METHODS OF MODULATING BCKDH

Abstract

An agent capable of increasing the activity of branched-chain alpha-keto acid dehydrogenase (BCKDH) for use in increasing leptin levels. The invention also relates to the use of such agents for supporting satiety and for supporting weight maintenance and/or treating or preventing obesity.

Claims

1. Method for increasing leptin levels comprising administering an agent capable of increasing the activity of branched-chain alpha-keto acid dehydrogenase (BCKDH) to an individual in need of same.

2. Method according to claim 1 for use in supporting satiety.

3. Method according to claim 1, for use in supporting weight maintenance and/or treating or preventing obesity.

4. Method according to claim 1, wherein the agent increases the activity of the BCKDH E1 B subunit.

5. Method according to claim 1, wherein the agent is administered to a subject during or after a weight loss intervention.

6. Method according to claim 1, wherein the agent increases the level of BCKDH in a subject.

7. Method according to claim 1, wherein the agent does not affect the activity of branched-chain alpha-keto acid dehydrogenase kinase (BCKDH kinase).

8. Method according to claim 1, wherein the agent is selected from the group consisting of resveratrol and valproic acid.

9. Method according to claim 1, wherein the agent decreases the activity of branched-chain alpha-keto acid dehydrogenase kinase (BCKDH kinase).

10. Method according to claim 9, wherein the agent decreases the level of BCKDH kinase.

11. Method according to claim 9, wherein the agent is selected from the group consisting of -chloroisocaproic acid and -ketoisocaproic acid (KIC).

12. A method of identifying an agent capable of supporting weight maintenance and/or treating or preventing obesity in a subject comprising the steps: (a) contacting a preparation comprising a branched-chain alpha-keto acid dehydrogenase (BCKDH) polypeptide or polynucleotide with a candidate agent; and (b) detecting whether the candidate agent affects the activity of the BCKDH polypeptide or polynucleotide.

13. The method of claim 12, wherein the BCKDH is the BCKDH E1 B subunit.

14. The method of claim 12, wherein the method comprises contacting the preparation comprising BCKDH with a candidate agent and measuring the conversion of NAD+ to NADH.

15. A method of identifying an agent capable of supporting weight maintenance and/or treating or preventing obesity in a subject comprising the steps: (a) contacting a preparation comprising a branched-chain alpha-keto acid dehydrogenase kinase (BCKDH kinase) polypeptide or polynucleotide with a candidate agent; and (b) detecting whether the candidate agent affects the activity of the BCKDH kinase polypeptide or polynucleotide.

16. The method of claim 15, wherein the method comprises contacting the preparation comprising BCKDH kinase with a candidate agent in the presence of ATP and measuring the incorporation of phosphate into a substrate or measuring the conversion of ATP to ADP.

Description

DESCRIPTION OF THE DRAWINGS

[0064] FIG. 1

[0065] A Manhattan plot zooming in on a region located in the regulatory region of the BCKDHB gene of chromosome 6.

[0066] FIG. 2

[0067] Box plots indicating that protein expression stratified based on trans-acting SNP genotype did not underline a strong difference of expression.

[0068] FIG. 3

[0069] Variables distribution for leucine, isoleucine and valine before (1) and after (2) low caloric diet (LCD) intervention.

DETAILED DESCRIPTION OF THE INVENTION

[0070] Various preferred features and embodiments of the present invention will now be described by way of non-limiting examples.

[0071] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, biochemistry, molecular biology, microbiology and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements) Current Protocols in Molecular Biology, Ch. 9, 13 and 16, John Wiley & Sons; Roe, B., Crabtree, J. and Kahn, A. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; Polak, J. M. and McGee, J. OD. (1990) In Situ Hybridization: Principles and Practice, Oxford University Press; Gait, M. J. (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and Lilley, D. M. and Dahlberg, J. E. (1992) Methods in Enzymology: DNA Structures Part A: Synthesis and Physical Analysis of DNA, Academic Press. Each of these general texts is herein incorporated by reference.

Branched-Chain Alpha-Keto Acid Dehydrogenase (BCKDH)

[0072] Branched-chain alpha-keto acid dehydrogenase (BCKDH) is a protein complex located on the mitochondrial inner membrane which plays a key role in the catabolism of the branched-chain amino acids (BCAAs) valine, leucine and isoleucine. In BCAA catabolism, the BCKDH complex catalyses the oxidative decarboxylation of the branched-chain alpha-keto acid, which is a rate limiting step in the overall catabolic pathway.

[0073] The BCKDH complex consists of three subunits: [0074] 1. E1 subunit (EC 1.2.4.4), which exhibits branched-chain alpha-keto acid decarboxylase activity. The E1 subunit is comprised of A and B chains (also known as a and 3, respectively), which are encoded by the BCKDHA and BCKDHB genes; [0075] 2. E2 subunit (EC 2.3.1.168), which exhibits lipoamide acyltransferase activity; and [0076] 3. E3 subunit (EC 1.8.1.4), which exhibits lipoamide dehydrogenase activity.

[0077] The E2 subunit acts as the core of the BCKDH complex and is found in either 24 copies in octahedral symmetry or in 60 copies in icosahedral symmetry. Subunits E1 and E3 bind to E2 via non-covalent bonds, each with multiple copies.

[0078] Mutations in the BCKDH complex, in particular mutations located in the E1 B subunit have been associated with many disorders in humans, including Maple Syrup Urine Disease (MSUP; varsson, A. et al. (2000) Structure 8: 277-291).

[0079] In one embodiment, the BCKDH is human BCKDH.

[0080] An example amino acid sequence of the BCKDH E1 B subunit is the sequence deposited under NCBI Accession No. NP 000047.1.

[0081] An example amino acid sequence of the BCKDH E1 B subunit is:

TABLE-US-00001 (SEQIDNO:1) MAVVAAAAGWLLRLRAAGAEGHWRRLPGAGLARGFLHPAATVEDAAQ RRQVAHFTFQPDPEPREYGQTQKMNLFQSVTSALDNSLAKDPTAVIF GEDVAFGGVFRCTVGLRDKYGKDRVFNTPLCEQGIVGFGIGIAVTGA TAIAEIQFADYIFPAFDQIVNEAAKYRYRSGDLFNCGSLTIRSPWGC VGHGALYHSQSPEAFFAHCPGIKVVIPRSPFQAKGLLLSCIEDKNPC IFFEPKILYRAAAEEVPIEPYNIPLSQAEVIQEGSDVTLVAWGTQVH VIREVASMAKEKLGVSCEVIDLRTIIPWDVDTICKSVIKTGRLLISH EAPLTGGFASEISSTVQEECFLNLEAPISRVCGYDTPFPHIFEPFYI PDKWKCYDALRKMINY

[0082] An example nucleotide sequence encoding the BCKDH E1 B subunit is the sequence deposited under NCBI Accession No. NM_000056.4.

[0083] An example nucleotide sequence encoding the BCKDH E1 B subunit is:

TABLE-US-00002 (SEQIDNO:2) ATGGCGGTTGTAGCGGCGGCTGCCGGCTGGCTACTCAGGCTCAGGGC GGCAGGGGCTGAGGGGCACTGGCGTCGGCTTCCTGGCGCGGGGCTGG CGCGGGGCTTTTTGCACCCCGCCGCGACTGTCGAGGATGCGGCCCAG AGGCGGCAGGTGGCTCATTTTACTTTCCAGCCAGATCCGGAGCCCCG GGAGTACGGGCAAACTCAGAAAATGAATCTTTTCCAGTCTGTAACAA GTGCCTTGGATAACTCATTGGCCAAAGATCCTACTGCAGTAATATTT GGTGAAGATGTTGCCTTTGGTGGAGTCTTTAGATGCACTGTTGGCTT GCGAGACAAATATGGAAAAGATAGAGTTTTTAATACCCCATTGTGTG AACAAGGAATTGTTGGATTTGGAATCGGAATTGCGGTCACTGGAGCT ACTGCCATTGCGGAAATTCAGTTTGCAGATTATATTTTCCCTGCATT TGATCAGATTGTTAATGAAGCTGCCAAGTATCGCTATCGCTCTGGGG ATCTTTTTAACTGTGGAAGCCTCACTATCCGGTCCCCTTGGGGCTGT GTTGGTCATGGGGCTCTCTATCATTCTCAGAGTCCTGAAGCATTTTT TGCCCATTGCCCAGGAATCAAGGTGGTTATACCCAGAAGCCCTTTCC AGGCCAAAGGACTTCTTTTGTCATGCATAGAGGATAAAAATCCTTGT ATATTTTTTGAACCTAAAATACTTTACAGGGCAGCAGCGGAAGAAGT CCCTATAGAACCATACAACATCCCACTGTCCCAGGCCGAAGTCATAC AGGAAGGGAGTGATGTTACTCTAGTTGCCTGGGGCACTCAGGTTCAT GTGATCCGAGAGGTAGCTTCCATGGCAAAAGAAAAGCTTGGAGTGTC TTGTGAAGTCATTGATCTGAGGACTATAATACCTTGGGATGTGGACA CAATTTGTAAGTCTGTGATCAAAACAGGGCGACTGCTAATCAGTCAC GAGGCTCCCTTGACAGGCGGCTTTGCATCGGAAATCAGCTCTACAGT TCAGGAGGAATGTTTCTTGAACCTAGAGGCTCCTATATCAAGAGTAT GTGGTTATGACACACCATTTCCTCACATTTTTGAACCATTCTACATC CCAGACAAATGGAAGTGTTATGATGCCCTTCGAAAAATGATCAACTA TTGA

[0084] In one embodiment, the BCKDH E1 B subunit comprises an amino acid sequence that has at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1, preferably wherein the amino acid sequence substantially retains the natural function of the protein represented by SEQ ID NO: 1.

