Johns Hopkins University and the Institute of Bioinformatics (www.netpath.org/)), Rn T-cell receptor NetPath 11, Rn IL-4 NetPath 16, Rn IL-7 NetPath 19, Rn eicosanoid synthesis, Rn insulin signaling, Rn cholesterol biosynthesis, Rn fatty acid synthesis BiGCaT (University of Maastricht (www.bigcat.unimass.n1/index.html)), Rn Krebs-TCA cycle, Rn mitochondrial fatty acid betaoxidation, Rn striated muscle contraction, leukocyte transendothelial migration, T cell receptor signaling pathway, tight junction, B cell receptor signaling pathway, complement and coagulation cascades, Fc epsilon RI signaling pathway, toll-like receptor signaling pathway, PPAR signaling pathway, biosynthesis of steroids, glycolysis/gluconeogenesis, arachidonic acid metabolism, pyruvate metabolism, and riboflavin metabolism; (2) in liver, lipid metabolic process, fatty acid metabolic process, lipid biosynthetic process, fatty acid biosynthetic process, steroid metabolic process, proteolysis, carbohydrate metabolic process, glucose metabolic process, gluconeogenesis, amino acid metabolic process, amine metabolic process, nitrogen compound metabolic process, one-carbon compound metabolic process, xenobiotic metabolic process, sodium ion transport, multicellular organismal development, regulation of cell growth, myelination, regulation of progression through cell cycle, antigen processing and presentation, Rn adipogenesis, Rn fatty acid synthesis BiGCaT, Rn glycolysis and gluconeogenesis, Rn nuclear receptors in lipid metabolism and toxicity, Rn fatty acid beta oxidation 1 BiGCaT, Rn fatty acid omega oxidation BiGCaT, PPAR signaling pathway, metabolism of xenobiotics by cytochrome P450, fatty acid metabolism, alanine and aspartate metabolism, arginine and proline metabolism, pyruvate metabolism, glutamate metabolism, nitrogen metabolism, cysteine metabolism, and tyrosine metabolism; and (3) in muscle, immune response, defense response, inflammatory response, and Rn circadian exercise.
Thus, a number of genes have been identified that are differentially expressed in animals manifesting a lean phenotype, resulting from LP-promoting treatments including a high protein diet, administration of CLA and/or increased exercise. Polynucleotides and fragments thereof that form these genes, as well as their encoded proteins and fragments, can be used, for example, in diagnostic or prognostic assays to measure a shift to a lean phenotype, or assays useful for screening test substances for their effectiveness to promote or support a lean phenotype.
In certain embodiments, expression of at least one differentially expressed gene is measured. In preferred embodiments, expression of two or more differentially expressed genes is measured, providing a gene expression pattern or gene expression profile. More preferably, measurement of a multiplicity of differentially expressed genes is performed, providing additional information for a gene expression pattern or profile.
In various embodiments of the invention, changes in gene expression may be measured in one or both of two ways: (1) measuring transcription through detection of mRNA produced by a particular gene; and (2) measuring translation through detection of protein produced by a particular transcript.
Decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, such as, for example, PCR (including, without limitation, RT-PCR and qPCR), RNase protection, Northern blotting, microarray, macroarray, and other hybridization methods. The genes that are assayed or interrogated according to the invention are typically in the form of mRNA or reverse transcribed mRNA. The genes may be cloned and/or amplified. The cloning itself does not appear to bias the representation of genes within a population. However, it may be preferable to use polyA+ RNA as a source, as it can be used with fewer processing steps.
Thus, in one aspect, the invention provides a combination comprising a plurality of polynucleotides or proteins expressed therefrom that are differentially expressed in animals exhibiting a lean phenotype resulting from one or more lean phenotype-promoting treatments comprising (1) administration of CLA, (2) consumption of a high protein diet, and (3) increased exercise, wherein the polynucleotides are selected from genes encoding proteins listed in Table 6 or Table 10, or fragments thereof.
