WO2004064714A2 - Modele - Google Patents

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WO2004064714A2
WO2004064714A2 PCT/GB2004/000168 GB2004000168W WO2004064714A2 WO 2004064714 A2 WO2004064714 A2 WO 2004064714A2 GB 2004000168 W GB2004000168 W GB 2004000168W WO 2004064714 A2 WO2004064714 A2 WO 2004064714A2
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obesity
rats
compound
slob
compounds
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PCT/GB2004/000168
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WO2004064714A3 (fr
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Iain Robinson
Paul Letissier
Randip Bains
Molly Strom
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Medical Research Council
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0362—Animal model for lipid/glucose metabolism, e.g. obesity, type-2 diabetes

Definitions

  • the invention relates to an animal model for visceral obesity.
  • the invention relates to an animal model for visceral obesity which displays normal levels of insulin sensitivity, allowing independent study of the factors that affect obesity and insulin sensitivity.
  • Obesity is closely linked to insulin resistance and the metabolic syndrome in human subjects and in many animal models of obesity.
  • tubby tub
  • agouti NM2
  • MCH methylcholine
  • CART peptides melanocortin-4 ligands, uncoupling proteins (UCPl-3), carboxypeptidase E, NPY, their related transcripts or homologs or their receptors (Coleman et al, 1990; Miller et al, 1993; Good et al, 1997; Klebig et al,
  • transgenic rats using a cosmid (cVO14) of rat DNA containing the rat vasopressin (ANP) and rat oxytocin (OXT) genes (Ivell & Richter, 1984), into which reporter genes were inserted, namely human growth hormone (hGH) in the ANP gene and bovine OXT mostly replacing the rat OXT gene.
  • hGH human growth hormone
  • transgene construct contained larger amounts of flanking sequences, which when analyzed, lead us to discover a novel gene termed 5'OT-EST, present in normal rat D ⁇ A, and present in a mutated form in the cosmid used to generate these rats.
  • Rats bearing multiple copies of this cosmid as a concatamer integrant exhibited an unexpected and novel late onset obesity and infertility dominant phenotype that would not be predicted from the known D ⁇ A sequences present in this cosmid.
  • Rats from this transgenic line were termed SLOB rats (for Severe Late-onset OBesity; see International Patent Application WO00/09686).
  • the obesity phenotype included (i) a late onset, (ii) a highly selective visceral distribution of fat developing on a normal rodent diet (containing 4% fat) without hyperphagia (iii) an effect greatly preponderant in males, and (iv) a dominant pattern of inheritance.
  • a late onset a highly selective visceral distribution of fat developing on a normal rodent diet (containing 4% fat) without hyperphagia
  • iii an effect greatly preponderant in males
  • iv a dominant pattern of inheritance.
  • severe infertility in males but normal fertility in females.
  • the presence or absence of the transgene insert, established before weaning by analysis of D ⁇ A samples, would predict which animals would develop obesity.
  • This obesity phenotype showed some parallels to several forms of human obesity, including that associated with human syndrome-X (Reaven et al, 1988) for which a late- onset increase in abdominally distributed fat, affecting males much more severely than females (Gray et al 1997) may be mimicked in the SLOB rat.
  • the similarity of the phenotype to several forms of late-onset obesity in humans suggested that the discoveries described therein may have beneficial utility in diagnosis, assessment of existing treatments or development of novel treatments for these conditions.
  • SLOB rats show a metabolic phenotype which all differs from that seen in other models for obesity, such as those mentioned above.
  • SLOB rats show elevated plasma leptin concentrations, associated with excessive adiposity, but normal food intake and normal fasting insulin and blood glucose levels, unlike in the above models (e.g. Russell, 1993, 1994; Michaelis et al, 1995; Takiguchi et al, 1998).
  • SLOB rats In common with the rats reported by Ikeda et al (1994), SLOB rats also show reduced rat GH production and secretion. GH deficiency is associated with increased visceral fat in humans, but this can be alleviated by hGH treatment. However, isolated rat GH deficiency is an unlikely cause of obesity in SLOB rats since other severely GH-deficient dwarf rats (Charlton et al, 1988) do not develop obesity when housed under identical conditions to SLOB rats. Obesity can be induced in such dwarf rats (as in normal rats) 3 when placed on high fat diets for prolonged periods though females are more susceptible than males (Clark et al, 1996). Pituitary rat GH suppression is also seen in the non-obese JP59 rats of both sexes and in Tgr rats (Flavell et al, 1996) which do not develop obesity.
  • the most commonly used genetic models of obesity in the rat include the Zucker fa/fa rat, the Koletsky (f) obese rat, the JLA/cp diverent rat, and the OLETF rat, and their related sub strains (lida et al, 1996; Wu-peng et al, 1997; Takaya et al, 1996; Lee et al, 1997; Kahle et al, 1997. None of these show the male specificity, late onset or pattern of distribution of obesity seen in SLOB rats. Importantly, they all exhibit significant hyperglycaemia and insulin resistance, which distinguishes them from SLOB rats.
  • SLOB obesity phenotype e.g. male specificity, central distribution of adiposity, late onset and severity, and associated morbidity
  • SLOB obesity phenotype e.g. male specificity, central distribution of adiposity, late onset and severity, and associated morbidity
  • SLOB obesity phenotype e.g. male specificity, central distribution of adiposity, late onset and severity, and associated morbidity
  • Other forms of central obesity which may be most severely expressed in human males, with or without reduced fertility and which are associated with increased morbidity.
  • Rodent models of obesity may be of value in testing the ability of pharmaceutical preparations of novel agents, to be beneficial in delaying or preventing the occurrence, development, course, severity, progression, or exacerbation of obesity or infertility (Mathe, 1995; Fan et al, 1997). This is of particular advantage where a genetic test can identify which young individuals are predisposed to become obese in later life (Whitaker et al 1997).
  • SLOB rats thus have particular beneficial utility as a novel animal model of late-onset human visceral obesity, preponderant in males, especially since the phenotype is predictable and non-transgenic littermates are ideal controls.