[0085] In one embodiment, the BCKDH E1 B subunit-encoding nucleotide sequence comprises a nucleotide sequence that has at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 2, preferably wherein the protein encoded by the nucleotide sequence substantially retains the natural function of the protein represented by SEQ ID NO: 1.

[0086] In one embodiment, the BCKDH E1 B subunit-encoding nucleotide sequence comprises a nucleotide sequence that encodes an amino acid sequence that has at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1, preferably wherein the amino acid sequence substantially retains the natural function of the protein represented by SEQ ID NO: 1.

[0087] An example amino acid sequence of the BCKDH E1 A subunit is the sequence deposited under NCBI Accession No. NP 000700.1.

[0088] An example amino acid sequence of the BCKDH E1 A subunit is:

TABLE-US-00003 (SEQIDNO:3) MAVAIAAARVWRLNRGLSQAALLLLRQPGARGLARSHPPRQQQQFSS LDDKPQFPGASAEFIDKLEFIQPNVISGIPIYRVMDRQGQIINPSED PHLPKEKVLKLYKSMTLLNTMDRILYESQRQGRISFYMTNYGEEGTH VGSAAALDNTDLVFGQYREAGVLMYRDYPLELFMAQCYGNISDLGKG RQMPVHYGCKERHFVTISSPLATQIPQAVGAAYAAKRANANRVVICY FGEGAASEGDAHAGFNFAATLECPIIFFCRNNGYAISTPTSEQYRGD GIAARGPGYGIMSIRVDGNDVFAVYNATKEARRRAVAENQPFLIEAM TYRIGHHSTSDDSSAYRSVDEVNYWDKQDHPISRLRHYLLSQGWWDE EQEKAWRKQSRRKVMEAFEQAERKPKPNPNLLFSDVYQEMPAQLRKQ QESLARHLQTYGEHYPLDHFDK

[0089] An example nucleotide sequence encoding the BCKDH E1 A subunit is the sequence deposited under NCBI Accession No. NM_000709.3.

[0090] An example nucleotide sequence encoding the BCKDH E1 A subunit is:

TABLE-US-00004 (SEQIDNO:4) ATGGCGGTAGCGATCGCTGCAGCGAGGGTCTGGCGGCTAAACCGTGG TTTGAGCCAGGCTGCCCTCCTGCTGCTGCGGCAGCCTGGGGCTCGGG GACTGGCTAGATCTCACCCCCCCAGGCAGCAGCAGCAGTTTTCATCT CTGGATGACAAGCCCCAGTTCCCAGGGGCCTCGGCGGAGTTTATAGA TAAGTTGGAATTCATCCAGCCCAACGTCATCTCTGGAATCCCCATCT ACCGCGTCATGGACCGGCAAGGCCAGATCATCAACCCCAGCGAGGAC CCCCACCTGCCGAAGGAGAAGGTGCTGAAGCTCTACAAGAGCATGAC ACTGCTTAACACCATGGACCGCATCCTCTATGAGTCTCAGCGGCAGG GCCGGATCTCCTTCTACATGACCAACTATGGTGAGGAGGGCACGCAC GTGGGGAGTGCCGCCGCCCTGGACAACACGGACCTGGTGTTTGGCCA GTACCGGGAGGCAGGTGTGCTGATGTATCGGGACTACCCCCTGGAAC TATTCATGGCCCAGTGCTATGGCAACATCAGTGACTTGGGCAAGGGG CGCCAGATGCCTGTCCACTACGGCTGCAAGGAACGCCACTTCGTCAC TATCTCCTCTCCACTGGCCACGCAGATCCCTCAGGCGGTGGGGGCGG CGTACGCAGCCAAGCGGGCCAATGCCAACAGGGTCGTCATCTGTTAC TTCGGCGAGGGGGCAGCCAGTGAGGGGGACGCCCATGCCGGCTTCAA CTTCGCTGCCACACTTGAGTGCCCCATCATCTTCTTCTGCCGGAACA ATGGCTACGCCATCTCCACGCCCACCTCTGAGCAGTATCGCGGCGAT GGCATTGCAGCACGAGGCCCCGGGTATGGCATCATGTCAATCCGCGT GGATGGTAATGATGTGTTTGCCGTATACAACGCCACAAAGGAGGCCC GACGGCGGGCTGTGGCAGAGAACCAGCCCTTCCTCATCGAGGCCATG ACCTACAGGATCGGGCACCACAGCACCAGTGACGACAGTTCAGCGTA CCGCTCGGTGGATGAGGTCAATTACTGGGATAAACAGGACCACCCCA TCTCCCGGCTGCGGCACTATCTGCTGAGCCAAGGCTGGTGGGATGAG GAGCAGGAGAAGGCCTGGAGGAAGCAGTCCCGCAGGAAGGTGATGGA GGCCTTTGAGCAGGCCGAGCGGAAGCCCAAACCCAACCCCAACCTAC TCTTCTCAGACGTGTATCAGGAGATGCCCGCCCAGCTCCGCAAGCAG CAGGAGTCTCTGGCCCGCCACCTGCAGACCTACGGGGAGCACTACCC ACTGGATCACTTCGATAAGTGA

[0091] In one embodiment, the BCKDH E1 A subunit comprises an amino acid sequence that has at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 3, preferably wherein the amino acid sequence substantially retains the natural function of the protein represented by SEQ ID NO: 3.

[0092] In one embodiment, the BCKDH E1 A subunit-encoding nucleotide sequence comprises a nucleotide sequence that has at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 4, preferably wherein the protein encoded by the nucleotide sequence substantially retains the natural function of the protein represented by SEQ ID NO: 3.

[0093] In one embodiment, the BCKDH E1 A subunit-encoding nucleotide sequence comprises a nucleotide sequence that encodes an amino acid sequence that has at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 3, preferably wherein the amino acid sequence substantially retains the natural function of the protein represented by SEQ ID NO: 3.

[0094] The BCKDH E1 A and B subunits are mitochondrial proteins and their sequences may include mitochondrial-targeting signal sequences. Such signal sequences may be cleaved upon targeting of the protein to the mitochondrion, thus the protein may naturally exist in a mature form lacking the signal sequence. The skilled person is readily able to determine such signal sequences using appropriate bioinformatic and molecular biology techniques. For example, residues 1-50 of SEQ ID NO: 1 and residues 1-45 of SEQ ID NO: 3 may act as signal sequences.

Branched-Chain Alpha-Keto Acid Dehydrogenase Kinase (BCKDH Kinase)

[0095] The activity of BCKDH is regulated by a phosphorylation/dephosphorylation cycle. Branched-chain alpha-keto acid dehydrogenase kinase (BCKDH kinase) inactivates the BCKDH complex by phosphorylation of the BCKDH E1 A subunit, while BCKDH phosphatase activates the complex by dephosphorylating the BCKDH E1 A subunit.

[0096] In one embodiment, the BCKDH kinase is human BCKDH kinase.

[0097] An example amino acid sequence of the BCKDH kinase is the sequence deposited under NCBI Accession No. NP_005872.2.

[0098] An example amino acid sequence of the BCKDH kinase is:

TABLE-US-00005 (SEQIDNO:5) MILASVLRSGPGGGLPLRPLLGPALALRARSTSATDTHHVEMARERS KTVTSFYNQSAIDAAAEKPSVRLTPTMMLYAGRSQDGSHLLKSARYL QQELPVRIAHRIKGFRCLPFIIGCNPTILHVHELYIRAFQKLTDFPP IKDQADEAQYCQLVRQLLDDHKDVVTLLAEGLRESRKHIEDEKLVRY FLDKTLTSRLGIRMLATHHLALHEDKPDFVGIICTRLSPKKIIEKWV DFARRLCEHKYGNAPRVRINGHVAARFPFIPMPLDYILPELLKNAMR ATMESHLDTPYNVPDVVITIANNDVDLIIRISDRGGGIAHKDLDRVM DYHFTTAEASTQDPRISPLFGHLDMHSGAQSGPMHGFGFGLPTSRAY AEYLGGSLQLQSLQGIGTDVYLRLRHIDGREESFRI

[0099] An example nucleotide sequence encoding the BCKDH kinase is the sequence deposited under NCBI Accession No. NM 005881.3.