In one embodiment, the polynucleotides are differentially expressed in each of the lean phenotype promoting treatments comprising (1) administration of CLA, (2) consumption of a high protein diet, and (3) increased exercise, and the polynucleotides are selected from genes encoding proteins listed in Table 6, or fragments thereof. In more specific embodiments, the polynucleotides are selected from genes encoding proteins listed in tissue-specific subsets of Table 6 selected from Table 6A, Table 6B, Table 6C, Table 6D, Table 6E, Table 6F, and Table 6G, or fragments thereof. In other embodiments, the polynucleotides are differentially expressed in adipose tissue and encode proteins involved in functions selected from those recited hereinabove and set forth in Table 7. In other embodiments, the polynucleotides are differentially expressed in liver and encode proteins involved in the functions recited hereinabove and set forth in Table 8. In yet another embodiment, the polynucleotides are differentially expressed in muscle and encode proteins involved in lipid metabolism, as set forth in Table 9.
In another embodiment, the polynucleotides are differentially expressed in the lean phenotype promoting treatment comprising consumption of a high protein diet, and the polynucleotides are selected from genes encoding proteins listed in Table 10, or fragments thereof. In more specific embodiments, the polynucleotides are selected from genes encoding proteins listed in tissue-specific subsets of Table 10 selected from: Table 10A, Table 10B, Table 10C, Table 10D, Table 10E, Table 10F, and Table 100, or fragments thereof. In other embodiments, the polynucleotides are differentially expressed in adipose tissue and encode proteins involved in functions selected from those recited above, and listed in Table 11. In other embodiments, the polynucleotides are differentially expressed in liver and encode proteins involved in functions selected from those recited above and set forth in Table 12. In yet another embodiment, the polynucleotides are differentially expressed in muscle and encode proteins involved in functions selected from those recited above and listed in Table 13.
In one embodiment, the combination comprises two or more polynucleotides or proteins expressed from the polynucleotides. Preferably, the combination comprises a plurality of polynucleotides or proteins expressed from polynucleotides, generally about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more polynucleotides or proteins, or fragments thereof, as appropriate for a particular Group and use. When the combination comprises one or more fragments, the fragments can be of any size that retains the properties and function of the original polynucleotide or protein, preferably from about 30%, 60%, or 90% of the original.
The polynucleotides and proteins can be from any animal, preferably canines and felines, most preferable canines. Homologs of the polynucleotides and proteins from different animal species are obtainable by standard information mining and molecular methods well known to the skilled artisan. For example, the name, or description of function of a gene or protein may be entered into one of several publicly available databases, which will generate a list of sources providing information about that gene from different species, including sequence information. One such database is the “Information Hyperlinked over Proteins (iHOP) database, which is accessible on the internet via the url: ihop-net.org. Alternatively, a public database accession number of a known gene or protein may be utilized to access sequence information for that gene or protein and to search for homologs or orthologs in other species using a sequence comparison search. For example, the GenBank accession number of a gene or protein from mouse may be entered into the National Institutes of Health's National Center for Biotechnology Information (NCBI) database, thereby accessing DNA or polypeptide sequences for that mouse gene. Using the same database, a BLAST search may be performed on the mouse DNA or protein sequence, or fragments thereof of sufficient length to define the gene or protein, to identify sequences of sufficient homology from other species, e.g., a canine. Accession numbers of the sequences from the other species of interest may then be entered into the database to obtain information pertaining to those full-length nucleotide or protein sequences, as well as other descriptive information.
In another aspect, the invention provides a composition comprising two or more probes for detecting differential gene expression in animals exhibiting a lean phenotype resulting from one or more lean phenotype-promoting treatments comprising (1) administration of CLA, (2) consumption of a high protein diet, and (3) increased exercise, wherein the probes comprise (a) polynucleotides that specifically hybridize to two or more genes encoding proteins listed in Table 6 or Table 10, or fragments thereof, or (b) polypeptide binding agents that specifically bind to two or more polypeptides selected from proteins listed in Table 6 (genes differentially expressed in all three treatments) or Table 10 (genes differentially expressed in high protein diet treatment), or fragments thereof.
In certain embodiments, the probes specifically hybridize to genes encoding proteins listed in listed in tissue-specific subsets of Table 6 selected from Table 6A, Table 6B, Table 6C, Table 6D, Table 6E, Table 6F, and Table 6G, or fragments thereof, or specifically bind to polypeptides comprising proteins listed in listed in tissue-specific subsets of Table 6 selected from Table 6A, Table 6B, Table 6C, Table 6D, Table 6E, Table 6F, and Table 6G, or fragments thereof. In other embodiments, the probes specifically hybridize to, or specifically bind to, polynucleotides encoding proteins, or polypeptides comprising proteins having certain functions or biochemical roles, as listed in Table 7, Table 8 or Table 9.