  • the hypertriglyceridemia and insulin resistance commonly observed in human obesity are clearly established risk factors for cardiovascular disease (Brewer, 1999).
  • High triglyceride levels are implicated in the progression of atherosclerosis via reductions in HDL cholesterol and elevations of atherogenic small-dense LDL, and may be directly linked to diabetic hyperglycemia and insulin resistance via Randle's cycle whereby fatty acids and glucose compete as an energy source in a variety of tissues.
  • Obesity and compromised glucose handling are so often associated that it is difficult to separate the mechanisms involved. For example, in a population of hyperglycaemic subjects of more than 20 years of age, at least 90% are > 10% overweight (Norman & Litwick, 1997).
  • the most commonly associated physical finding with maturity-onset diabetes is obesity, and when investigated, many obese individuals show some degree of insulin resistance, often exhibiting elevated fasting insulin levels with or without an inappropriately raised level of blood glucose.
  • SLOB rats are a candidate for such a model since they are not diabetic, and have normal fasting plasma glucose and insulin levels (WOOO/09686).
  • WOOO/09686 normal fasting plasma glucose and insulin levels
  • a method for identifying a candidate compound or compounds capable of modulating factors which influence obesity independently of insulin resistance comprising the steps of administering the compound or compounds to a transgenic non-human animal expressing, as a result of transgene expression, a 5'OT-EST polypeptide or mutant 5'OT-EST polypeptide.
  • SLOB animals which overexpress 5'OT-EST, display an obese phenotype without being insulin resistant.
  • compounds which influence obesity in the SLOB model do so by means which are not related to the modulation of insulin resistance.
  • the investigator can conclude, therefore, that compounds which successfully increase or reduce obesity in the SLOB model are compounds which influence obesity independently of insulin resistance.
  • the method according to the invention comprises the steps of: a) exposing a transgenic non-human animal expressing, as a result of transgene expression, a 5'OT-EST polypeptide or mutant 5'OT-EST polypeptide to the compound or compounds to be tested; b) determining the effect of the compound or compounds on the obesity phenotype; and c) selecting the compound or compounds which are capable of modulating the obesity phenotype in the desired manner; and d) classifying said selected compound or compounds as compound(s) which influence obesity independently of insulin resistance.
  • the obesity is visceral obesity, for example male-specific visceral obesity.
  • the method of the invention is thus useful for studying the mechanisms of obesity which develops when the animal is fed a low-fat diet, without the complications of changes in insulin resistance. Surprisingly, the separation of obesity and insulin sensitivity is maintained even when the animal is fed a high-fat diet. Surprisingly, despite the increased insulin sensitivity seen in SLOB animals in comparison with wild-type (non-obese) animals, the animals are even more responsive than wild-type animals to agents which modulate insulin sensitivity.
  • a candidate compound or compounds capable of modulating factors which influence insulin resistance independently of obesity comprising administering the compound or compounds to a transgenic non-human animal expressing, as a result of transgene expression, a 5'OT-EST polypeptide or mutant 5'OT-EST polypeptide.
  • the foregoing method comprises the steps of: a) exposing a transgenic non-human animal expressing, as a result of transgene expression, a 5'OT-EST polypeptide or mutant 5'OT-EST polypeptide to the compound or compounds to be tested; b) determining the effect of the compound or compounds on insulin sensitivity; and c) selecting the compound or compounds which are capable of modulating insulin sensitivity in the desired manner; and d) classifying said selected compound or compounds as compound(s) which influence insulin sensitivity independently of obesity.
  • the invention provides a method for identifying a compound or compounds which act differentially on insulin sensitivity and obesity, comprising the steps of: a) exposing a transgenic non-human animal expressing, as a result of transgene expression, a 5'OT-EST polypeptide or mutant 5'OT-EST polypeptide to the compound or compounds to be tested; b) determining the effect of the compound or compounds on insulin sensitivity; c) determining the effect of the compound or compounds on obesity; d) selecting the compound or compounds which are capable of modulating insulin sensitivity and/or obesity in the desired manner; and e) classifying said selected compound(s) according to their ability to influence insulin sensitivity, obesity or both insulin sensitivity and obesity.
  • SLOB rats have been shown to be more responsive to agents which modulate the absorption of dietary fat than wild-type animals.
  • a method for identifying a compound or compounds which modulate the absorption of dietary fat comprising the steps of: a) exposing a transgenic non-human animal expressing, as a result of transgene expression, a 5'OT-EST polypeptide or mutant 5'OT-EST polypeptide to the compound or compounds to be tested; b) determining the effect of the compound or compounds on obesity; and c) selecting the compound or compounds which are capable of modulating obesity in the desired manner.
  • SLOB rats have been shown to produce elevated levels of adiponectin before they become overtly obese and show a greater response to agents known to elevate plasma adiponectin and also increase insulin sensitivity, compared to wild-type animals.
  • a method for identifying a compound or compounds which modulate adiponectin levels and increase insulin sensitivity comprising the steps of: a) exposing a transgenic non-human animal expressing, as a result of transgene expression, a 5'OT-EST polypeptide or mutant 5'OT-EST polypeptide to the compound or compounds to be tested; b) determining the effect of the compound or compounds on adiponectin levels, insulin sensitivity and obesity c) selecting the compound or compounds which are capable of modulating adiponectin levels, insulin sensitivity and obesity.
  • Figure 1 Bar chart showing comparative weight gain of SLOB and non-transgenic male and female rats.
  • Figure 2 Bar chart showing weight gain in male and female SLOB and wild-type rats, specifically in the supra-renal and epididymal/ovarian fat pads at 15 months.
  • Figure 3 Bar chart showing leptin levels in normal and SLOB male and female rats at 15 months.
  • Figure 4 Graph showing insulin tolerance test in SLOB and normal rats after administration of insulin.
  • Figure 5 Graph showing insulin tolerance test in SLOB and normal rats after administration of glucose.
  • Figure 6 Bar chart showing weight gain in male and female SLOB and wild-type rats, specifically in the supra-renal and epididymal/ovarian fat pads in rats fed on low fat and high fat diets.
  • Figure 7 Bar chart showing basal plasma glucose levels after insulin administration in low fat and high fat fed SLOB and normal rats.