[0100] An example nucleotide sequence encoding the BCKDH kinase is:

TABLE-US-00006 (SEQIDNO:6) ATGATCCTGGCGTCGGTGCTGAGGAGCGGTCCCGGGGGCGGGCTTCC GCTCCGGCCCCTCCTGGGACCCGCACTCGCGCTCCGGGCCCGCTCGA CGTCGGCCACCGACACACACCACGTGGAGATGGCTCGGGAGCGCTCC AAGACCGTCACCTCCTTTTACAACCAGTCGGCCATCGACGCGGCAGC GGAGAAGCCCTCAGTCCGCCTAACGCCCACCATGATGCTCTACGCTG GCCGCTCTCAGGACGGCAGCCACCTTCTGAAAAGTGCTCGGTACCTG CAGCAAGAACTTCCAGTGAGGATTGCTCACCGCATCAAGGGCTTCCG CTGCCTTCCTTTCATCATTGGCTGCAACCCCACCATACTGCACGTGC ATGAGCTATATATCCGTGCCTTCCAGAAGCTGACAGACTTCCCTCCG ATCAAGGACCAGGCGGACGAGGCCCAGTACTGCCAGCTGGTGCGACA GCTGCTGGATGACCACAAGGATGTGGTGACCCTCTTGGCAGAGGGCC TACGTGAGAGCCGGAAGCACATAGAGGATGAAAAGCTCGTCCGCTAC TTCTTGGACAAGACGCTGACTTCGAGGCTTGGAATCCGCATGTTGGC CACGCATCACCTGGCGCTGCATGAGGACAAGCCTGACTTTGTCGGCA TCATCTGTACTCGTCTCTCACCAAAGAAGATTATTGAGAAGTGGGTG GACTTTGCCAGACGCCTGTGTGAGCACAAGTATGGCAATGCGCCCCG TGTCCGCATCAATGGCCATGTGGCTGCCCGGTTCCCCTTCATCCCTA TGCCACTGGACTACATCCTGCCGGAGCTGCTCAAGAATGCCATGAGA GCCACAATGGAGAGTCACCTAGACACTCCCTACAATGTCCCAGATGT GGTCATCACCATCGCCAACAATGATGTCGATCTGATCATCAGGATCT CAGACCGTGGTGGAGGAATCGCTCACAAAGATCTGGACCGGGTCATG GACTACCACTTCACTACTGCTGAGGCCAGCACACAGGACCCCCGGAT CAGCCCCCTCTTTGGCCATCTGGACATGCATAGTGGCGCCCAGTCAG GACCCATGCACGGCTTTGGCTTCGGGTTGCCCACGTCACGGGCCTAC GCGGAGTACCTCGGTGGGTCTCTGCAGCTGCAGTCCCTGCAGGGCAT TGGCACGGACGTCTACCTGCGGCTCCGCCACATCGATGGCCGGGAGG AAAGCTTCCGGATCTGA

[0101] In one embodiment, the BCKDH kinase comprises an amino acid sequence that has at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 5, preferably wherein the amino acid sequence substantially retains the natural function of the protein represented by SEQ ID NO: 5.

[0102] In one embodiment, the BCKDH kinase-encoding nucleotide sequence comprises a nucleotide sequence that has at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 6, preferably wherein the protein encoded by the nucleotide sequence substantially retains the natural function of the protein represented by SEQ ID NO: 5.

[0103] In one embodiment, the BCKDH kinase-encoding nucleotide sequence comprises a nucleotide sequence that encodes an amino acid sequence that has at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 5, preferably wherein the amino acid sequence substantially retains the natural function of the protein represented by SEQ ID NO: 5.

[0104] BCKDH kinase is a mitochondrial protein and its sequence may include a mitochondrial-targeting signal sequence. Such a signal sequence may be cleaved upon targeting of the protein to the mitochondrion, thus the protein may naturally exist in a mature form lacking the signal sequence. The skilled person is readily able to determine such signal sequences using appropriate bioinformatic and molecular biology techniques. For example, residues 1-30 of SEQ ID NO: 5 may act as a signal sequence.

Weight Loss and Weight Maintenance

[0105] Weight loss may refer to a reduction in parameters such as weight (e.g. in kilograms), body mass index (kg/m.sup.2), waist-hip ratio (e.g. in centimetres), fat mass (e.g. in kilograms), hip circumference (e.g. in centimetres) or waist circumference (e.g. in centimetres).

[0106] Weight loss may be calculated by subtracting the value of one or more of the aforementioned parameters at the end of an intervention (e.g. a diet and/or exercise regimen) from the value of the parameter at the onset of the intervention.

[0107] The degree of weight loss may be expressed as a percent change of one of the aforementioned weight phenotype parameters (e.g. a percent change in a subject's body weight (e.g. in kilograms) or body mass index (kg/m.sup.2)). For example, a subject may lose at least 10% of their initial body weight, at least 8% of their initial body weight, or at least 5% of their initial body weight. By way of example only, a subject may lose between 5 and 10% of their initial body weight.

[0108] In one embodiment, a degree of weight loss of at least 10% of initial body weight results in a considerable decrease in the risk of obesity-related co-morbidities.

[0109] Weight maintenance may refer to the maintenance in parameters such as weight (e.g. in kilograms), body mass index (kg/m.sup.2), waist-hip ratio (e.g. in centimetres) fat mass (e.g. in kilograms), hip circumference (e.g. in centimetres) or waist circumference (e.g. in centimetres). Weight maintenance may refer to, for example, maintaining weight lost following an intervention (e.g. a diet and/or exercise regimen).

[0110] The degree of weight maintenance may be calculated by determining the change in one or more of the afore-mentioned parameters over a period of time. The period of time may be, for example, at least 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 weeks.

[0111] Weight maintenance supported by the agents of the invention may result in, for example, a change (e.g. gain) of less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% in one or more of the afore-mentioned parameters over a period of time.

[0112] The degree of weight maintenance may be expressed as the weight regained during a period following attainment of weight loss, for example as a percentage of the weight lost during attainment of weight loss.

[0113] Weight maintenance supported by the agents of the invention may result through suppression of a subject's appetite following administration of the agent. The subject may therefore have a reduced appetite compared to the appetite in the absence of the agent of the invention.

[0114] Weight maintenance supported by the agents of the invention may result through control of a subject's appetite following administration of the agent. The subject may therefore maintain control over their appetite and therefore maintain their weight, for example following a period of weight loss intervention.

[0115] In particular, the agents of the invention may support weight maintenance through appetite suppression or control during and/or following a period of weight loss intervention (e.g. a diet or exercise regime).

[0116] In one aspect, the invention provides the non-therapeutic use of an agent of the invention to maintain a healthy body composition, for example after a period of weight loss.

Obesity

[0117] Overweight is defined for an adult human as having a body mass index (BMI) between 25 and 30.

[0118] Body mass index means the ratio of weight in kg divided by the height in metres, squared.

[0119] Obesity is a condition in which the natural energy reserve, stored in the fatty tissue of animals, in particular humans and other mammals, is increased to a point where it is associated with certain health conditions or increased mortality. Obese is defined for an adult human as having a BMI greater than 30.

[0120] Normal weight is defined for an adult human as having a BMI of 18.5 to 25, whereas underweight may be defined as a BMI of less than 18.5.

[0121] Obesity is a chronic metabolic disorder that has reached epidemic proportions in many areas of the world and is the major risk factor for serious co-morbidities such as type 2 diabetes mellitus, cardiovascular disease, dyslipidaemia and certain types of cancer (World Health Organ. Tech. Rep. Ser. (2000) 894: i-xii, 1-253).

[0122] Obesity-related disorder refers to any condition which an obese individual is at an increased risk of developing. Obesity-related disorders include diabetes (e.g. type 2 diabetes), stroke, high cholesterol, cardiovascular disease, insulin resistance, coronary heart disease, metabolic syndrome, hypertension and fatty liver.

Methods of Screening

[0123] The invention provides agents that are capable of increasing the activity of BCKDH and/or decreasing the activity of BCKDH kinase, and additionally provides methods for identifying such agents.

[0124] The agents of the invention may be identified by methods that provide either qualitative or quantitative results. Furthermore, such methods may be used to characterise as well as identify agents of the invention.

[0125] The candidate agents may be any agents of potential interest, for example peptides, polypeptides (e.g. antibodies), nucleic acids or small molecules. Preferably, the candidate agents are compounds or mixtures of potential therapeutic interest. Preferably, the candidate agents are of low toxicity for mammals, in particular humans. In some embodiments, the candidate agents may comprise nutritional agents and/or food ingredients, including naturally-occurring compounds or mixtures of compounds such as plant or animal extracts.

[0126] The candidate agents may form part of a library of agents, for example a library produced by combinatorial chemistry or a phage display library. In one embodiment, the candidate agents form part of a library of plant bioactive molecules.

BCKDH Activity

[0127] The invention also provides methods for identifying agents that are capable of increasing the activity of BCKDH and agents that are identified by such methods. The activity of BCKDH may be analysed directly, for example by analysing the enzymatic activity of the BCKDH.

[0128] A number of techniques are known in the art for measuring BCKDH activity. These techniques may be applied to BCKDH that has been isolated from a cell. The BCKDH may have been expressed using recombinant techniques. Preferably, the BCKDH has been purified.

[0129] In one embodiment, BCKDH is determined spectrophotometrically by monitoring the production of NADH from NAD.sup.+, for example in the presence of -ketoisovaleric acid, a substrate for BCKDH. Such assay techniques are described in, for example, Hawes, J. W. et al. (2000) Methods Enzymol. 324: 200-207.