In other embodiments, the probes specifically hybridize to genes encoding proteins listed in tissue-specific subsets of Table 10 selected from Table 10A, Table 10B, Table 10C, Table 10D, Table 10E, Table 10F, and Table 100, or fragments thereof, or specifically bind to polypeptides comprising proteins listed in listed in tissue-specific subsets of Table 10 selected from Table 10A, Table 10B, Table 10C, Table 10D, Table 10E, Table 10F, and Table 10G, or fragments thereof. In other embodiments, the probes specifically hybridize to, or specifically bind to, polynucleotides encoding proteins, or polypeptides comprising proteins having certain functions or biochemical roles, as listed in Table 11, Table 12 or Table 13.
Preferably, the composition comprises a plurality of probes, generally about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 500 or more probes for detecting the polynucleotides or proteins, or fragments thereof, as appropriate for a particular Group and use. It will be understood by the skilled artisan that multiple different probes for a single target gene or protein may be utilized, in order to refine the sensitivity or accuracy of an assay utilizing the probes. For example, several oligonucleotide probes, specifically hybridizing to different sequences on a target polynucleotide, may be employed. Likewise, several antibodies, immunologically specific for different epitopes on a target protein, may be utilized.
One or more oligonucleotide or polynucleotide probes for interrogating a sample may be prepared using the sequence information for any of the genes listed herein, from any species, preferably canine or feline. The probes should be of sufficient length to specifically hybridize substantially exclusively with appropriate complementary genes or transcripts. In certain embodiments, the oligonucleotide probes will be at least about 10, 12, 14, 16, 18, 20 or 25 nucleotides in length. In some embodiments, longer probes of at least about 30, 40, 50, 60, 70, 80, 90 or 100 nucleotides are desirable, and probes longer than about 100 nucleotides may be suitable in some embodiments. The probes may comprise full length sequences encoding functional proteins. The nucleic acid probes are made or obtained using methods known to skilled artisans, e.g., in vitro synthesis from nucleotides, isolation and purification from natural sources, or enzymatic cleavage of the polynucleotides of the invention.
Hybridization complexes comprising nucleic acid probes hybridized to a polynucleotide of the invention may be detected by a variety of methods known in the art. In certain embodiments of the invention, immobilized nucleic acid probes may be used for the rapid and specific detection of polynucleotides and their expression patterns. Typically, a nucleic acid probe is linked to a solid support and a target polynucleotide (e.g., a gene, a transcription product, an amplicon, or, most commonly, an amplified mixture) is hybridized to the probe. Either the probe, or the target, or both, can be labeled, typically with a fluorophore or other tag, such as streptavidin. Where the target is labeled, hybridization may be detected by detecting bound fluorescence. Where the probe is labeled, hybridization is typically detected by quenching of the label. Where both the probe and the target are labeled, detection of hybridization is typically performed by monitoring a color shift resulting from proximity of the two bound labels. A variety of labeling strategies, labels, and the like, particularly for fluorescent based applications, are known in the art.
In another embodiment, the probes comprise polypeptide binding agents that specifically bind to polypeptides produced by expression of one or more of the polypeptides listed herein, or fragments thereof. Such protein binding probes may be prepared using the sequence information available for any of the proteins identified in Table 6 and Table 10, or fragments thereof.
Assay techniques that can be used to determine levels of a protein in a sample are also well known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western blot analysis and ELISA assays. In the assay methods utilizing antibodies, both polyclonal and monoclonal antibodies are suitable for use in the invention. Such antibodies may be immunologically specific for a particular protein, or an epitope of the protein, or a protein fragment, as would be well understood by those of skill in the art. Methods of making polyclonal and monoclonal antibodies immunologically specific for a protein or peptide are also well known in the art.
Preferred embodiments of the invention may utilize antibodies for the detection and quantification of proteins produced by expression of the genes described herein. Though proteins may be detected by immunoprecipitation, affinity separation, Western blot analysis and the like, a preferred method utilizes ELISA-type methodology wherein the antibody is immobilized on a solid support and a target protein or peptide is exposed to the immobilized antibody. Either the probe, or the target, or both, can be labeled. A variety of labeling strategies, labels, and the like, are known in the art.