  • Figure 8 Graph of insulin tolerance test performed in normal and SLOB rats treated with Rosiglitazone, or untreated normal and SLOB rats.
  • Figure 9 Bar chart showing suprarenal and epididymal/ovarian fat pad weight in Rosiglitazone treated and untreated normal and SLOB rats.
  • Figure 10 Graph and bar chart showing weight gain of Orlistat treated and control normal and SLOB rats.
  • Figure 11 Bar chart showing food intake in Orlistat treated and untreated normal and SLOB rats.
  • Figure 12 Bar chart showing plasma adiponectin levels in Rosiglitazone treated and untreated normal and SLOB rats.
  • 5'OT-EST is the polypeptide represented in SEQ. ID. Nos. 2, 4 or 6 (rat, human and mouse respectively). Preferably, it is the human sequence. However, the term also covers alternative peptides homologous to 5'OT-EST, such as polypeptides derived from other species, including other mammalian species.
  • mutants of 5'OT-EST include polypeptides which differ only in minor, insignificant ways from wild-type 5'OT-EST, for example polypeptides having conservative amino acid replacements or additions or deletions. Preferred, however, are mutants which are able to confer, on animals expressing them, an obese phenotype as defined herein.
  • An example of such a mutant is the 5'OT-EST - xdel polypeptide set forth in SEQ. ID. No. 8. Further mutants may be obtained as described herein, and defined according to their functional effects in transgenic animals or host cells.
  • a "transgenic animal” is an animal whose genome has been functionally altered by genetic manipulation. In the context of the present invention, this includes animals bearing and expressing a 5 'OT-EST or mutant 5 'OT-EST transgene, animals from which 5 'OT-EST sequences have been deleted or in which they have been modified, and animals which are transiently transformed to express a (mutant) 5 'OT-EST transgene such as by transduction with viral sequences.
  • Transformation refers to the functional insertion of a gene by nucleic acid transfer, or the functional deletion of a gene, in a cell or organism. The term thus includes transfection, transduction and any other techniques useful for transferring nucleic acids into cells or organisms. Cells transfo ⁇ ned according to the invention express a novel genotype as a result of the transformation. For the avoidance of doubt, unless otherwise required by the specific context, reference herein to an entity in the singular includes the plural thereof. Thus, the expressions "a gene” and “one or more genes” are equivalent.
  • references to 5'OT-EST preferably include mutants of 5'OT-EST (5'OT-EST).
  • Fractors which influence obesity include behavioural, metabolic, endocrine and other genetic factors which result in the development of obesity.
  • Compound(s) identified in accordance with the invention may act, for example, to modify appetite, to modulate fat deposition in the body or change its distribution, as well as to alter metabolic function.
  • the compound or compounds identified according to the invention modulate obesity independently of insulin resistance.
  • any modulations in the levels of insulin sensitivity are 50% or less of the effects on obesity, expressed as percentage change in the level of insulin sensitivity or obesity itself. For example, therefore, if obesity measured as an increase in mass of the suprarenal fat pads increases by 25%, then insulin sensitivity decreases by no more that 12.5% as measured by ITT.
  • the effects on insulin sensitivity are 40%, 30%, 20%, 10% or less of the effects on obesity.
  • Modulating means increasing or decreasing in overall amount of a measurable quantity. Moreover, it may refer to a change in a property which is expressed as a local change such as a change in fat distribution which is not associated with an overall change in the amounts of fat in the animal. Similarly, a “change” in the obesity phenotype can be any change in the amount, type or distribution of fat in the animal, as further defined below.
  • a “cosmid” is a bacteriophage-based vector as commonly known in the art.
  • a “compound or compounds”, as referred to in connection with the methods according to the invention, may be any chemical entity, including inorganic ' compounds, organic compounds, nucleic acids, polypeptides, and the like. The term may include elements when subjected to testing.
  • references herein to "obesity" and obese animals are preferably references to the SLOB phenotype observed in SLOB rats according to the invention, characterised in being ter alia male-specific, late onset, with fat deposition concentrated in the abdominal area and associated with sterility.
  • Animals are "exposed" to a compound or compounds to be tested, in accordance with the invention, by administration of the compound or compounds.
  • the compound(s) can be administered topically, systemically, locally or otherwise, by injection, inhalation, ingestion, topical application of liquids, powders or creams etc., and the like.
  • the amounts of compounds to be administered to the animals can be determined empirically using methods known in the art and further described below.
  • animal models of obesity are used to test compounds for activity in modulating obesity itself, insulin resistance, plasma adiponectin levels or dietary fat absorption.
  • the animal models are preferably non- human mammalian models, and advantageously include rodent models. Rat models are especially preferred.
  • SLOB animals may be created de novo as described in international patent application WOOO/09686.
  • the SLOB phenotype may be also fransferred between animals by crossing, or other means.
  • Transgenic animals such as mice bearing 5 'OT-EST or mutants thereof or in which 5 'OT-EST has been disrupted, may be usefully intercrossed with other animal strains with defined mutations, or with undefined genetic backgrounds associated with propensity for the development of obesity.
  • Comparison of the resulting progeny with or without the 5 'OT-EST transgene will provide additional information on the alterations in occurrence, development, course, severity, progression, exacerbation, amelioration or cure of the obesity phenotype when expressed in these other genetic backgrounds, and analysed as described above. Such intercrossing may then be envisaged to enhance the utility of the resulting progeny exhibiting the obesity phenotype.
  • Examples of this use include (without being limited to) interbreeding with Zucker fa/fa rats (lida et al, 1996), diverent (cp) rats (Kahle et al, 1997), OLETF rats (Takiguchi et al, 1998), ZDF rats, tfm rats, spontaneously hypertensive or salt-sensitive rats (Michaelis et al, 1995) or other dwarf rats such as dw/dw (Charlton et al, 1988) or dr/dr rats (Takeuchi et al, 1991).
  • An example of the utility of this approach is given by (Michaelis et al, 1995).
