Patent Application
20150126438
This application incorporates by reference the Sequence Listing contained in the following ASCII text file being submitted concurrently herewith:
a) File name: SequenceListing.txt; created Nov. 15, 2012, 33 KB in size.
The prevalence of obesity and type 2-diabetes is increasing worldwide and threatens to shorten lifespan. Impaired insulin action in peripheral tissues is a major pathogenic factor. Insulin stimulates glucose uptake in adipose tissue through the Glut4-glucose transporter and alterations in adipose-Glut4 expression or function regulate systemic insulin sensitivity. Downregulation of adipose tissue-Glut4 occurs early in diabetes development. Complications from obesity and type-2 diabetes include vascular disease, which detracts from quality of life further. In addition, cancer, a prevalent and devastating disorder can be characterized by changes in metabolic flux, e.g., via the so-called Warburg effect, by which cancer cells substantially upregulate the level of glycolysis. In view of the prevalence of these disorders and their relation to changes in metabolism, a need exists for methods of detecting and monitoring disease states and/or treatment programs in subjects with diabetes-related disorders, obesity, vascular disease, or cancer, as well as tools and method of identifying agents that modulate key metabolic pathways.
The invention provides, inter alia, methods of monitoring disease states and/or treatment response for a variety of metabolic disorders, such as obesity and type 2 diabetes (and diabetes-related disorders) as well as common complications such as vascular disease, and other disorders associated with changes in metabolic flux, such as cancer. The invention also provides isolated nucleic acids and proteins encoding novel isoforms of ChREBP, termed ChREBP β expression products. The invention also provides reporter molecules and methods of screening candidate therapeutic agents for a variety of metabolic disorders as well as cancer and vascular disease. The invention is based, at least in part, on Applicants' surprising discovery of a new mechanism for glucose-regulation of ChREBP: Glucose-mediated activation of the canonical ChREBP isoform (ChREBPα) induces the expression of a novel, potent isoform (ChREBPβ) that is transcribed from an alternative promoter. Expression of ChREBPβ in human adipose tissue predicts insulin sensitivity indicating that it may be an effective target for treating diabetes. Furthermore, in view of the metabolic shift to glycolysis in cancer cells (the Warburg effect) and observations regarding a role for ChREBP α in cell proliferation (see Tong et al., Proc Natl Acad Sci USA. 106(51):21660-5 (2009) (exploring the role of ChREBP α in glucose-dependent anabolic synthesis and cell proliferation)), Applicants' observations on the substantially elevated activity of ChREBP β relative to ChREBP α make levels of ChREBP β expression products important biomarkers for cancer and a target for therapeutic interventions.
Accordingly, in a first aspect, the invention provides methods of assessing the disease state and/or treatment response of a mammalian subject for a disease or disorder selected from, for example, obesity, type 2 diabetes, impaired glucose tolerance, impaired fasting glucose, metabolic syndrome, insulin resistance, vascular disease or cancer. The methods typically include the step of determining the level of a ChREBP β expression product in a biological sample isolated from the mammalian subject, where the level of the ChREBP β expression product is indicative of the subject's disease state and/or treatment response for the disease or disorder. Another aspect of the invention provides methods of treatment for any of the forgoing disorders by providing a suitable prophylaxis or treatment to a subject assessed by the methods provided by the invention, which, in some embodiments, can include either an agonist or antagonist of a ChREBP β expression product.
In another aspect, the invention provides an isolated nucleic acid comprising a ChREBP β expression product or a fragment thereof, which, in some embodiments, may be characterized by comprising a sequence that has at least 60% identity to SEQ ID NO: 1 or a fragment thereof or a similar sequence, e.g., which hybridizes to the complement of SEQ ID NO: 1 under highly stringent hybridization conditions. In another aspect, the invention provides an isolated oligonucleotide capable of specifically amplifying the foregoing nucleic acids. A further aspect of the invention provides a siRNA molecule that specifically targets any of the foregoing nucleic acids.