In another aspect, the invention provides a device comprising a solid support to which is affixed an array comprising a plurality of probes for detecting differential gene expression in animals exhibiting a lean phenotype resulting from one or more lean phenotype-promoting treatments comprising (1) administration of CLA, (2) consumption of a high protein diet, and (3) increased exercise. In particularly preferred embodiments of the invention, expression patterns or profiles of a plurality of genes differentially expressed in lean versus normal phenotypes as defined herein, are observed utilizing arrays of probes for detecting target polynucleotides or proteins. The device may be used to detect differential expression of genes encoding the gene products set forth in Table 6 or table 10, or in subsets thereof, namely tissue-specific subsets as listed in Table 6A-G and Table 10A-G, or functional subsets as set forth in Tables 7, 8 and 9 (for three-treatment analysis) and Tables 11, 12 and 13 (for high protein diet analysis). In a preferred embodiment, the device is uses to detect differential expression of genes from canines or felines.
In one embodiment, arrays of oligonucleotide or polynucleotide probes may be utilized, whereas another embodiment may utilize arrays of antibodies or other proteins that bind specifically to the differentially expressed gene products. Such arrays may be custom made according to known methods, such as, for example, in-situ synthesis on a solid support or attachment of pre-synthesized probes to a solid support via micro-printing techniques. In preferred embodiments, arrays of nucleic acid or protein-binding probes are custom made to specifically detect transcripts or proteins produced by two or more of the differentially expressed genes or gene fragments described herein.
In a further aspect, the invention provides a method for detecting differential expression of one or more genes differentially expressed in animals exhibiting a lean phenotype resulting from one or more lean phenotype-promoting treatments comprising (1) administration of CLA, (2) consumption of a high protein diet, and (3) increased exercise, as compared with normal or untreated animals. The method generally comprises: (a) providing probes comprising (i) polynucleotides that specifically hybridize to two or more genes encoding proteins listed in Table 6 or Table 10, or fragments thereof; or (ii) polypeptide binding agents that specifically bind to two or more polypeptides selected from proteins listed in Table 6 or Table 10, or fragments thereof, (b) adding the probes to a sample comprising mRNA or proteins from an animal exhibiting the lean phenotype, in a manner enabling hybridization or binding of the probes to the mRNA or proteins in the sample, thereby forming hybridization or binding complexes in the sample (c) optionally, adding the probes to another sample comprising mRNA or proteins from a normal animal, in a manner enabling hybridization or binding of the probes to the mRNA or proteins in the second sample, thereby forming hybridization or binding complexes in the other sample; (d) detecting the hybridization complexes in the sample or samples; and (e) comparing the hybridization or binding complexes from the first sample with the hybridization or binding complexes from a standard or, optionally, from the other sample, wherein at least one difference between the amount of hybridization or binding in the sample as compared with the standard or the optional other sample indicates differential expression of the one or more genes differentially expressed in animals exhibiting the lean phenotype, as compared to animals that do not exhibit the phenotype.
The method may be used to detect differential expression of genes encoding the gene products set forth in Table 6 or table 10, or in subsets thereof, namely tissue-specific subsets as listed in Table 6A-G and Table 10A-G, or functional subsets as set forth in Tables 7, 8 and 9 (for three-treatment analysis) and Tables 11, 12 and 13 (for high protein diet analysis). In a preferred embodiment, the device is uses to detect differential expression of genes from canines or felines. In particular embodiments, the probes are bound to a substrate, preferably in an array.
Step (c) and part of steps (d) and (e) are optional and are used if a relatively contemporaneous comparison of two or more test systems is to be conducted. However, in a preferred embodiment, the standard used for comparison is based upon data previously obtained using the method.
These probes are exposed to a sample to form hybridization or binding complexes that are detected and compared with those of a standard. The differences between the hybridization or binding complexes from the sample and standard indicate differential expression of polynucleotides and therefore genes differentially expressed in the lean phenotype versus normal phenotype in the sample. In a preferred embodiment, probes are made to specifically detect polynucleotides or fragments thereof produced by one or more of the genes or gene fragments identified by the invention. Methods for detecting hybridization complexes are known to skilled artisans.