  • a related example includes intercrossing mice carrying transgenes or deletions affecting 5 'OT-EST with other strains of mice in which genes already known to be involved in obesity or male fertility have been deleted by homologous recombination or introduced by transgenesis (singly or in combination).
  • Examples of these are already known to include (but are not limited to) leptin, tubby and related genes, NPY, insulin, GLP-1, IGF-1, IGF-II, MCH, CRH, POMC, CCK, orexins or hypocretins, CART peptides, agouti protein, as well as the genes or alternate products structurally related to or homologous with, the above peptides.
  • NPY receptors such as subtype 5
  • bombesin-receptor 3 such as subtype 5
  • uncoupling proteins such as UCPl-3, carboxypeptidase E, or PPARs or adrenergic receptor subtype 3 or TNF alpha or, all of which have been implicated in obesity.
  • the transfer of the SLOB phenotype onto these genetic backgrounds may also alter the occurrence, development, course, severity, progression, exacerbation, amelioration or cure of the specific phenotypes expressed in the strain with which SLOB animals are bred.
  • Such intercrossed lines for example with those genetic strains as outlined above and in which the SLOB phenotype is present in full or in a modified form, may also be useful for the applications to screen for anti-obesity agents as outlined above, and are considered within the scope of this application provided their particular utility is enhanced by the presence of the SLOB transgene or phenotype.
  • the exchange of SLOB rat genetic material may be achieved by methods other than conventional breeding, e.g. by administration of suitable vectors containing constructs expressing proteins of interest, or by transgenesis, or nuclear transfer techniques.
  • suitable vectors containing constructs expressing proteins of interest or by transgenesis, or nuclear transfer techniques.
  • Such examples include, but are not limited to, constructs containing the gene products or analogues of other genes already thought to be active in obesity, whose effects may be advantageously studied in SLOB rats due to the predictable development of their phenotype.
  • Such genes and their products include but are not limited to those examples specifically mentioned above.
  • Such derived animals in which the SLOB phenotype is present in full or in a modified form may also be useful to screen for anti-obesity agents as outlined above, and are considered within the scope of this application provided their particular utility is enhanced by the presence of the SLOB transgene or phenotype.
  • SLOB rats or animals from other strains bearing the SLOB transgene and exhibiting a specific late-onset visceral obesity may prove of particular value when used in a similar way to screen for the beneficial effects of reducing or eliminating other gene products by their silencing or elimination as described above using transgenesis, or homologous recombination, or by adenoviral delivery of antisense nucleotides. Examination of any alterations in the occurrence, development, course, severity, or progression of the SLOB phenotype in these genetic backgrounds would be of utility in identifying the role, if any, of such disrupted genes in the expression of the SLOB phenotype.
  • SLOB animals are useful for screening compounds for effects in modulating obesity.
  • SLOB rats, or animals of other strains bearing the SLOB transgene may be particularly useful in studies employing administration of natural or recombinant proteins, peptides or other agents or their derivatives already known or suspected to be involved in some forms of obesity (e.g. growth hormones, or reproductive hormones, steroid hormones, enzymes involved in their metabolism activation or inactivation, their homologs, analogs, antagonists, inhibitors or secretagogues, or leptin, its homologs, analogs and antagonists) or other natural or pharmacological agents already known to be active and/or of therapeutic value in these conditions (e.g.
  • compounds may be administered to SLOB rats, or animals of other strains bearing the SLOB transgene or their non-transgenic littermates, by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, or subcutaneous injection or infusion, or implant), nasal, pulmonary, rectal, sublingual, or topical routes of administration, and can be formulated in dosage forms appropriate for each route of administration e.g. in soluble form, or in suspension, or in other suitable pharmaceutical formulations.
  • parenteral e.g., intramuscular, intraperitoneal, intravenous, or subcutaneous injection or infusion, or implant
  • nasal, pulmonary, rectal, sublingual, or topical routes of administration e.g. in soluble form, or in suspension, or in other suitable pharmaceutical formulations.
  • the effects of such compounds on the SLOB phenotype may be assessed by carcass analysis, measurement of growth, body weight, body fat distribution, as well as other measures of analytes in body fluids or tissues relevant to obesity (Mathe, 1995; Shillabeer, 1992). These include, but are not limited to, cholesterol, triglycerides, fatty acids, lipoproteins, and other dietary constituents or metabolites, as well as metabolic hormones, such as leptin, insulin, glucagon, catecholamines or glucocorticoids. Other relevant parameters include cardiovascular measures (Reaven, 1988, Gray & Yudkin, 1997).
  • systolic or diastolic blood pressure may include measures of systolic or diastolic blood pressure, cardiac output, or vascular resistance, together with morphological changes to organ systems known to be affected by cardiovascular or obesity disorders, including, but not limited to, the heart, major or minor blood vessels, their muscle or endothelial layers, and their elasticity or fragility. See for example McNamee et al (1994).
  • Compounds identified as effective in such screening or analysis based on the use of SLOB rats, or animals of other strains bearing the SLOB transgene, would be predicted to be particularly useful in treatment of late-onset visceral obesity, in particular where they occur in combination, and disorders related to these conditions with a view to delaying or preventing the occurrence, development, course, severity or progression of the phenotype, avoiding its exacerbation, and preferably promoting its amelioration or cure in animals of commercial importance, or more preferably in humans.
  • compounds having converse but also therapeutically valuable activities may be developed based on screening or analysis as above in SLOB rats, or animals of other strains bearing the SLOB transgene, but which are intended to promote the occurence, development, or progression of increased fat deposition or increased calorie intake or decreased energy consumption,
  • disorders in humans include, but are not limited to, wasting, or anorexia, or cachexia, associated with prolonged illness, or malabsorptive states or catabolic states associated with other diseases, such as, but not limited to, inflammatory conditions, Crohns disease, or AIDS wasting, or burns, or cancer, or bone disease.
  • the development and late-onset of obesity in SLOB rats, or animals of other strains bearing the SLOB transgene is particularly useful in studying the chronic effects of novel food additives or formulations designed to prevent or exacerbate the deposition of fat in animals of commercial importance, of destined for use in human food products or dietary aids.
  • novel food additives or formulations designed to prevent or exacerbate the deposition of fat in animals of commercial importance, of destined for use in human food products or dietary aids.