Another aspect of the invention provides n isolated polypeptide comprising a ChREBP β expression product or a fragment thereof as well as isolated antibodies or antigen-binding fragments thereof that specifically bind such polypeptides.
In yet another aspect, the invention provides screening methods for identifying an agent that modulates the expression of a ChREBP β target gene. The screening methods typically include the step of determining the level of a ChREBP β expression product reporter in a cell contacted with a candidate agent, where a change in the level of the ChREBP β expression product reporter in the cell relative to a control cell not contacted with the candidate agent indicates that the agent modulates the expression of a ChREBP β target gene. In another aspect, the invention also provides isolated nucleic acids comprising a ChREBP β promoter. In certain embodiments, the promoter can provide a useful research tool and includes a nucleic acid sequence that has at least 60% identity to SEQ ID NO: 3 in operative association with a heterologous sequence, e.g., a suitable reporter molecule.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.
The foregoing will be apparent from the following more particular description of example embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments of the present invention.
a, mRNA expression of fatty acid synthetic enzymes, and
c, Nuclear abundance of mature Srebp1 and Lamin B1 (loading control) protein in AG4OX and WT adipose tissue from 6-week-old females. Each lane comprises pooled nuclear lysates from 3-4 mice. mRNA of,
A description of example embodiments of the invention follows.
The following definitions will be adhered to throughout the application.
“ChREBP β expression product” or simply “ChREBP β” refers to a non-recombinant nucleic acid encoding an N-terminally truncated isoform of ChREBP that exhibits enhanced transcriptional activation relative to non-recombinant ChREBP protein that is not N-terminally truncated—termed “ChREBP α,” here; “ChREBP β” and “ChREBP β expression product” also encompasses a protein expression product encoded by non-recombinant nucleic acid which encodes an N-terminally truncated isoform of ChREBP that exhibits enhanced transcriptional activation relative to ChREBP α. In a particular embodiment, ChREBP α is exemplified by the reference sequence NP—116569. In some embodiments a ChREBP β expression product has (or, in the case of a nucleic acid, encodes) an N-terminal truncation, relative to ChREBP α, of at least about 10, 20, 30, 40, 50, 75, 100, 125, 150, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, or 180 amino acids. In certain embodiments, the ChREBP β expression product is encoded by a transcript lacking the first exon of a ChREBP transcript encoding a ChREBP α protein (such as the reference sequence NM—032951.2). In more particular embodiments the ChREBP β expression product is encoded by a transcript comprising an alternative first exon, termed “exon 1b” herein, preferably wherein the exon 1b does not encode protein sequence. In more particular embodiments, the exon 1b comprises a nucleic acid sequence having at least 60% identity to SEQ ID NO: 1 or 5. ChREBP β expression products will typically retain the function of structurally conserved regions, including those depicted in
“Abnormal levels of a ChREBP β expression product” may be either aberrantly high or aberrantly low, depending on the particular subject, disorder, or tissue, and may be assessed by a variety of means including concentration of the expression product that is normalized to volume and/or, for example, amount or concentration of total protein, a reference nucleic acid, protein or other metabolite, as well as a ratio of concentration to ChREBP α expression products. Abnormal levels are determined relative to suitable controls, which can be assessed by any means including, for example, paired samples from a single patient (e.g., samples obtained at different times, e.g., before and after developing a disorder; as well as a pair of samples from different tissues, which may be obtained at the same or different times) as well as reference values previously compiled from samples determined—by any means—to be associated with, e.g., a diseased or normal state. For example, reference values for one or more disorders and/or treatments may be compiled and used to develop binary or probabilistic classification algorithms that are then used to classify a sample from a patient as, e.g., normal or abnormal.
“Gene expression” or “expression product” encompasses both nucleic acid (e.g., mRNA or cDNA derived from it) and protein expression of a gene. Nucleic acid expression products may or may not include subsequences that do not encode and/or get translated into protein.