In one embodiment, the method further comprises exposing the animal or sample to a test substance before performing the assay. Then, the comparison is indicative of whether the test substance altered the expression of genes differentially expressed in treated versus untreated animals.
The assays described herein for the detection of lean phenotype-associated transcription and translation products are useful in methods for identifying a lean phenotype in an animal, or the absence of a lean phenotype in the animal. Such methods may be useful for implementing, facilitating, or guiding a weight-loss regimen (especially for loss of fat mass, and particular for loss of fat while preserving or improving lean body mass) or physical fitness regimen, or for diagnostic purposes to identify animals at risk for obesity or obesity-associated health risks or diseases. Such methods comprise obtaining a sample of cells or tissue from an animal manifesting a lean phenotype as defined herein, or an overweight or obese animal. Such cells or tissues can include, without limitation, adipose, muscle, or liver tissues. The cell or tissue sample is then analyzed for modulated expression of one or more genes associated with a lean phenotype, via detection of mRNA or protein, or for a particular lean phenotype-associated gene expression profile using a gene- or protein-array as described herein. The results of the analysis will reveal whether the animal is manifesting a lean phenotype or transitioning to a lean phenotype. The methods can also provide information regarding the efficacy of an animal's weight loss or physical fitness regimen, or to monitor an animal's relative risk for obesity or obesity-associated health risks or diseases over time. In these situations, the method is carried out at intervals during the animal's weight loss or physical fitness regimen, or life generally, wherein a change in expression or pattern of expression of a target gene associated with a lean phenotype is indicative of the animal's progress, improvement, or continued risk.
In another aspect, the invention provides a method of determining if a test substance is likely to be useful in promoting a lean phenotype when administered to an animal. The method typically comprises (a) determining a first gene expression profile by measuring the transcription or translation products of two or more polynucleotides selected from genes encoding proteins listed in Table 6 or Table 10, or fragments thereof, in a test system in the absence of the test substance; (b) determining a second gene expression profile by measuring the transcription or translation products of two or more polynucleotides selected from genes encoding proteins listed in Table 6 or Table 10, or fragments thereof, in a test system in the presence of the test substance; and (c) comparing the first gene expression profile with the second gene expression profile, wherein a change in the second gene expression profile as compared with the first gene expression profile indicates that the test substance is likely to be useful in promoting a lean phenotype when administered to an animal.
In certain embodiments, the method may further include the step of comparing at least the second gene expression profile with a reference or standard gene expression profile obtained by measuring the transcription or translation products of two or more polynucleotides selected from genes encoding proteins listed in Table 6 or Table 10, or fragments thereof, in a test system in the presence of a reference substance known to promote a lean phenotype when administered to animals. Such a substance may be, for example, CLA.
In one embodiment, the test system comprises a population of cultured cells. A nucleic acid construct comprising a lean phenotype-associated gene according to the invention is introduced into cultured host cells. The host cells can be mammalian cell lines, such as but are not limited, to NIH3T3, CHO, HFLA, and COS, although non-mammalian cells such as yeast, bacteria and insect cells can also be used. The coding sequences of the genes are operably linked to appropriate regulatory expression elements suitable for the particular host cell to be utilized. The nucleic acid constructs can be introduced into the host cells according to any acceptable means in the art, including but not limited to, transfection, transformation, calcium phosphate precipitation, electroporation and lipofection. Such techniques are well known and routine in the art. Transformed cells can be also used to identify compounds that modulate expression of the lean phenotype-associated genes.
Gene expression assays can be carried out using a gene construct comprising the promoter of a selected lean phenotype-associated gene operably linked to a reporter gene. The reporter construct may be introduced into a suitable cultured cell, including, without limitation, the standard host cell lines described above, or cells freshly isolated from an animal such as adipose, muscle, or liver cells. The assay is performed by monitoring expression of the reporter gene in the presence or absence of a test substance such as a test compound.