  • Such compounds may be administered as above and their effects on the development, course, severity, progression, exacerbation, amelioration or cure of the SLOB obesity phenotype assessed as described above.
  • Additives or formulations shown to reduce the development of visceral obesity in this model may have utility in human food products or dietary aids or find beneficial medicinal use in reducing fat absorption.
  • SLOB rats, or animals of other strains bearing the SLOB transgene, and/or their nontransgenic littermates may prove useful in experiments designed to identify obesity-related differences in gene expression, RNA transcripts, proteins, or other biochemical measures, such as, but not limited to lipids, peptides, carbohydrates, amino acids or compounds or precursors or metabolites thereof, or their distribution, in whole animals, or in samples of biological fluids taken from SLOB rats, or animals of other strains bearing the SLOB transgene.
  • These fluids may include, but are not limited to: serum, plasma, lymph fluid, syno ial fluid, follicular fluid, seminal fluid, amniotic fluid, milk, whole blood, urine, cerebrospinal fluid, saliva, sputum, tears, perspiration, mucus, tissue culture medium, tissue extracts, and cellular extracts.
  • tissue derived from such animals are preferably (but not limited to) endocrine tissues, such as pancreas, adrenals, or pituitary gland, adipose tissues from different locations, preferably but not limited to, inguinal, omental, perirenal, subcutaneous, mammary, periorbital or other regions, thermogenic fat, brown or white adipose tissue in other locations, areas of the CNS though to be involved in obesity, preferably but not limited to the hypothalamus, and other tissues, preferably, but not restricted to liver, gastrointestinal tract, gonads, heart, musculoskeletal system, immune system, kidney, connective tissue including skin, epithelial or endothelial tissues.
  • endocrine tissues such as pancreas, adrenals, or pituitary gland
  • adipose tissues from different locations preferably but not limited to, inguinal, omental, perirenal, subcutaneous, mammary, periorbital or other regions, thermo
  • SLOB rats The development of obesity itself in SLOB rats, or animals of other strains bearing the SLOB transgene would be predicted to induce secondary changes in other obesity related parameters and regulators.
  • These include, but are not limited to, blood pressure, pituitary hormones, sperm development, maturation, and/or motility, lipid mobilising enzymes or receptors, or agents controlling these.
  • the latter include, but are not limited to leptin and its receptors, melanocortin, NPY, catecholamines, adrenal or gonadal or pituitary hormones, gut hormones such as insulin and glucagon, growth hormone and other growth factors such as members of the GH and IGF-1 families, their binding proteins and receptors.
  • They may also include drugs of several classes that have be thought useful in obesity. Examples of such classes include agents affecting the serotonin system or the fat cell free fatty acid uptake or release or metabolism.
  • SLOB rats have now been shown to retain enhanced responsiveness to insulin sensitizers and to lipase inhibitors, and are of particular utility in screening for drugs affecting these parameters and distinguishing these effects from effects on obesity.
  • This example may also include the use of SLOB rats, or animals of other strains bearing the SLOB transgene, to study morphological alterations in any tissue or cells of the cardiovascular system, including but not limited to, the heart and major blood vessels, other blood vessels carrying either arterial or venous blood, and any or all cells comprising these tissues.
  • SLOB rats or animals of other strains bearing the SLOB transgene, develop obesity without diabetes or hypercortisolism, have been shown now to retain normal or even enhanced insulin sensitivity, and elevated adiponectin levels. These rats may thus prove particularly beneficial in studying the developmental changes in these secondary parameters induced by other means, in the development of hypertension or cardiovascular disease or hypercortisolism (Russell et al, 1993), all which are known to be associated with obesity in humans (Reaven, 1988), but separable from diabetes in SLOB rats, or animals of other strains bearing the SLOB transgene. Examples of such means includes (but is not limited to) variation in dietary components or quantity.
  • the added contribution to obesity of diabetes or insulin resistance may be separately studied by treatment of SLOB rats, or animals of other strains bearing the SLOB transgene with diabetogenic agents, such as GH or cortisol. Similar effects on cardiac output or peripheral resistance or blood pressure may be studied by adminstration of other agents including, but not limited to, angiotensin-converting enzyme inhibitors or cardiac glycosides, or beta adrenergic receptor 3 agonists or antagonists.
  • Morphological changes may also be seen in adipose tissues or cells, or the other tissues in the body containing fat, such as the liver and related cells, or the skeleton, or in other organs or tissues. Differences in these measures detected specifically in SLOB rats (or animals of other strains bearing the SLOB transgene) and their alteration by elimination, blockade, endogenous stimulation, or exogenous administration of anti-obesity or other agents affective in obesity or related disorders would provide novel approaches to evaluate, improve and perfect existing or novel therapeutic approaches to obesity in other animals of commercial importance, and more preferably, in humans. An obvious example is the ready source of adipocyte cells and products from specific fat depots that are differentially increased in SLOB rats, or animals of other strains bearing the SLOB transgene.
  • responses to agents affecting fat cell metabolism or fat storage or lipogenesis or lipolysis or lipid-lowering agents may be studied with particular advantage to discern effects on visceral or peripheral fat tissues and to seek differential effects on fat from different depots in SLOB rats. More preferably, these responses following treatment with insulin sensitizing agents, agents causing insulin resistance, or agents inhibiting fat absorption may be studied with particular utility in tissues from SLOB rats, or animals of other strains bearing the SLOB transgene.
  • the compound(s) tested in accordance with the invention may be administered according to known procedures to SLOB animals. Dosage periods may be adjusted to provide the optimum response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the observed effects on metabolism and obesity.
  • the compound(s) may be administered in a convenient manner such as by the oral, intravenous (where water soluble), intramuscular, subcutaneous, infranasal, infradermal or suppository routes or implanting (e.g. using slow release molecules).
  • the compound(s) may be required to be coated in a material to protect said ingredients from the action of enzymes, acids and other natural conditions which may inactivate said compound(s).
  • the compound(s) may be coated by, or administered with, a material to prevent its inactivation.