BCKDH Kinase Activity

[0130] The invention also provides methods for identifying agents that are capable of decreasing the activity of BCKDH kinase and agents that are identified by such methods. The activity of BCKDH kinase may be analysed directly, for example by analysing the enzymatic activity of the BCKDH kinase.

[0131] The ability of a candidate agent to reduce the activity of a protein, for example an enzyme such as a kinase, may be expressed in terms of an IC50 value. The IC50 is the concentration of an agent that is required to give rise to a 50% reduction in the activity of the protein (e.g. a 50% reduction in enzymatic activity). The calculation of IC50 values is well known in the art. Preferably, the agents of the invention have an IC50 value for inhibition of BCKDH kinase of less than 100 M, more preferably less than 10 M, for example less than 1 M, less than 100 nM or less than 10 nM.

[0132] A number of techniques are known in the art for measuring kinase activity. These techniques may be applied to a kinase, for example BCKDH kinase, that has been isolated from a cell. The BCKDH kinase may have been expressed using recombinant techniques. Preferably, the BCKDH kinase has been purified.

[0133] In one embodiment, kinase activity is determined by monitoring the incorporation of phosphate into a substrate, for example radiolabelled phosphate from [-.sup.32P]-labelled ATP into a BCKDH substrate or suitable fragment thereof. Such assay techniques are described in, for example, Hastie, C. J. et al. (2006) Nat. Protocols 1: 968-971.

[0134] In another embodiment, kinase activity is determined by monitoring the amount of ADP that is produced in a kinase reaction (e.g. monitoring the rate of ADP production). Such assay systems (such as the commercial ADP-Glo Kinase Assay produced by Promega) may be based on the reconversion of ADP (produced in the kinase reaction) to ATP, which may be detected, for example via the production of a luminescent signal by a luciferase. In such an assay, the luminescent signal correlates with kinase activity. Such assays are particularly suitable for determining the effects of candidate agents on the activity of a broad range of purified kinases and are well suited to use in high-throughput screening.

[0135] In another embodiment, kinase activity is determined by monitoring the amount of ATP that remains at certain time points during a reaction (e.g. monitoring the rate of ATP consumption). In such assays, the signal correlates with the amount of ATP present, which inversely correlates with the kinase activity. Such assay systems (such as the commercial Kinase-Glo Kinase Assay by Promega) may be based on the production of a luminescent signal by a luciferase.

BCKDH and BCKDH Kinase Binding

[0136] The invention also provides methods of identifying agents which are capable of binding to BCKDH (in particular BCKDH E1 B subunit) and/or BCKDH kinase and, alternatively or additionally, characterising such binding. For example, the method may allow measurement of absolute or relative binding affinity, and/or enthalpy and entropy of binding. Binding affinity may be expressed in terms of the equilibrium dissociation (K.sub.d) or association (K.sub.a) constant.

[0137] A number of assay techniques are known in the art for identifying binding between a candidate agent and a protein. The assay technique employed is preferably one which is amenable to automation and/or high throughput screening of candidate agents. The assay may be performed on a disposable solid support such as a microtitre plate, microbead, resin or similar.

[0138] For example, target BCKDH (in particular BCKDH E1 B subunit) and/or BCKDH kinase may be immobilised on a solid support, for example a microbead, resin, micotitre plate or array. Candidate agents may then be contacted with the immobilised target protein. Optionally, a wash procedure may be applied to remove weakly or non-specifically binding agents. Any agents binding to the target protein may then be detected and identified. To facilitate the detection of bound agents, the candidate agents may be labelled with a readily detectable marker. The marker may comprise, for example, a radio label, an enzyme label, an antibody label, a fluorescent label, a particulate (e.g. latex or gold) label or similar.

[0139] Alternatively, the above procedure may be reversed and the candidate agents may be immobilised and the target BCKDH (in particular BCKDH E1 B subunit) and/or BCKDH kinase may be contacted with said immobilised agents. Optionally, a wash procedure may be applied to remove weakly or non-specifically bound target protein. Any agents to which BCKDH (in particular BCKDH E1 B subunit) and/or BCKDH kinase the binds may then be detected and identified. To facilitate the detection of binding, the BCKDH (in particular BCKDH E1 B subunit) and/or BCKDH kinase may be labelled with a readily detectable marker as described above.

[0140] In addition to the assays described above, other suitable assay techniques are known in the art. Examples of such techniques include radioassays, fluorescence assays, ELISA, fluorescence polarisation, fluorescence anisotropy, isothermal titration calorimetry (ITC), surface plasmon resonance (SPR) and the like. These assays may be applied to identify agents which bind to BCKDH (in particular BCKDH E1 B subunit) and/or BCKDH kinase. Indeed, platforms for the automation of many of these techniques are widely known in the art to facilitate high-throughput screening.

[0141] More than one assay techniques may be used to provide a detailed understanding of a candidate agent's binding to BCKDH (in particular BCKDH E1 B subunit) and/or BCKDH kinase. For example, assays which provide qualitative binding information may be used as a first step in the method, followed by further assays using different techniques to provide quantitative binding data and/or data on the effect on activity of the target protein.

[0142] The assay techniques described above may be adapted to perform competition binding studies. For example, these techniques are equally suitable to analyse the binding of a protein to substrate or cofactor in the presence of a candidate agent. It will therefore be possible to use the above techniques to screen and identify agents that modulate the binding between a protein and its substrate or cofactor, thus having an effect on the protein's activity. For example, these assays will enable the detection of binding between the BCKDH E1 subunit and thiamine diphosphate, or between BCKDH kinase and ATP, in the presence of a candidate agent.

[0143] Preferably, the agents of the invention will bind with high affinity. For example, the agents of the invention will bind to BCKDH (in particular BCKDH E1 B subunit) and/or BCKDH kinase with a K.sub.d of less than 100 M, more preferably less than 10 M, for example less than 1 M, less than 100 nM or less than 10 nM.

[0144] Binding affinity may be measured using standard techniques known in the art, e.g. surface plasmon resonance, ELISA and so on (for instance as described above), and may be quantified in terms of either dissociation (K.sub.d) or association (K.sub.a) constants.

[0145] Bioinformatics-based approaches, such as in silico structure-guided screening, may also be used to identify agents of the invention.

BCKDH and BCKDH Kinase Levels

[0146] The invention provides agents for increasing BCKDH (in particular BCKDH E1 B subunit) levels and/or decreasing BCKDH kinase levels. Levels of the relevant protein may be equated with levels of expression of the protein in a cell or organism. Protein levels may be analysed directly or indirectly, for example by analysis of levels of mRNA encoding the protein.

[0147] Methods for analysing the expression of BCKDH (in particular BCKDH E1 B subunit) and/or BCKDH kinase may be employed in the invention to screen the effect of a candidate agent on the protein's levels.

[0148] A number of techniques are known in the art for determining the expression level of a protein. These techniques may be applied to test the effect of candidate agents on the expression level of BCKDH (in particular BCKDH E1 B subunit) and/or BCKDH kinase. The technique employed is preferably one which is amenable to automation and/or high throughput screening of candidate agents.

[0149] For example, screens may be carried out using cells harbouring polynucleotides encoding BCKDH (in particular BCKDH E1 B subunit) and/or BCKDH kinase operably linked to a reporter moiety. The reporter moiety may be operably linked to endogenous BCKDH(in particular BCKDH E1 B subunit) and/or BCKDH kinase-encoding genes. Alternatively, exogenous copies of BCKDH (in particular BCKDH E1 B subunit) and/or BCKDH kinase operably linked to a reporter moiety may be inserted into a cell. In this embodiment, the cell may be engineered to be deficient for natural BCKDH and/or BCKDH kinase expression. The reporter moieties linked to BCKDH and/or BCKDH kinase may be different and distinguishable from one another. Suitable reporter moieties include fluorescent labels, for example fluorescent proteins such as green, yellow, cherry, cyan or orange fluorescent proteins.

[0150] By operably linked it is to be understood that the components described are in a relationship permitting them to function in their intended manner.

[0151] Such cells may be contacted with candidate agents and the level of expression of BCKDH (in particular BCKDH E1 B subunit) and/or BCKDH kinase may be monitored by analysing the level of reporter moiety expression in the cell. Fluorescent reporter moieties may be analysed by a number of techniques known in the art, for example flow cytometry, fluorescence-activated cell sorting (FACS) and fluorescence microscopy. Expression of BCKDH (in particular BCKDH E1 B subunit) and/or BCKDH kinase may be analysed separately or simultaneously within the same cell. Expression levels of BCKDH (in particular BCKDH E1 B subunit) and/or BCKDH kinase may be compared before and after contact with the candidate agent. Alternatively, expression levels of BCKDH (in particular BCKDH E1 B subunit) and/or BCKDH kinase may be compared between cells contacted with a candidate agent and control cells.

[0152] Other methods may be used for analysing the expression of proteins, for example BCKDH (in particular BCKDH E1 B subunit) and/or BCKDH kinase. Protein expression may be analysed directly. For example, expression may be quantitatively analysed using methods such as SDS-PAGE analysis with visualisation by Coomassie or silver staining. Alternatively expression may be quantitatively analysed using Western blotting or enzyme-linked immunosorbent assays (ELISA) with antibody probes which bind the protein product. BCKDH (in particular BCKDH E1 B subunit) and/or BCKDH kinase labelled with reporter moieties, as described above, may also be used in these methods. Alternatively, protein expression may be analysed indirectly, for example by studying the amount of mRNA corresponding to the protein that is transcribed in a cell. This can be achieved using methods such as quantitative reverse transcription PCR and Northern blotting.