In a preferred embodiment, the test system comprises animals. Typically, a test substance is administered to an animal and the gene expression profile of the animal is analyzed to determine the effect of the test substance on transcription or the translation of the genes or gene products of the invention. Gene expression can be analyzed in situ or ex vivo to determine the effect of the test substance. In another embodiment, a test substance is administered to an animal and the activity of a protein expressed from a gene is analyzed in situ or ex vivo according to any means suitable in the art to determine the effect of the test substance on the activity of the proteins of interest. In addition, where a test substance is administered to an animal, the physiological, systemic, and physical effects of the compound, as well as potential toxicity of the compound can also be evaluated. Test substances can be administered to the animals for periods appropriate for the test substance, the animal, and the objective, including long term administration, administration on a regular basis, and on an extended regular basis. Administration can be via any suitable route, including, but are not limited to, oral, rectal, nasal, topical, intradermal, subcutaneous, intravenous, intramuscular, and intraparenteral modes of administration. Oral administration is preferred, most preferably oral administration as a food component.
Test substances can be any substance that may have an effect on polynucleotides or genes differentially expressed in animals exhibiting a lean phenotype. Suitable test substances include, but are not limited to, amino acids; proteins, peptides, polypeptides, nucleic acids, oligonucleotides, polynucleotides, small molecules, macromolecules, vitamins, minerals, simple sugars; complex sugars; polysaccharides; carbohydrates; medium-chain triglycerides (MCTs); triacylglycerides (TAGS); n-3 (omega-3) fatty acids including DHA, EPA, ALA; n-6 (omega-6) fatty acids including LA, γ-linolenic acid (GLA) and ARA; SA, conjugated linoleic acid (CLA); choline sources such as lecithin; fat-soluble vitamins including vitamin A and precursors thereof such as carotenoids (e.g., (β-carotene), vitamin D sources such as vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol), vitamin E sources such as tocopherols (e.g., α-tocopherol) and tocotrienols, and vitamin K sources such as vitamin K1 (phylloquinone) and vitamin K2 (menadione); water-soluble vitamins including B vitamins such as riboflavin, niacin (including nicotinamide and nicotinic acid), pyridoxine, pantothenic acid, folic acid, biotin and cobalamin; and vitamin C (ascorbic acid); antioxidants, including some of the vitamins listed above, especially vitamins E and C; also bioflavonoids such as catechin, quercetin and theaflavin; quinones such as ubiquinone; carotenoids such as lycopene and lycoxanthin; resveratrol; and α-lipoic acid; L-carnitine; D-limonene; glucosamine; S-adenosylmethionine; and chitosan. In a preferred embodiment, test substances are nutrients that may be added to food or consumed as a supplement. Substances identified by the foregoing method are also contemplated as part of the invention.
In a further aspect, the invention provides a computer system comprising a database containing information identifying expression levels of one or more polynucleotides that are differentially expressed in animals administered a substance that affects one or more of food intake, satiety, lipid metabolism, and fat utilization, wherein the polynucleotides are selected from genes encoding proteins listed in Table 6 or Table 10, or fragments thereof, and a user interface that enables a user to access or manipulate the information in the database. The system comprises a database containing information identifying the expression level of one or more polynucleotides selected from genes encoding proteins listed in Table 6 or Table 10 and/or polypeptides that specifically bind to the proteins listed in Table 6 or Table 10, and a user interface to interact with the database, particularly to input, manipulate, and review the information for different animals or categories of animals. In one embodiment, the database further contains information identifying the activity level of one or more polypeptides listed in Table 6 or Table 10. In another, the database further comprises sequence information for one or more of the polynucleotides or polypeptides as listed in Table 6 or Table 10, preferably from a variety of species. In other embodiments, the database contains additional information pertaining to the description of the genes in one or more animal species. The computer system is any electronic device capable of containing and manipulating the data and interacting with a user, e.g., a typical computer or an analytical instrument designed to facilitate using the invention and outputting the results relating to the status of an animal.
In another aspect, the invention provides kits comprising a container containing a collection of two or more probes for detecting differential gene expression of the lean phenotype resulting from one or more lean phenotype-promoting treatments comprising (1) administration of CLA, (2) consumption of a high protein diet, and (3) increased exercise. The kits comprise in separate containers in a single package or in separate containers in a virtual package, as appropriate for the use and kit component, two or more probes for detecting differential gene expression in animals exhibiting a lean phenotype resulting from one or more lean phenotype-promoting treatments comprising (1) administration of CLA, (2) consumption of a high protein diet, and (3) increased exercise, wherein the probes comprise: (a) polynucleotides that specifically hybridize to two or more genes encoding proteins listed in Table 6 or Table 10, or fragments thereof, or (b) polypeptide binding agents that specifically bind to two or more polypeptides selected from proteins listed in Table 6 or Table 10, or fragments thereof; and further comprises at least one of (1) instructions for how to use the probes in a gene expression assay for detecting differential gene expression in animals exhibiting a lean phenotype resulting from one or more lean phenotype-promoting treatments comprising administration of CLA, consumption of a high protein diet, and/or increased exercise, (2) reagents and equipment for using the probes, and (3) a composition known to induce the lean phenotype. Preferably, the probes are affixed to a solid support at known locations. Suitable reference substances known to induce the lean phenotype include CLA, for example.