  • the compound(s) may be administered in an adjuvant, co-administered with enzyme inhibitors or in liposomes.
  • Adjuvant is used in its broadest sense and includes any immune stimulating compound such as interferon.
  • Adjuvants contemplated herein include resorcinols, non-ionic surfactants such as polyoxyethylene oleyl ether and n- hexadecyl polyethylene ether.
  • Enzyme inhibitors include pancreatic trypsin.
  • Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes.
  • the compound(s) may also be administered parenterally or intraperitoneally.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • the form are preferably sterile and fluid to the extent that easy syringability exists. It is advantageously stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene gloycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the compound(s) in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilisation.
  • dispersions are prepared by incorporating the sterilised active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.
  • the compound(s) When the compound(s) is suitably protected as described above, it may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsules, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet.
  • the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The amount of compound(s) in such therapeutically useful compositions in such that a suitable dosage will be obtained.
  • the tablets, troches, pills, capsules and the like may also contain the following: a binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring.
  • a binder such as gum tragacanth, acacia, corn starch or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, alginic acid and the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermin
  • any material may be present as coatings or to otherwise modify the physical form of the dosage unit.
  • tablets, pills, or capsules may be coated with shellac, sugar or both.
  • a syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the active compound may be incorporated into sustained-release preparations and formulations.
  • pharmaceutically acceptable carrier and/or diluent includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such as active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired.
  • compositions containing supplementary active ingredients are compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form.
  • dosages are determined by reference to the usual dose and manner of administration of the said ingredients.
  • Rat tail biopsies up to 1 cm, are taken from 10-14 day old rats and placed in 50mM Tris.HCl ⁇ H8, lOOmM EDTA, lOOmM NaCl ('tail mix').
  • Genomic DNA is prepared following a standard procedure (Hogan et al 1986) involving incubation with protemase K, RNase A, phenol extraction and precipitation with isopropanol.
  • Genomic DNA from other tissue such as liver may be prepared by the same method, though this requires additional homogenisation in a larger volume (typically 5ml) of tail mix using a Kinematica Polytron PT 3000 homogenizer prior to the preparation of DNA from a smaller aliquot of homogenate.
  • a polymerase chain reaction may be used to amplify fragments of DNA using 50 ⁇ l of a reaction mix which contains lOmM Tris, pH8.3, 20mM KCl, 0.2mM 5 dNTPs, 200nM primers, 50-250ng template DNA, 2.5units Amplitaq DNA polymerase and l-3mM MgCl 2 (the optimal conditions for each amplification is determined empirically). Conditions vary for each template target, but a typical amplification might be to place the reaction mix in a thermal cycler (MJ Research Inc.), denature for 2 minutes and then subject the reaction to 34 cycles of 94 °C for 1 minute, 58 °C for 1
  • the male preponderant visceral obesity phenotype in SLOB rats may be assessed by comparing relative weights of supra-renal and epididymal fat pads in males or ovarian fat pads in females, and may be reflected in leptin concentrations in blood, measured by radioiummunoassay (RIA). Accordingly, 12 male and 12 female Fi rats from the above breeding experiment were culled at 15 months of age (6 T and 6
  • Figure 3 (b) shows that plasma leptin levels are significantly elevated in both SD/SLOB male (p ⁇ 0.001) and female rats (p ⁇ 0.05), with levels in T males almost twice those of T females and four-fold higher than levels in either male or female normal NT animals.
  • the dominant SLOB visceral obesity phenotype described earlier in JP17 Wistar rats may be fransferred by breeding to another rat strain such as SD, and maintains its sexual dimorphism in expression in the transgenic, but not non- transgenic progeny.
  • a mild initJ-I -h retardation is also evident, as previously shown for JP17 Wistar SLOB rats.
  • Transferring the SLOB phenotype to other normal, spontaneous mutant or genetically modified rat strains could have utility where these strains may be more or less prone to develop other forms of obesity which differ in onset, extent or regional distribution, or mechanism of induction (e.g. hormonal or dietary).
  • the example shown here is SD rats which are highly sensitive to diet induced obesity without hyperphagia (Levin et al 1986).
  • Other examples include, but are not limited to, leptin- resistant Zucker rats, BB rats, AS rats, and Brown Norway rats.
  • the genetic interchange may be achieved by conventional breeding, but may also be transferred by other reproductive techniques examples of which include, but are not limited to, in vitro fertilization, embryo transfer or nuclear transfer, where nuclei, sperm and/or eggs from SLOB rats are used. Examples of methods for transgenesis in rats are given in Charreau et al 1996.
  • the specific visceral obesity phenotype is transferred onto other rat strains which may be more or less prone to conditions commonly associated with obesity, but which may differ in their mechanism of induction and/or timing of expression.
  • examples of such conditions include, but are not limited to, hypertension and/or lack of CD36 (several strains of stroke prone or spontaneously hypertensive (SHR) rats, or Dahl rats, or insulin resistant rats (e.g. Zucker diabetic fatty ZDF rats), or rat models of Syndrome X generated by several mechanisms, examples of which mclude, but are not limited to, maternal undernutrition, hormonal treatments, dietary manipulation or other genetic manipulations.
  • the utility of introducing the SLOB obesity phenotype into these rat strains is to exacerbate or ameliorate these conditions, and to be able to study and separately analyze their development and severity when expressed in combination with the SLOB phenotype on the same or other genetic backgrounds.
  • Procedure Food was removed from animals at 9 am on the morning of the procedure day, and at approximately 2 pm, rats were anaesthetised with Sagatal (Rhone Merieux Limited, Harlow, Essex) at a dose of 60mg/kg i.p. bodyweight and the left jugular vein was canulated with 20cm Esco silicone tubing with an internal diameter of 0.5mm (Bibby Sterilin Ltd., Stone, UK).
  • tail veins were also cannulated using polythene tubing (Portex) attached to a 0.6 x 0.25mm sterile needle (Sherwood Medical, West Wales.UK).
  • mice received 0.4U/kg bodyweight of insulin (Human Actrapid, Novo Nordisk), administered via the jugular vein cannula.