“Level of expression,” “expression level,” “gene expression level” and the like, refers to the amount of a gene expression product (e.g., mRNA or protein) and may be normalized by any suitable means.
“ChREBP β expression product reporter” is an expression product—at the nucleic acid or protein level—whose level is modulated following induction of a ChREBP β expression product in a cell or non-cellular system and encompasses both recombinant and non-recombinant products.
“ChREBP β target gene” is a non-recombinant ChREBP β expression product reporter.
A “ChREBP β promoter” is a nucleic acid comprising an exon 1b of a ChREBP β expression product and further comprising at least one of an E-box element or a ChoRE, both of which are exemplified in
A “subject” refers to a mammal, including primates (e.g., humans or monkeys), cows, sheep, goats, horses, dogs, cats, rabbits, guinea pigs, rats, mice or other bovine, ovine, equine, canine, feline, rodent or murine species. Examples of suitable subjects include, but are not limited to, human patients (e.g., obese, diabetic, non-diabetic, having a diabetes-related disorder, cancer or vascular disease) and in more particular embodiments human patients (e.g., obese, non-obese) who have, or are at risk for developing, insulin resistance, type 2 diabetes, metabolic syndrome, vascular disease or cancer. Examples of high-risk groups for the development of metabolic syndrome, insulin resistance or type 2 diabetes include medically overweight and obese individuals. In preferred embodiments, the subject is human. In particular embodiments, the subjects to be tested or treated by the methods provided by the invention have, or are at increased risk for developing obesity or a diabetes-related disorder, cancer or vascular disease. In more particular embodiments, the vascular disease may be secondary to either obesity and/or a diabetes-related disorder. Similarly, the diabetes-related disorder may be secondary to obesity, or vice-versa. While subjects may be of any stage of life and any age, e.g., neonate, infant, toddler, child, young adult, adult, or geriatric; in particular embodiments the subject is an adult, e.g. a human adult, i.e. 18 years old, or older, e.g., 18-70, 20-60, 25-55, 25-50, 30-50, 25-65 years old, as well as greater than 30, 40, 50, 60, 70, 80 or 90 years old.
As used herein, the terms “treat,” “treating,” or “treatment,” mean to counteract a medical condition (e.g., obesity, a diabetes-related disorder, cancer, vascular disease) to the extent that the medical condition is improved according to a clinically-acceptable standard. For example, an improvement in a medical condition related to obesity can be determined according to one or more of the following: 1) reduction of body weight, 2) reduction of body mass index (BMI), 3) reduction of waist-to-hip ratio (WHR); improvement relative to diabetes can include 1) improved glucose tolerance, 2) reduced glycated hemoglobin, 3) improved insulin sensitivity, 4) improved glycemia; improvement in cancer can include: 1) reduced tumor growth, 2) tumor shrinking, 3) remission, 4) reduction in metastases, 5) reduced glucose uptake or utilization; improvement in vascular disease can include 1) reduced blood pressure, 2) lowered LDL cholesterol, 3) increased HDL cholesterol, 4) lowered triglycerides, 5) reduced atherosclerotic burden, 6) improved cardiac output.
The terms “prevent,” “preventing,” or “prevention,” as used herein, mean reducing the probability/likelihood, progression, onset, risk or severity of a disorder—including, for example, obesity or a diabetes-related disorder, cancer or vascular disease—in a subject. In general, a subject undergoing a preventative regimen most likely will be categorized as being “at-risk” for a given disorder, e.g., the risk for the subject developing obesity, a diabetes-related condition, vascular disease or cancer is higher than the risk for an individual represented by the relevant baseline population.
As used herein, a “therapeutically effective amount” is an amount sufficient to achieve the desired therapeutic or prophylactic effect under the conditions of administration, such as an amount sufficient to inhibit (e.g., reduce, prevent), e.g., obesity, diabetes-related disorder, vascular disease or cancer. The effectiveness of a therapy can be determined by one skilled in the art using standard measures and routine methods.