When the kit comprises a virtual package, the kit is limited to instructions in a virtual environment in combination with one or more physical kit components. In one embodiment, the kit contains probes and/or other physical components and the instructions for using the probes and other components are available via the internes. The kit may contain additional items such as a device for mixing samples, probes, and reagents and device for using the kit, e.g., test tubes or mixing utensils.
In another aspect, the invention provides a means for communicating information about or instructions for one or more of the compositions and methods described herein. Such means typically comprise documents, digital storage media, optical storage media, audio presentations, visual displays or the like, containing the information or instructions. For example, the communication means may be a displayed web site, a kiosk, brochure, product label, package insert, advertisement, handout, public announcement audiotape, videotape, DVD, CD, computer-readable chip, computer-readable card, computer-readable disk, computer memory, or any combination thereof. Useful information includes one or more of (1) methods for promoting the health and wellness of animals and (2) contact information for the animal's caregivers to use if they have a question about the invention and its use. Useful instructions include techniques for using the probes, instructions for performing a gene expression assay, and administration amounts and frequency for the substances. The communication means is useful for instructing on the benefits of using the invention.
Various aspects of the invention can be further illustrated by the following examples. It will be understood that these examples are provided merely for purposes of illustration and do not limit the scope of the invention disclosed herein unless otherwise specifically indicated.
The experimental results set forth in this example demonstrate that animals can be induced to transition to a lean phenotype in three ways: (1) dietary supplementation with CLA, (2) high protein diet, and (3) increased exercise.
Four-week old male Sprague Dawley rats were allowed to acclimate for two weeks on a control diet comprising AIN-93M (American Institute of Nutrition purified diet formula for maintenance of mature rodents). Rats were divided into four groups of 12. Group 1 was fed the control diet supplemented with CLA (Table 1). Group 2 was fed a modified diet increased in protein and decreased in carbohydrate (Table 1). Group 3 was fed the control diet, and was given the opportunity to engage in supplemental exercise. Specifically, a running wheel was placed in the cage of each rat in group 3, and daily use of the wheel was monitored by recording each turn of the wheel using a sensor connected to a computer. Group 4 was fed the control diet (Table 1). The energy content of the control and test diets is shown in Table 2.
Measurements were taken at 7 and 60 days after initiation of the feeding or exercise protocols. At the completion of the 60-day feeding protocol, all animals were sacrificed and analyses were performed.
Body composition analyses were performed on the test animals. Compared with the control group, none of the test regimens (CLA supplementation, exercise or high dietary protein) had any substantial effect on (1) food intake or final body weight, (2) stripped carcass weight, (3) total organ weight, digestive tract weight, heart weight, kidney weight, liver weight, lung weight or spleen weight (with the exception of the high protein regimen, which resulted in increased kidney and spleen weight), (4) protein and fat content of the carcass, or (5) protein and fat content of the organs (except that high protein regimen increased the protein content and decreased the fat content of the organs).
By comparison, as can be seen from Table 3, each of the three treatments decreased total fat pad weight and retroperitoneal fat pad weight, as compared with the Control group. Thus, the data showed that CLA supplementation, increased exercise or high protein diet reduced body fat, resulting in the lean body mass (LBM) phenotype.
Biochemical analyses of blood were performed on the test animals; results are shown in Table 4.