[0153] Similar techniques may also be used for the analysis of leptin protein expression.

Agents

[0154] The invention provides agents that are capable of increasing the activity of BCKDH and/or decreasing the activity of BCKDH kinase, and additionally provides methods for identifying such agents.

[0155] The agents of the invention may be, for example, peptides, polypeptides (e.g. antibodies), nucleic acids (e.g. siRNAs, shRNAs, miRNAs and antisense RNAs) or small molecules. Preferably, the agents are of low toxicity for mammals, in particular humans. In some embodiments, the agents may comprise nutritional agents and/or food ingredients, including naturally-occurring compounds or mixtures of compounds such as plant or animal extracts.

[0156] In one embodiment, the agent of the invention is resveratrol. Resveratrol (3,5,4-trihydroxy-trans-stilbene) is a stilbenoid and phytoalexin that is naturally produced by a number of plants in response to injury or when under attack by pathogens. Food sources of resveratrol include the grape skins, blueberries, raspberries and mulberries.

[0157] In one embodiment, the agent of the invention is valproic acid. Valproic acid is a medicament used in the treatment of epilepsy, bipolar disorder and migraines. It may prevent seizures in subjects with absence seizures, partial seizures and generalised seizures. The structure of valproic acid is:

##STR00001##

[0158] In one embodiment, the agent of the invention is -chloroisocaproic acid. -Chloroisocaproic acid is an analogue of leucine and is the most potent known inhibitor of BCKDH kinase (Skimomura, Y. et al. (2006) J. Nutr. 136: 250S-253S). The structure of -chloroisocaproic acid is:

##STR00002##

[0159] In one embodiment, the agent of the invention is -ketoisocaproic acid (KIC). -Ketoisocaproic acid, the transamination product of leucine is a known physiological inhibitor of BCKDH kinase (Shimomura, Y. et al. (2006) J. Nutr. 136: 250S-253S). The structure of -ketoisocaproic acid is:

##STR00003##

[0160] Example agents that affect the activity of BCKDH particularly through affecting the activity of the BCKDH E1 B subunit include the agents recited in Table 1a (Davis A P, et al. The Comparative Toxicogenomics Database: update 2017. Nucleic Acids Res. 2016 Sep. 19).

TABLE-US-00007 TABLE 1a Agents that increase the activity of branched-chain alpha-keto acid dehydrogenase E1 B subunit (BCKDHB). Chemical Chemical Name ID CAS RN Interaction Actions 2,4-dinitrotoluene C016403 121-14-2 affects expression Ammonium Chloride D000643 12125-02-9 affects expression Antirheumatic Agents D018501 increases expression Benzo(a)pyrene D001564 50-32-8 affects expression/ affects reaction Benzo(a)pyrene D001564 50-32-8 increases expression Cuprizone D003471 370-81-0 increases expression Diethylnitrosamine D004052 55-18-5 increases expression Methylmercuric chloride C004925 115-09-3 increases expression pirinixic acid C006253 50892-23-4 increases expression potassium chromate(VI) C027373 7789-00-6 increases expression Tetrachlorodibenzodioxin D013749 1746-01-6 affects expression Valproic Acid D014635 99-66-1 affects expression Valproic Acid D014635 99-66-1 increases expression Vancomycin D014640 1404-90-6 increases expression

[0161] Example agents that affect the activity of BCKDH particularly through affecting the activity of the BCKDH E1 A subunit include the agents recited in Table 1b (Davis A P, et al. The Comparative Toxicogenomics Database: update 2017. Nucleic Acids Res. 2016 Sep. 19).

TABLE-US-00008 TABLE 1b Agents that increase the activity of branched-chain alpha-keto acid dehydrogenase E1 A subunit (BCKDHA). Chemical Chemical Name ID CAS RN Interaction Actions 1,12-benzoperylene C006718 191-24-2 increases expression 17-ethynyl-5-androstene- C524733 affects binding 3,7,17-triol 2,4-dinitrotoluene C016403 121-14-2 affects expression Acetaminophen D000082 103-90-2 affects expression Acetaminophen D000082 103-90-2 increases expression Amiodarone D000638 1951-25-3 increases expression Ammonium Chloride D000643 12125-02-9 affects expression Atrazine D001280 1912-24-9 increases expression Bisphenol A C006780 80-05-7 increases expression Carbamazepine D002220 298-46-4 affects expression Carbon Tetrachloride D002251 56-23-5 increases expression Chloroprene D002737 126-99-8 increases expression Clofibrate D002994 637-07-0 increases expression Ethinyl Estradiol D004997 57-63-6 increases expression Ethinyl Estradiol D004997 57-63-6 affects cotreatment/ increases expression Fluorouracil D005472 51-21-8 affects expression Furan C039281 110-00-9 affects binding Ketamine D007649 6740-88-1 increases expression Methylmercuric chloride C004925 115-09-3 increases expression Pirinixic acid C006253 50892-23-4 increases expression Streptozocin D013311 18883-66-4 affects expression Tetrachlorodibenzodioxin D013749 1746-01-6 affects expression Tetrachlorodibenzodioxin D013749 1746-01-6 affects cotreatment/ increases expression Tetrachlorodibenzodioxin D013749 1746-01-6 increases expression Tetracycline D013752 60-54-8 increases expression Topotecan D019772 123948-87-8 affects response to substance Tunicamycin D014415 11089-65-9 increases expression Valproic Acid D014635 99-66-1 affects expression Vancomycin D014640 1404-90-6 increases expression Vinclozolin C025643 50471-44-8 increases expression

[0162] Example agents that affect the activity of BCKDH kinase include the agents recited in Table 2 (Davis A P, et al. The Comparative Toxicogenomics Database: update 2017. Nucleic Acids Res. 2016 Sep. 19).

TABLE-US-00009 TABLE 2 Agents that decrease the activity of branched-chain alpha-keto acid dehydrogenase kinase (BCKDH kinase). Chemical Chemical Name ID CAS RN Interaction Actions Acetaminophen D000082 103-90-2 affects expression Ammonium Chloride D000643 12125-02-9 affects expression Arbutin D001104 497-76-7 decreases expression Atrazine D001280 1912-24-9 decreases expression Bisphenol A C006780 80-05-7 affects expression Cacodylic Acid D002101 75-60-5 decreases expression Clofibrate D002994 637-07-0 decreases expression Clofibric Acid D002995 882-09-7 affects cotreatment/ affects expression Cobaltous chloride C018021 7646-79-9 decreases expression Copper D003300 7440-50-8 affects binding/ decreases expression Copper Sulfate D019327 7758-98-7 decreases expression Dibutyl Phthalate D003993 84-74-2 decreases expression Diethylnitrosamine D004052 55-18-5 affects cotreatment/ affects expression Formaldehyde D005557 50-00-0 decreases expression Hydrogen Peroxide D006861 7722-84-1 affects expression Hypochlorous Acid D006997 7790-92-3 decreases expression Ketolides D048628 decreases expression Methoxyacetic acid C013598 625-45-6 affects expression NSC 689534 C558013 affects binding/ decreases expression Ochratoxin A C025589 303-47-9 decreases expression Procymidone C035988 32809-16-8 decreases expression Sodium bichromate C016104 10588-01-9 decreases expression Tetrachlorodibenzodioxin D013749 1746-01-6 affects reaction/ decreases expression Tetrachlorodibenzodioxin D013749 1746-01-6 affects expression Tetrachlorodibenzodioxin D013749 1746-01-6 decreases expression Thapsigargin D019284 67526-95-8 decreases expression Tunicamycin D014415 11089-65-9 decreases expression Valproic Acid D014635 99-66-1 affects expression

[0163] The agents for use according to the invention may be, for example, present as salts or esters, in particular pharmaceutically acceptable salts or esters.

siRNAs, shRNAs, miRNAs and Antisense DNAs/RNAs

[0164] Expression of BCKDH kinase may be modulated using post-transcriptional gene silencing (PTGS). Post-transcriptional gene silencing mediated by double-stranded RNA (dsRNA) is a conserved cellular defence mechanism for controlling the expression of foreign genes. It is thought that the random integration of elements such as transposons or viruses causes the expression of dsRNA which activates sequence-specific degradation of homologous single-stranded mRNA or viral genomic RNA. The silencing effect is known as RNA interference (RNAi) (Ralph et al. (2005) Nat. Medicine 11: 429-433). The mechanism of RNAi involves the processing of long dsRNAs into duplexes of about 21-25 nucleotide (nt) RNAs. These products are called small interfering or silencing RNAs (siRNAs) which are the sequence-specific mediators of mRNA degradation. In differentiated mammalian cells, dsRNA>30 bp has been found to activate the interferon response leading to shut-down of protein synthesis and non-specific mRNA degradation (Stark et al. (1998) Ann. Rev. Biochem. 67: 227-64). However, this response can be bypassed by using 21 nt siRNA duplexes (Elbashir et al. (2001) EMBO J. 20: 6877-88; Hutvagner et al. (2001) Science 293: 834-8) allowing gene function to be analysed in cultured mammalian cells.