The term “obese” or “obesity” refers to the condition of a subject having a body mass index (BMI) of about 30 kg/m2 or higher, e.g., a BMI of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 kg/m2, or more. In particular embodiments, an obese subject has a BMI within the ranges defined as “obese” by the Center for Disease Control. See, URL //www.cdc.gov/obesity/defining.html. For example, in some embodiments, an adult who has a BMI >=30.0 kg/m2 is obese.
“Diabetes-related disorders” include T2D, impaired fasting glucose, impaired glucose tolerance, insulin resistance and metabolic syndrome.
“Type 2 diabetes” or “T2D” (OMIM 125853), in some embodiments, is defined as provided by the World Health Organization and the International Diabetes Federation in “Definition and diagnosis of diabetes mellitus and intermediate hyperglycaemia,” published in 2006, which is incorporated by reference in its entirety. In more particular embodiments, a diabetic subject exhibits a fasting plasma glucose of >=126 mg/dL or a 2-hour plasma glucose (2 hours after oral administration of 75 grams of glucose) >=200 mg/dL. In some embodiments a diabetic or pre-diabetic subject exhibits elevated levels of glycated hemoglobin, e.g., greater than 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6%, or more of total hemoglobin.
“Insulin resistance,” which may be identified by any means known in the art, and is characterized by a reduced ability of insulin to lower blood glucose levels.
The term “metabolic syndrome” refers to a group of symptoms that occur together and increase the risk for coronary artery disease, stroke and type 2 diabetes. In some embodiments the subject has central obesity (waist circumference >=80 cm for women; >=90 cm for Asian men, including ethnic South and Central Americans, and >=94 cm for all other males), BMI>30 kg/m2, raised triglycerides (>=150 mg/dL, or specific treatment for this lipid abnormality), reduced HDL cholesterol (<40 mg/dL in males, <50 mg/dL in females or specific treatment for this lipid abnormality), raised blood pressure (sBP>=130 mm HG or dBP>=85 mm HG or treatment of previously diagnosed hypertension) or raised fasting plasma glucose (FPG>=100 mg/dL or previous type 2 diabetes diagnosis), including combinations thereof. In more particular embodiments, the subject to be treated by the methods provided by the invention has or is at increased risk for metabolic syndrome, as defined by the International Diabetes Federation in “The IDF consensus worldwide definition of the metabolic syndrome,” published in 2006, which is incorporated by reference in its entirety, i.e., the subject has central obesity (as described above, and/or BMI>30 kg/m2) AND any two of raised triglycerides, reduced HDL cholesterol, raised blood pressure, or raised fasting plasma glucose.
“Cancer” refers to mammalian cancers, in some embodiments, human cancers, and carcinomas, sarcomas, adenocarcinomas, lymphomas, leukemias, et cetera, including solid and lymphoid cancers, kidney, breast, lung, kidney, bladder, colon, ovarian, prostate, pancreas, stomach, brain, head and neck, skin, uterine, testicular, esophagus, and liver cancer, including hepatocarcinoma, lymphoma, including non-Hodgkin's lymphomas (e.g., Burkitt's, Small Cell, and Large Cell lymphomas) and Hodgkin's lymphoma, leukemia, and multiple myeloma. Cancers embraced in the current application include both metastatic and non-metastatic cancers. In certain embodiments, a cancer cell may exhibit one or more of loss of contact inhibition when cultured, abnormal karyotype, abnormal cellular morphology or altered metabolic state consonant with what is termed the Warburg effect. Additional states that may be related to cancer and that can be diagnosed, monitored and/or treated by the methods provided by the invention include precancerous lesions and neoplasias.
“Vascular disease” is a pathological state of large, medium, or small sized arteries and may be triggered by endothelial cell dysfunction (e.g. including aneurisms, blockage, collapse) in central, peripheral or cerebral vasculature and can include angina, as well as severe complications such as stroke (ischemia), myocardial infarct (heart attack), arrhythmia, congestive heart failure, or ischemia resulting in gangrene or amputation.