After seven days of the respective treatments, it was found that the increased exercise treatment reduced blood insulin as compared with the CLA supplementation and the high protein diet. The high protein diet treatment increased blood glucagon level compared with the control, the CLA supplementation and the exercise treatment. Blood glucose was not affected by any treatment after 7 days. After 60 days of the respective treatments, it was found that the CLA supplementation and the high protein diet reduced blood glucose level compared with the control diet and the exercise treatment. The CLA supplementation and high protein diet also reduced blood insulin compared with the control diet and the exercise treatment. Blood glucagon was highest in the high protein diet group as compared with the other groups. These data show that CLA supplementation, exercise or the high protein diet altered the test animals' biochemical profiles in a manner consistent with the LBM phenotype.
This example sets forth the initial analysis of gene expression profiles in liver, muscle and/or adipose of the control and test animals comprising three treatments that promote a LBM phenotype, as described in the previous example at day 60.
Messenger RNA was prepared in accordance with standard techniques applicable to the various tissues. Affymetrix GeneChip® Rat Genome 230 2.0 Arrays were interrogated with mRNA from adipose (subcutaneous), liver and quadricep muscle from 6 rats from each of the four groups: (1) control, (2) CLA supplementation, (3) high protein diet, and (4) increased exercise regimen (total 72 samples, utilizing 72 GeneChip® arrays).
The RMA (Robust Multi-chip Analysis) procedure was used to normalize; correlation matrix and PCA were used to assess the quality control. Two-way Analysis of Variance (ANOVA) for repeated measures was used for a first step analysis (gene by gene). Then as a second step, the GEA (Global Error Assessment) procedure was applied, the robust statistic then replaced the error term of the ANOVA calculated in the first step. Genes were selected based on p<0.01 for overall treatment effect within tissue and then p<0.01 for a specific treatment versus control within that tissue.
The aforementioned processes identified several hundred genes that were differentially expressed between the control group and at least one of the three treatments, in at least one of the three tissues that were analyzed. Table 5 below shows a breakdown of results from an analysis of (1) the high protein diet treatment alone and (2) a combination of all three treatments, i.e., genes found to be differentially expressed in each of the three treatments as compared with the control group.
This example sets forth the further analysis of gene expression profiles in liver, muscle and/or adipose of the control and test animals comprising three treatments that promote a LBM phenotype, as described in Example 1 at day 60 and initially analyzed in Example 2.
Table 6 sets forth database identifiers and the names of the genes found to be differentially expressed in each of the three treatments.
Biological pathway analysis was conducted using commercially available mapping programs and public databases. The major pathways represented by the differentially expressed genes in adipose, liver, and muscle are set forth below in Tables 7, 8 and 9. As stated above, these genes were found to be differentially expressed as a result any one of the three treatments.
laevis)
laevis)
This example sets forth the further analysis of gene expression profiles in liver, muscle and/or adipose of the control and test animals comprising the high protein treatment described in Example 1 at day 60 and initially analyzed in Example 2.
Table 10 sets forth database identifiers and the names of the genes found to be differentially expressed in the high protein treatment.
laevis) (predicted)
norvegicus]
musculus]
norvegicus]
musculus]
norvegicus]
norvegicus]
gallus]
norvegicus]
norvegicus]
norvegicus]
norvegicus]
norvegicus]
musculus]
musculus]
musculus]
norvegicus]
laevis)
S. pombe) (S. cerevisiae)
norvegicus]
musculus]
norvegicus]
norvegicus]
musculus]
norvegicus]
Biological pathway analysis was conducted using commercially available mapping programs and public databases. The major pathways represented by the differentially expressed genes in adipose, liver, and muscle are set forth below in Tables 11, 12 and 13. As stated above, these genes were found to be differentially expressed as a result of the high protein diet treatment.
y
b (predicted)
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18653
)
indicates data missing or illegible when filed
The specification has disclosed typical preferred embodiments of the invention. Although specific terms are employed, they are used in a generic and descriptive sense only and not for purposes of limitation, the scope of the invention being set forth in the claims. Clearly, many modifications and variations of the invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims the invention may be practiced otherwise than as specifically described.
This application is a continuation of U.S. patent application Ser. No. 12/737,745 filed on Mar. 18, 2011, which is the National Stage of International Application No. PCT/US2009/04582 filed on Aug. 10, 2009, which claims the benefit of U.S. Provisional Application Ser. No. 61/190,369 filed Aug. 28, 2008, the disclosure of which is incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 61190369 | Aug 2008 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12737745 | Mar 2011 | US |
| Child | 14314088 | US |