[0165] shRNAs consist of short inverted RNA repeats separated by a small loop sequence. These are rapidly processed by the cellular machinery into 19-22 nt siRNAs, thereby suppressing the target gene expression.

[0166] Micro-RNAs (miRNAs) are small (22-25 nucleotides in length) noncoding RNAs that can effectively reduce the translation of target mRNAs by binding to their 3 untranslated region (UTR). Micro-RNAs are a very large group of small RNAs produced naturally in organisms, at least some of which regulate the expression of target genes. Founding members of the micro-RNA family are let-7 and lin-4. The let-7 gene encodes a small, highly conserved RNA species that regulates the expression of endogenous protein-coding genes during worm development. The active RNA species is transcribed initially as an 70 nt precursor, which is post-transcriptionally processed into a mature 21 nt form. Both let-7 and lin-4 are transcribed as hairpin RNA precursors which are processed to their mature forms by Dicer enzyme.

[0167] The antisense concept is to selectively bind short, possibly modified, DNA or RNA molecules to messenger RNA in cells and prevent the synthesis of the encoded protein.

[0168] Methods for the design of siRNAs, shRNAs, miRNAs and antisense DNAs/RNAs to modulate the expression of a target protein, and methods for the delivery of these agents to a cell of interest are well known in the art. Furthermore, methods for specifically modulating (e.g. reducing) expression of a protein in a certain cell type within an organism, for example through the use of tissue-specific promoters are well known in the art.

Antibodies

[0169] The term antibody, as used herein, refers to complete antibodies or antibody fragments capable of binding to a selected target, and includes Fv, ScFv, F(ab) and F(ab).sub.2, monoclonal and polyclonal antibodies, engineered antibodies including chimeric, CDR-grafted and humanised antibodies, and artificially selected antibodies produced using phage display or alternative techniques.

[0170] In addition, alternatives to classical antibodies may also be used in the invention, for example avibodies, avimers, anticalins, nanobodies and DARPins.

[0171] Methods for the production of antibodies are known by the skilled person. Alternatively, antibodies may be derived from commercial sources.

[0172] If polyclonal antibodies are desired, a selected mammal (e.g. mouse, rabbit, goat or horse) may be immunised. Serum from the immunised animal may be collected and treated according to known procedures. If the serum contains polyclonal antibodies to other antigens, the polyclonal antibodies may be purified by immunoaffinity chromatography. Techniques for producing and processing polyclonal antisera are known in the art.

[0173] Monoclonal antibodies directed against antigens (e.g. proteins) used in the invention can also be readily produced by the skilled person. The general methodology for making monoclonal antibodies by hybridomas is well known. Immortal antibody-producing cell lines can be created by cell fusion and also by other techniques such as direct transformation of B-lymphocytes with oncogenic DNA or transfection with Epstein-Barr virus. Panels of monoclonal antibodies produced against antigens can be screened for various properties, for example for isotype and epitope affinity.

[0174] An alternative technique involves screening phage display libraries where, for example, the phage express scFv fragments on the surface of their coat with a large variety of complementarity determining regions (CDRs). This technique is well known in the art.

[0175] Antibodies, both monoclonal and polyclonal, which are directed against antigens, are particularly useful in diagnosis, and those which are neutralising are useful in passive immunotherapy. Monoclonal antibodies in particular may be used to raise anti-idiotype antibodies. Anti-idiotype antibodies are immunoglobulins which carry an internal image of the antigen of the infectious agent against which protection is desired.

[0176] Techniques for raising anti-idiotype antibodies are known in the art. These anti-idiotype antibodies may also be useful for treatment, as well as for an elucidation of the immunogenic regions of antigens.

Introduction of Polypeptides and Polynucleotides into Cells

[0177] An agent for use in the invention may be, for example, a polypeptide or a polynucleotide. Polynucleotides and polypeptides may also need to be introduced into cells as part of the methods or screening assays of the invention.

[0178] Where the invention makes use of a polypeptide, the polypeptides may be administered directly to a cell (e.g. the polypeptide itself may be administered), or by introducing polynucleotides encoding the polypeptide into cells under conditions that allow for expression of the polypeptide in a cell of interest. Polynucleotides may be introduced into cells using vectors.

[0179] A vector is a tool that allows or facilitates the transfer of an entity from one environment to another. In accordance with the invention, and by way of example, some vectors used in recombinant nucleic acid techniques allow entities, such as a segment of nucleic acid (e.g. a heterologous DNA segment, such as a heterologous cDNA segment), to be transferred to a target cell. The vector may serve the purpose of maintaining the heterologous nucleic acid (e.g. DNA or RNA) within the cell, facilitating the replication of the vector comprising a segment of nucleic acid or facilitating the expression of the protein encoded by a segment of nucleic acid. Vectors may be non-viral or viral. Examples of vectors used in recombinant nucleic acid techniques include, but are not limited to, plasmids, chromosomes, artificial chromosomes and viruses. The vector may also be, for example, a naked nucleic acid (e.g. DNA). In its simplest form, the vector may itself be a nucleotide of interest.

[0180] The vectors used in the invention may be, for example, plasmid or virus vectors and may include a promoter for the expression of a polynucleotide and optionally a regulator of the promoter.

[0181] Vectors comprising polynucleotides used in the invention may be introduced into cells using a variety of techniques known in the art, such as transduction and transfection. Several techniques suitable for this purpose are known in the art, for example infection with recombinant viral vectors, such as retroviral, lentiviral, adenoviral, adeno-associated viral, baculoviral and herpes simplex viral vectors; direct injection of nucleic acids and biolistic transformation.

[0182] Non-viral delivery systems include but are not limited to DNA transfection methods. Here, transfection includes a process using a non-viral vector to deliver a gene to a target cell.

[0183] Transfer of the polypeptide or polynucleotide may be performed by any of the methods known in the art which may physically or chemically permeabilise the cell membrane. Cell-penetrating peptides may also be used to transfer a polypeptide into a cell.

[0184] In addition, the invention may employ gene targeting protocols, for example the delivery of DNA-modifying agents.

[0185] The vector may be an expression vector. Expression vectors as described herein comprise regions of nucleic acid containing sequences capable of being transcribed. Thus, sequences encoding mRNA, tRNA and rRNA are included within this definition.

[0186] Expression vectors preferably comprise a polynucleotide for use in the invention operably linked to a control sequence that is capable of providing for the expression of the coding sequence by the host cell. A regulatory sequence operably linked to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequence. The control sequence may be modified, for example by the addition of further transcriptional regulatory elements to make the level of transcription directed by the control sequence more responsive to transcriptional modulators.

Polynucleotides

[0187] Polynucleotides of the invention may comprise DNA or RNA. They may be single-stranded or double-stranded. It will be understood by a skilled person that numerous different polynucleotides can encode the same polypeptide as a result of the degeneracy of the genetic code. In addition, it is to be understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides of the invention to reflect the codon usage of any particular host organism in which the polypeptides of the invention are to be expressed.

[0188] The polynucleotides may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or lifespan of the polynucleotides of the invention.

[0189] Polynucleotides, such as DNA polynucleotides, may be produced recombinantly, synthetically or by any means available to the skilled person. They may also be cloned by standard techniques.

[0190] Longer polynucleotides will generally be produced using recombinant means, for example using polymerase chain reaction (PCR) cloning techniques. This will involve making a pair of primers (e.g. of about 15 to 30 nucleotides) flanking the target sequence which it is desired to clone, bringing the primers into contact with mRNA or cDNA, for example mRNA or cDNA obtained from an animal or human cell, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture with an agarose gel) and recovering the amplified DNA. The primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable vector.

Proteins

[0191] As used herein, the term protein includes single chain polypeptide molecules as well as multiple-polypeptide complexes where individual constituent polypeptides are linked by covalent or non-covalent means. As used herein, the terms polypeptide and peptide refer to a polymer in which the monomers are amino acids and are joined together through peptide or disulfide bonds.

Variants, Derivatives, Analogues, Homologues and Fragments

[0192] In addition to the specific proteins and nucleotides mentioned herein, the present invention also encompasses variants, derivatives, analogues, homologues and fragments thereof.

[0193] In the context of the present invention, a variant of any given sequence is a sequence in which the specific sequence of residues (whether amino acid or nucleic acid residues) has been modified in such a manner that the polypeptide or polynucleotide in question retains at least one of its endogenous functions. A variant sequence can be obtained by addition, deletion, substitution, modification, replacement and/or variation of at least one residue present in the naturally occurring polypeptide or polynucleotide.

[0194] The term derivative as used herein, in relation to proteins or polypeptides of the invention, includes any substitution of, variation of, modification of, replacement of, deletion of and/or addition of one (or more) amino acid residues from or to the sequence, providing that the resultant protein or polypeptide retains at least one of its endogenous functions.

[0195] The term analogue as used herein, in relation to polypeptides or polynucleotides, includes any mimetic, that is, a chemical compound that possesses at least one of the endogenous functions of the polypeptides or polynucleotides which it mimics.