  • animals received 0.5g/kg bodyweight of D-glucose injected via the tail vein catheter.
  • Blood samples were withdrawn via the jugular cannula before, and at 5, 10, 15, 30, 45, 60 and 90 minutes after administration of glucose or insulin. 250 ⁇ l of blood was collected in tubes containing heparin and immediately centrifuged to separate plasma, which was then analysed for insulin and/or glucose as appropriate.
  • Plasma glucose concentrations were measured using a Beckman Glucose Analyser, plasma insulin concentrations were determined using a commercial RIA kit (Linco Research Inc., St. Charles, MO).
  • Initial blood glucose levels were the same, and both T and NT groups showed the same degree hypoglycaemia in response to insulin within the first 30 minutes.
  • the plasma glucose levels in the SLOB T group remained lower for much longer than in the NT normal group, being significantly lower at 45, 60 and 90 minutes, at which time they had not recovered to pre-inj ection values, whereas the NT group had achieved normoglycaemia at this time point.
  • ITTs were performed in another group of male SLOB (T) rats that had been pre-treated for 4 weeks with a subcutaneous infusion of recombinant human growth hormone (200 ⁇ g/d, Genotropin, Pharmacia AB, Milton Keynes, UK) delivered from 2-week osmotic minipumps (Alzet, Palo Alto, CA) inserted subcutaneously under brief fluo thane anaesthesia, and replaced after 2 weeks.
  • This dose of hGH is sufficient to stimulate growth and increase lean body mass in these animals, but does not reduce their visceral obesity (not shown).
  • the ITT results are shown in Figure 4 for comparison with the non-GH-treated SLOB rats.
  • GH treatment did not alter initial blood glucose levels, had no effect on the maximal hypoglycaemic response, and did not shorten the period of prolonged hypoglycaemia seen in SLOB rats, so that the blood glucose curve was entirely comparable to that of the untreated SLOB (T) animals ( Figure 4), and different from NT animals.
  • GTT glucose tolerance test
  • Procedure Groups of 6 SLOB (T) and normal (NT) rats were anaesthetised and cannulated as described above. After an initial blood sample they were then given an i.v injection of 0.5g/kg glucose and samples taken at different times thereafter, and these were taken for assay of glucose and insulin.
  • Figure 7 shows that ingestion of the 30% fat diet had no significant effect on basal glucose levels, nor did it affect the fall in glucose levels following insulin induced hypoglycaemia at 30minutes post injection in the T SLOB rats, whereas it significantly blunted the response at this time point in NT rats. Blood sugar levels recovered more rapidly in the T rats on 30% fat diet than on 4% fat diet, reaching normal values at 90 minutes, similar to those in both high fat and low fat fed NT groups ( Figure 7). Surprisingly, therefore, even when fed a high fat diet, obese SLOB (T) rats are more responsive than NT rats on a high fat diet and do not become insulin-resistant despite their worsening obesity on ingestion of 30% fat.
  • T obese SLOB
  • Thiazolidinediones enhance insulin action and lower blood glucose concentrations in obese, insulin-resistant animals and patients with glucose intolerance or non-insulin- dependent-diabetes (NIDDM) (Saltiel and Olefsky, 1996).
  • NIDDM non-insulin- dependent-diabetes
  • PPAR- ⁇ peroxisome-proliferator activator receptor- ⁇
  • NEFA non-esterified fatty acid
  • Rosiglitazone Rosiglitazone (Avandia®) which lowers glucose and lipid levels in patients with type 2 diabetes by activating the nuclear receptor PPAR- ⁇ (Spiegelman, 1998). See Komers & Vrana (1998) for a review of the role of thiazolininediones in investigating metabolic diseases including Syndrome X.
  • Rosiglitazone improves glucose tolerance, lowers hyperinsulinaemia and up-regulates insulin receptors in peripheral tissues (Fujiwara et al, 1998; Smith et al, 1993), preventing the progression from insulin resistance to overt diabetes in this model (Smith et al, 2000).
  • This drug may be beneficial in conditions of obesity and insulin resistance, but dissecting mechanisms governing the effectiveness of such drugs is difficult, since obesity commonly occurs in rodent models of insulin resistance, whilst insulin resistance commonly occurs in most models of obesity. It would be an advantage to be able to test such drugs in a rodent model of obesity in which such mechanisms could be clearly separated.
  • Rosiglitazone treatment has a dramatic effect in SLOB (T) rats, to greatly enhance their insulin sensitivity, and maintaining much lower blood glucose levels than in untreated SLOB rats in response to the same dose of insulin, the differences vs the untreated group being significant at all time points after 15 minutes.
  • SLOB rats show markedly increased responsiveness compared to normal rats when treated with an insulin sensitizer such as Rosiglitazone, and this occurred with no change in visceral or peripheral fat accumulation, as assessed by fat pad weights.
  • an insulin sensitizer such as Rosiglitazone
  • SLOB rats therefore, it is possible to maintain normal or even heightened responsiveness to insulin sensitizing agents, and to study these effects without any changes in fat accumulation.
  • the difference between visceral and peripheral fat depots persists in SLOB rats even on Rosiglitazone treatment.
  • One advantage is that SLOB rats are surprisingly sensitive to such agents.
  • SLOB rats may be used with advantage to screen agents in which the contributions of effects on insulin resistance and on obesity may be separately evaluated.
  • High dietary fat intakes are associated with obesity, coronary disease, and cancer. Consequently, an obvious approach to combat obesity is to reduce the availability of absorbable fat in the diet. This may be done by reducing overall calorie intake, or by reducing the fat composition of the diet. Another approach is to treat with drugs that reduce fat absorption by preventing the breakdown of fat and/or reducing its absorption from the gastrointestinal tract.
  • An example of a commonly used anti-obesity drug in this category is Orlistat (Xenical®), an inhibitor of gastric and pancreatic Upases, which is minimally absorbed after oral ingestion, but which has been and shown to reduce bodyweight by inhibiting absorption of ingested dietary fat by up to 30% (Hogan et al, 1987).
  • Orlistat treatment caused weight loss despite an increase in overall intake of food (Hogan et al, 1987).