“Highly stringent hybridization” conditions refers to at least about 6×SSC and 1% SDS at 65° C., with a first wash for 10 minutes at about 42° C. with about 20% (v/v) formamide in 0.1×SSC, and with a subsequent wash with 0.2×SSC and 0.1% SDS at 65° C.
The term “antibody,” as used herein, refers to an immunoglobulin or a part thereof, and encompasses any polypeptide comprising an antigen-binding site regardless of the source, species of origin, method of production, and characteristics. Antibodies for use in the methods provided by the invention include, for example, human, orangutan, mouse, rat, goat, sheep, rabbit and chicken antibodies. Antibodies may be polyclonal, monoclonal, monospecific, polyspecific, non-specific, humanized, camelized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, and CDR-grafted antibodies. For the purposes of the present invention, antibodies also include, unless otherwise stated, antibody fragments such as Fab, F(ab′)2, Fv, scFv, Fd, dAb, VHH (also referred to as nanobodies), and other antibody fragments that retain the antigen-binding function. Antibodies will have at least 3 CDRs and, in more particular embodiments, 4, 5, or 6 CDRS.
The present invention relates to novel forms of carbohydrate responsive-element binding protein, ChREBP, also known as MlxIPL (Mlx interacting protein-like, see OMIM 605678), MIO, MONDOB, WBSCR14, WS-bHLH and bHLHd14. ChREBP sequences from a variety of organisms are known, as shown in TABLE A.
Human
Mus musculus
Bos taurus
Pongo abelii
Pan troglodytes
Gallus gallus
Canis lupus familiaris
Rattus norvegicus
Sus scrofa
Cricetulus griseus
The GeneIDs in TABLE A can be used to retrieve reference protein and mRNA sequences from the NCBI (National Center for Biotechnology Information) database, as well as genomic sequences, along with annotations of each of these sequences for ChREBP α expression products—i.e. expression products that may include, e.g., differentially spliced variants—but none of which include the unprecedented ChREBP β expression products discovered by Applicants. For example, Table B shows reference protein and mRNA sequences available under some of the GeneIDs in Table A.
The present invention is based, at least in part, on Applicants' unexpected and surprising discovery of novel forms of ChREBP, termed ChREBP β expression products, as defined above. The working examples of the application summarize observations made in mouse and human cells and provide exemplary mRNAs, proteins and promoters for human and mouse ChREBP β, which are provided in SEQ ID NOs: 1-8 as summarized in TABLE C, below.
Based on Applicants' observations, variants of the exemplary ChREBP β expression products listed in TABLE C, including ChREBP β expression products from other species, can be readily detected.
For example, before the present invention, the ENTREZ database annotated 4 splice variants of human ChREBP α, which result in slightly different protein sequences in the C-terminus of the proteins, but none of which encompass the ChREBP β expression products provided by the invention. Therefore, in certain embodiments, ChREBP β expression products encompasses N-terminal truncations of each of above human splice variants, concordant with Applicants' observations in mouse. In a similar fashion, ChREBP β expression products from other species can be easily identified by performing sequence alignments (pairwise or multiple sequence alignments—by any means), and making reference to, for example, the reference nucleic acid and protein structures illustrated in
Methods for sequence alignments and comparison are widely known and include FASTA (Lipman and Pearson, Science, 227: 1435-41 (1985) and Lipman and Pearson, PNAS, 85: 2444-48), BLAST (McGinnis & Madden, Nucleic Acids Res., 32:W20-W25 (2004) (current BLAST reference, describing, inter alia, MegaBlast); Zhang et al., J. Comput. Biol., 7(1-2):203-14 (2000) (describing the “greedy algorithm” implemented in MegaBlast); Altschul et al., J. Mol. Biol., 215:403-410 (1990) (original BLAST publication)), Needleman-Wunsch (Needleman and Wunsch, J. Molec. Bio., 48 (3): 443-53(1970)), Sellers (Sellers, Bull. Math. Biol., 46:501-14 (1984), and Smith-Waterman (Smith and Waterman, J. Molec. Bio., 147: 195-197 (1981)), and other algorithms (including those described in Gerhard et al., Genome Res., 14(10b):2121-27 (2004)), which are incorporated by reference.