[0196] Typically, amino acid substitutions may be made, for example from 1, 2 or 3, to 10 or 20 substitutions, provided that the modified sequence retains the required activity or ability. Amino acid substitutions may include the use of non-naturally occurring analogues.

[0197] Proteins used in the present invention may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent protein. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues as long as the endogenous function is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include asparagine, glutamine, serine, threonine and tyrosine.

[0198] Conservative substitutions may be made, for example according to the table 3 below. Amino acids in the same block in the second column and preferably in the same line in the third column may be substituted for each other:

TABLE-US-00010 TABLE 3 Conservative substitutions of Amino Acids ALIPHATIC Non-polar G A P I L V Polar - uncharged C S T M N Q Polar - charged D E K R H AROMATIC F W Y

[0199] The term homologue means an entity having a certain homology with the wild type amino acid sequence or the wild type nucleotide sequence. The term homology can be equated with identity.

[0200] In the present context, a homologous sequence is taken to include an amino acid sequence which may be at least 50%, 55%, 65%, 75%, 85% or 90% identical, preferably at least 95% or 97% or 99% identical to the subject sequence. Typically, the homologues will comprise the same active sites etc. as the subject amino acid sequence. Although homology can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties/functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.

[0201] In the present context, a homologous sequence is taken to include a nucleotide sequence which may be at least 50%, 55%, 65%, 75%, 85% or 90% identical, preferably at least 95% or 97% or 99% identical to the subject sequence. Although homology can also be considered in terms of similarity, in the context of the present invention it is preferred to express homology in terms of sequence identity.

[0202] Preferably, reference to a sequence which has a percent identity to any one of the SEQ ID NOs detailed herein refers to a sequence which has the stated percent identity over the entire length of the SEQ ID NO referred to.

[0203] Homology comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate percent homology or identity between two or more sequences.

[0204] Percent homology may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid or nucleotide in one sequence is directly compared with the corresponding amino acid or nucleotide in the other sequence, one residue at a time. This is called an ungapped alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.

[0205] Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion in the amino acid or nucleotide sequence may cause the following residues or codons to be put out of alignment, thus potentially resulting in a large reduction in percent homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall homology score. This is achieved by inserting gaps in the sequence alignment to try to maximise local homology.

[0206] However, these more complex methods assign gap penalties to each gap that occurs in the alignment so that, for the same number of identical amino acids or nucleotides, a sequence alignment with as few gaps as possible, reflecting higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps. Affine gap costs are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestfit package the default gap penalty for amino acid sequences is 12 for a gap and 4 for each extension.

[0207] Calculation of maximum percent homology therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A.; Devereux et al. (1984) Nucleic Acids Research 12: 387). Examples of other software that can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al. (1999) ibidCh. 18), FASTA (Atschul et al. (1990) J. Mol. Biol. 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al. (1999) ibid, pages 7-58 to 7-60).

[0208] However, for some applications, it is preferred to use the GCG Bestfit program. Another tool, BLAST 2 Sequences, is also available for comparing protein and nucleotide sequences (FEMS Microbiol. Lett. (1999) 174(2):247-50; FEMS Microbiol. Lett. (1999) 177(1):187-8).

[0209] Although the final percent homology can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix (the default matrix for the BLAST suite of programs). GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see the user manual for further details). For some applications, it is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.

[0210] Once the software has produced an optimal alignment, it is possible to calculate percent homology, preferably percent sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.

[0211] Fragments are also variants and the term typically refers to a selected region of the polypeptide or polynucleotide that is of interest either functionally or, for example, in an assay. Fragment thus refers to an amino acid or nucleic acid sequence that is a portion of a full-length polypeptide or polynucleotide.

[0212] Such variants may be prepared using standard recombinant DNA techniques such as site-directed mutagenesis. Where insertions are to be made, synthetic DNA encoding the insertion together with 5 and 3 flanking regions corresponding to the naturally-occurring sequence either side of the insertion site may be made. The flanking regions will contain convenient restriction sites corresponding to sites in the naturally-occurring sequence so that the sequence may be cut with the appropriate enzyme(s) and the synthetic DNA ligated into the cut. The DNA is then expressed in accordance with the invention to make the encoded protein. These methods are only illustrative of the numerous standard techniques known in the art for manipulation of DNA sequences and other known techniques may also be used.

Codon Optimisation

[0213] The polynucleotides used in the invention may be codon-optimised. Codon optimisation has previously been described in WO 1999/41397 and WO 2001/79518. Different cells differ in their usage of particular codons. This codon bias corresponds to a bias in the relative abundance of particular tRNAs in the cell type. By altering the codons in the sequence so that they are tailored to match with the relative abundance of corresponding tRNAs, it is possible to increase expression. By the same token, it is possible to decrease expression by deliberately choosing codons for which the corresponding tRNAs are known to be rare in the particular cell type. Thus, an additional degree of translational control is available. Codon usage tables are known in the art for mammalian cells, as well as for a variety of other organisms.

Method of Treatment

[0214] It is to be appreciated that all references herein to treatment include curative, palliative and prophylactic treatment. The treatment of mammals, particularly humans, is preferred. Both human and veterinary treatments are within the scope of the present invention.

Administration

[0215] Although the agents for use in the invention can be administered alone, they will generally be administered in admixture with a pharmaceutical carrier, excipient or diluent, particularly for human therapy.

[0216] In some embodiments, the agent is a nutritional agent, food additive or food ingredient, and may thus be formulated in a suitable food composition. Thus, the agent may be administered, for example, in the form of a food product, drink, pet food product, food supplement, nutraceutical or nutritional formula.

Dosage

[0217] The skilled person can readily determine an appropriate dose of one of the agents of the invention to administer to a subject without undue experimentation. Typically, a physician will determine the actual dosage which will be most suitable for an individual patient and it will depend on a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy. There can of course be individual instances where higher or lower dosage ranges are merited, and such are within the scope of the invention.

Subject

[0218] A subject refers to either a human or non-human animal.

[0219] Examples of non-human animals include vertebrates, for example mammals, such as non-human primates (particularly higher primates), dogs, rodents (e.g. mice, rats or guinea pigs), pigs and cats. The non-human animal may be a companion animal.

[0220] Preferably, the subject is a human.

EXAMPLES

Example 1

[0221] This study relates to a protein quantitative trait loci (pQTL) analysis performed on Diogenes weight loss intervention data. The Diogenes study is a pan-European, randomised and controlled dietary intervention study investigating the effects of dietary protein and glycaemic index on weight loss and weight maintenance in obese and overweight families in eight European centres (Larsen et al. (2009) Obesity Rev. 11: 76-91). In brief, the Diogenes study subjected screened participants to a low-calorie diet (LCD) phase (CID1), in which the overweight/obese subjects followed an 8 week Modifast diet (approximately 800 kCal/day), followed by a weight maintenance phase (CID2).

[0222] This is the first study that has tested the association between common variants genotyped on an Illumina chip and proteins expression change during intervention focusing on proteins associated to body mass index (BMI) change.

[0223] In this study, SNPs not observed on the Illumina chip were imputed using the Minimac3 tool using the European 1000 Genome population as the reference genome. Analysis was performed using best-guess genotypes (calculated on the basis of the 80% best-guess genotypes) from variants passing specific QC thresholds: MAF0.01 and significant deviation from Hardy-Weinberg equilibrium (P>1.0e6). Based on these new data, a new pQTL analysis was performed to extract more association signals in previously associated regions or identify new regions.

Materials and Methods

Data

[0224] The cohort included 498 participants with information at CID1 and CID2 for 1129 Somalogic proteins extracted from plasma. Genetic data imputation led to 4020756 SNPs that passed QC processes. Protein expression information was obtained using Somalogic technology. Data were pre-processed and controlled for quality. Gene expression (rnaSEQ technology, information available for almost 15000 transcripts from adipose tissue after quality control) and metabolomics data were also available and used this study.

Methods

[0225] Association between BMI and each protein expression change during the low-calorie diet (LCD) was tested using a linear regression (univariate regression). BMI change was first regressed on confounding cofactors (sex, age and centre), and residuals were regressed against delta protein expression. P-values were corrected for multiple testing using Benjamini-Hochberg standard false discovery rate correction.

[0226] pQTL association between SNPs and protein expression was performed using a linear mixed model (LMM). LMM is an emerging method of choice for association mapping, which allows for correction of genetic population heterogeneity (geographic population structure generated by the different recruitment centres across Europe). The basic approach is to build a genetic relationship matrix (GRM) modelling genome-wide sample structure (the genetic background of all patients in the study) using all (or part of the) SNPs available on the chip (imputed SNPs were not used during this step). Its contribution to protein expression variance is estimated using a random-effects model and association statistic is computed accounting for this component of variance. LMM association methods are effective in preventing false-positive associations between genetic variants and traits in studies of human and model organisms. In the present study, the dependent variable was the protein expression residuals from regression on age, sex and centre, and the independent variables were the SNPs. For each protein, a genome-wide association study (GWAS) was performed testing each SNP independently.

[0227] GCTA software was used for LMM computation with the loco option that excludes all SNPs belonging to the same chromosome than the SNP under study to avoid multi-collinearity. If the SNP under study, and all SNPs in linkage disequilibrium were used in the GRM, the log likelihood of the null model would be higher than it should be and lead to deflation of the test statistic and loss of power. This phenomenon is called proximal contamination.