  • female Sprague Dawley rats treated with Orlistat also reduced in bodyweight despite an increase in food intake (Ackroff et al., 1996). This latter study also showed that Orlistat caused a reduction in the effectiveness of dietary fat to cause obesity, but did not make the fat diet aversive to the rats.
  • SLOB rats do not require a high fat diet to develop obesity, which occurs in middle age in rats consuming 4% fat (previous patent)
  • the onset, magnitude and rate of development of obesity may be accelerated by increasing the fat content of the diet fed to SLOB rats. It was therefore not clear whether the SLOB rats would be more or less responsive than non-transgenic rats of the same strain, to treatment with agents such as Orlistat that reduce the availability of dietary fat for absorption. It would be an advantage to identify a rodent model of obesity which showed an enhanced responsiveness to such agents.
  • adiponectin has recently become a good candidate.
  • Administration of adiponectin increases insulin sensitivity (Yamauchi et al., 2001) whilst adiponectin- deficient mice become insulin resistant (Kubota et al, 2002); low plasma adiponectin levels are also associated with insulin resistance, Type 2 diabetes and obesity in humans (Hu et al., 1996).
  • the SLOB (T) rat thus provides a useful model to test the role of adiponectin in relation to the degree of insulin resistance in visceral obesity, since SLOB rats are unusual in maintaining insulin sensitivity despite severe visceral obesity.
  • adiponectin levels were significantly higher in young SLOB T males than in their NT littermates even before their obesity was apparent, suggesting that in this model, rising levels of this protein is an early predictive marker of visceral obesity, and may be one mechanism by which these animals are protected from the development of insulin resistance despite the large accumulation of visceral fat.
  • Data from the rosiglitazone experiment suggest that such a mechanism is not maximally operative in SLOB rats since their adiponectin levels could be increased further by rosiglitazone treatment, as has been reported in other studies (Yang et al., 2002).
  • Our data clearly show that adiponectin levels rise with insulin sensitivity, and are not decreased by adiposity. Plasma adiponectin levels were not different in the older non-obese SLOB T females, showing the sexually dimorphism in SLOB phenotype extends to plasma adiponectin also.
  • the SLOB rat may be uniquely useful for studying the mechanisms of obesity developing on a low fat diet without the complications of changes in insulin resistance. • This property is maintained even when SLOB rats are subjected to a high fat diet.
  • SLOB rats may have unique value in, screening the effectiveness of agents that affect obesity and insulin resistance differentially, and in separating the consequences of their mechanisms.
  • SLOB rats provide a highly sensitive screen for identifying drugs and their interaction with dietary components that otherwise induce obesity.
  • Hypertriglyceridemia Changes in the plasma lipoproteins associated with an increased risk of cardiovascular disease. Am. J. Cardiol. 83, 3F-12F.
  • Carboxypeptidase E is a regulated secretory pathway sorting receptor: genetic obliteration leads to endocrine disorders in Cpe(fat) mice.
  • Rosigliatazone (BRL49653), a PPAR-D -selective agonist, causes peroxisome proliferators-like liver effects in obese mice. Journal of Lipid Research , 40, 1177-1184.
  • Flavell DM. Wells, T., Wells, S.E., Carmignac, D.F., Thomas, G.B., Robinson, I.C.A.F. 1996 Dominant dwarfism in transgenic rats by targeting human growth hormone (GH) expression to hypothalamic GH-releasing factor neurons.
  • GH growth hormone
  • Hu, E., Liang, P., Spiegelman, B. M. 1996 AdipoQ is a novel adipose-specific gene dysregulated in obesity. J Biol Chem 271 (18), 10697-703. Huszar, D., Lynch, C. A., Fairchild, H. N., Dunmore, J. H., Fang, Q., Berkemeier, L. R, Gu, W., Kesterson, R. A., Boston, B. A., Cone, R. D., Smith, F. J., Campfield, L. A., Burn, P., and Lee, F. (1997). Targeted disruption of the melanocortin-4 receptor results in obesity in mice. Cell 88, 131-41.
  • Orexins and orexin receptors a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior. Cell 92: 573-585
  • BRL 49653 normalises glycaemic control in Zucker fa/fa rats by improving hepatic and peripheral tissue sensitivity to insulin. Diabetologia,

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Abstract

L'invention concerne une méthode d'identification d'un ou de plusieurs composés candidats qui peuvent moduler des facteurs agissant sur l'obésité, indépendamment de la résistance à l'insuline. La méthode consiste à administrer le(s) composé(s) de l'invention à un animal SLOB transgénique non humain exprimant, consécutivement à une expression transgénique, un polypeptide 5'OT-EST ou un polypeptide 5'OT-EST mutant.
PCT/GB2004/000168 2003-01-20 2004-01-20 Modele WO2004064714A2 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000009686A1 (fr) * 1998-08-12 2000-02-24 Medical Research Council Gene

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000009686A1 (fr) * 1998-08-12 2000-02-24 Medical Research Council Gene

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BAINS R K ET AL: "Transgenic SLOB rats showing Severe Late-onset male-specific visceral obesity" JOURNAL OF ENDOCRINOLOGY, vol. 164, no. Suppl., March 2000 (2000-03), page P201, XP008030956 & 19TH JOINT MEETING OF THE BRITISH ENDOCRINE SOCIETIES, WITH THE EUROPEAN FEDERATION OF ENDOCRINE SO; BIRMINGHAM, ENGLAND, UK; MARCH 13-16, 2000 ISSN: 0022-0795 *
KAHN B B ET AL: "Obesity and insulin resistance" JOURNAL OF CLINICAL INVESTIGATION, NEW YORK, NY, US, vol. 106, no. 4, August 2000 (2000-08), pages 473-481, XP002231945 ISSN: 0021-9738 *
WELLS SARA E ET AL: "Transgenesis and neuroendocrine physiology: A transgenic rat model expressing growth hormone in vasopressin neurones." JOURNAL OF PHYSIOLOGY (CAMBRIDGE), vol. 551, no. 1, 15 August 2003 (2003-08-15), pages 323-336, XP002281391 ISSN: 0022-3751 cited in the application *

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