[0228] GWASs pQTL were performed for all proteins. Results were extracted for proteins with delta expression associated to BMI change during intervention. No correction for multiple testing was applied. Our objective was to highlight/prioritise pQTL for further analysis using other omics information including transcriptomics and genetics (genome sequencing).

[0229] Results were plotted using locusZoom software implemented in a R script (launch_locuszoom.R) using 1000 Genomes European genetic data as reference (hg19).

[0230] Gene co-expression was evaluated using GeneMANIA (Zuberi, K. et al. (2013) Nucleic Acids Res. 41(Web Server issue): W115-22). By definition, two genes are linked (co-expressed) if their expression levels are similar across conditions in a gene expression study. Most of these data are collected from the Gene Expression Omnibus (GEO) and only data associated with a publication are collected.

[0231] A pipeline was written in R from Minimac imputed data output to results extraction including QC steps, parallelised pQTL GWASs, extraction of significant signals and plots.

[0232] Based on a set of top proteins for which change in expression during the LCD was associated to BMI change, pQTL results were extracted and investigated. Change in protein or expression, BMI or other covariates during the LCD implies change in expression/level before and after intervention unless specified.

Results

[0233] Before starting the analysis, for all proteins, SNPs with a FDR q-value<0.20 and MAF>0.05 were extracted. Their cis/trans acting effect was then evaluated according to their position +/500kb around the corresponding coding gene. A q-value measures the False Discovery Rate (FDR) incurred by accepting the given test and every test with a smaller p-value (and maybe even larger p-values, if they improve the FDR).

[0234] No cis-acting SNP reached a q-value <0.05 association cut-off for all 1129 proteins. Relaxing FDR cutoff to 0.20 did not identify any cis-acting SNPs, only trans-acting.

Protein Expression Change During LCD

[0235] After correction for multiple testing and assuming a p-value cutoff set to 5%, 55 proteins were positively and 52 negatively correlated to BMI during weight loss intervention. A correlation heat-map was built based on Kendal correlation tau correlation coefficient for all proteins associated to BMI during LCD Kendall tau rank correlation is a non-parametric test for statistical dependence between two ordinal (or rank-transformed) variables (similar to Spearman's), but which, unlike Spearman's, can handle ties. Hierarchical clustering was used to identify potential clusters. We did not observe clearly delineated large blocks of proteins possibly because of numerous false positive results in this large list.

[0236] Association with P<1.0e06 (after BH multiple testing correction) was observed for 9 proteins. A block of very significant correlation was observed between these 9 proteins the Kendall correlation level for protein pairs with p-value corrected for multiple testing under 5% after Bonferroni correction. Two anti-correlated blocks of proteins were observed including all but IL1 RAP gene coding-protein.

[0237] Table 4 provides an overview of these 9 proteins with Somalogic ID, name of coding gene, UNIPROT ID, direction of correlation with BMI and p-value (estimated p-value, PVAL; and multiple testing corrected, p-value, PBH, based on the Benjamin-Hochberg method (BH)). A first block of proteins including leptin, growth-hormone receptor, TIG2 (chemerin) and SAP was positively correlated with BMI change during the LCD while a second block including NRP1, SHBG, IGFBP-2, angiopoietin-2 and IL-1 R AcP was negatively correlated to BMI change.

TABLE-US-00011 TABLE 4 Top proteins associated to BMI change during the LCD. TARGET GENE UNIPROT Correlation PVAL PBH Growth GHR P10912 positive 5.2e26 5.2e26 hormone receptor Leptin LEP P41159 positive 3.9e21 3.9e21 SHBG SHBG P04278 negative 1.1e16 1.1e16 IGFBP-2 IGFBP2 P18065 negative 2.8e16 2.8e16 NRP1 NRP1 O14786 negative 8.5e13 8.5e13 TIG2 RARRES2 Q99969 positive 4.9e12 4.9e12 Angiopoietin-2 ANGPT2 O15123 negative 8.5e12 8.5e12 IL-1 R AcP IL1RAP Q9NPH3 negative 1.3e11 1.3e11 SAP APCS P02743 positive 1.3e09 1.3e09

[0238] The results below relate to proteins with promising pQTL results in genes likely involved in obesity and/or related traits. Other genes were discarded as not having GWA pQTL enriched signals and top SNPs not targeting genes of potential interest.

Leptin

[0239] pQTL Results

[0240] Leptin, the satiety hormone, is a hormone made by adipose cells that helps to regulate energy balance by inhibiting hunger. In obesity, a decreased sensitivity to leptin occurs, resulting in an inability to detect satiety despite high energy stores. Leptin levels fall during weight loss and increased brain activity occurs in areas involved in emotional, cognitive and sensory control of food intake. Restoration of leptin levels maintains weight loss and reverses the changes in brain activity. Thus, leptin is a critical factor linking reduced energy stores to eating behaviour (Ahima, R. S. (2008) J. Clin. Invest. 118: 2380-2383).

[0241] A QQ plot demonstrated the enrichment of association signal (genomic inflation factor (GIF)=1.0147153, 1.693076710.sup.5). This enrichment targets a specific region on chromosome 6

[0242] The top ten pQTL results for leptin includes SNPs in the same targeted region (Table 5 displays only the top 10 SNPs not in complete linkage disequilibrium (LD)). This region contains ncRNAs and is located in the regulatory region of the BCKDHB gene (http://www.genecards.org/cgi-bin/carddisp.pl?gene=BCKDHB&keywords=BCKDHB) FIG. 1 shows a Manhattan plot zooming in on this specific region of chromosome 6.

[0243] The BCKDHB enzyme complex is responsible for one step in the normal breakdown of leucine, isoleucine and valine. These three amino acids are obtained from the diet and are present in many kinds of food, particularly protein-rich foods such as milk, meat and eggs.

TABLE-US-00012 TABLE 5 Top 10 pQTL results for leptin. SnpsID Chr bp Freq b p rs1336257 6 81585576 0.2381 0.2255 1.111e07 rs507451 6 81571792 0.2397 0.2223 1.377e07 rs481481 6 81586692 0.2386 0.2238 1.408e07 rs9344031 6 81400749 0.09607 0.3105 1.476e07 rs4443477 6 81588425 0.238 0.2225 1.69e07 rs115586175 6 81433261 0.09401 0.3112 1.694e07 rs1981174 6 81588713 0.2385 0.2227 1.698e07 rs534800 6 81648445 0.1353 0.266 2.149e07 rs475407 6 81591054 0.237 0.2147 5.274e07 rs16892128 6 81391023 0.09544 0.2948 6.665e07

[0244] Protein expression stratified based on trans-acting SNP genotype did not underline a strong difference of expression despite significance shown in FIG. 2. However, since SNPs are generally surrogate markers of functional variant(s), it is likely that despite imputation the true underlying variant(s) is(are) not available.

BCKDHB Gene Expression Analysis

[0245] Expression data from rnaSEQ was available for the BCKDHB gene. This gene was significantly down-regulated during LCD intervention (P=1.1e11) after correction for multiple testing using Benjamini-Hochberg method. Association to BMI change was observed at the nominal level (P=0.016), but did not pass multiple testing correction (P=0.179). A significant and positive association was observed for a subset of 126 participants with proteomics and rnaSEQ data. BMI positively was associated (after correction for confounding cofactors) with leptin (P=1.6e8) and BCKDHB (P=2.4e2) expression, and leptin protein expression also displayed positive association with BCKDHB gene expression (P=2.6e3).

[0246] Leptin gene and protein expression were highly correlated (association pvalue=3.15e9) like BCKDHB and leptin (LEP) change during LCD (p=2.8e9) and to a lesser extent BCKDHB and leptin protein (p=0.0026). The direction of the association was the same (positive) for all paired of variable tested.

Integration with Metabolomics

[0247] The BCKD enzyme complex is active in mitochondria, where it is involved in the breakdown of leucine, isoleucine and valine to provide energy. Stroeve et al. (Stroeve, J. H. (2016) Obesity 24: 379-388) observed that valine contributed negatively to weight loss success.

[0248] From an analysis of metabolomics data extracted for leucine, valine and isoleucine, all 3 branched-chain amino acids (BCAAs) were differentially expressed during the LCD (non parametric Wilcoxon paired test, Table 6 and FIG. 3).

TABLE-US-00013 TABLE 6 Wilcoxon paired test comparing BCAA before/after intervention. BCAA P-value valine 2.5e11 isoleucine 2.7e07 leucine 1.5e09

[0249] BCKDHB gene expression was associated to valine and leucine change during the LCD Table 7, but not isoleucine.

TABLE-US-00014 TABLE 7 Association between BCKDHB and metabolic parameters. BCAA Estimate Pr(>|t|) P corrected valine 0.63 0.0091 0.013 leucine 0.65 0.0019 0.0058 isoleucine 0.22 0.22 0.22

[0250] All publications mentioned in the above specification are herein incorporated by reference.

[0251] Various modifications and variations of the described agents and methods of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention, which are obvious to those skilled in biochemistry and biotechnology or related fields, are intended to be within the scope of the following claims.