For simplicity, ChREBP β expression products claimed or used in methods provided by the invention may be described by reference (e.g. by percent identity or homology) to any of the suitable reference sequences in TABLE C as well as ChREBP β sequences readily deduced from any of the identifiers provided in TABLES A and B, above. For example, in certain embodiments, a protein ChREBP β expression product comprises an amino acid sequence having at least 60% identity to SEQ ID NO: 2 or 6, e.g., at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% identity to SEQ ID NO: 2 or 6. In particular embodiments, the protein ChREBP β expression product can be characterized as lacking a functional “LID” domain, as shown in
Accordingly, in one aspect, the invention provides isolated nucleic acid comprising a ChREBP β expression products or a fragment thereof, wherein the fragment is of a size suitable to distinguish it from other nucleic acids in a biological sample. In particular embodiments, the fragment is at least about 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3050, 3100 nucleotides, or more, in length and, in particular embodiments, comprises a nucleic acid that has at least 60% identity to SEQ ID NO: 1 or 5 or a fragment thereof, e.g. at least 60, 65, 70, 80, 85, 90, 95, 96, 97, 98, 99 or 100% identity to a fragment of at least about 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 nucleotides. In other embodiments, the nucleic acid comprises a sequence hybridizes under highly stringent conditions to SEQ ID NO: 1 or 5 or its complement.
In another aspect, the invention provides siRNAs that specifically target nucleic acid ChREBP β expression products and in particular embodiments, specifically target a nucleic acid ChREBP β expression product comprising a nucleic acid sequence that has at least 60% identity to SEQ ID NO: 1 or 5 or a fragment thereof, e.g. at least 60, 65, 70, 80, 85, 90, 95, 96, 97, 98, 99 or 100% identity to a fragment of at least about 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 nucleotides of SEQ ID NOs: 1 or 5. Methods of designing siRNAs are well known in the art and described, inter alia, in U.S. Pat. Nos. 7,078,196; 7,056,704, U.S. Patent Application Publication No. 20090209625 and International Publication Numbers WO 2001/075164 and WO 2002/044321, which are incorporated by reference.
In another aspect, the invention provides isolated polypeptides comprising a protein ChREBP β expression product or a fragment thereof. In particular embodiments, the fragment is at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 amino acids in length. In particular embodiments, the polypeptide comprises an amino acid sequence has at least 60% identity to SEQ ID NO: 2 or 6, e.g., at least least 60, 65, 70, 80, 85, 90, 95, 96, 97, 98, 99 or 100% identity. The invention also provides dominant negative ChREBP proteins based on protein ChREBP β expression products—e.g., those lacking a functional DNA binding domain, e.g., due to deletion or mutation. The DNA binding domain of ChREBP is contained in the bHLH domain depicted in
SEQ ID NO: 1 is a human ChREBP β exon 1b nucleic acid sequence.
SEQ ID NO: 2 is a human ChREBP β protein sequence.
SEQ ID NO: 3 is the genomic sequence 500 base pairs 5′ and 3′ to human ChREBP β exon 1b. The upstream e-box is nucleotides 418 to 423. The ChoRE is nucleotides 653 to 669. The exon 1b sequence is nucleotides 501 to 695. In the human genomic exon 1b region there is an additional e-box, nucleotides 693 to 698, and a potential ChoRE, nucleotides 1075 to 1091, that is conserved across primates, but not present in the mouse genomic sequence.
SEQ ID NO: 4 is a full-length human ChREBP β mRNA. The sequence of Exon 1b is nucleotides 1 to 195.
SEQ ID NO: 5 is a murine exon 1b nucleic acid sequence.
SEQ ID NO: 6 is a protein sequence of murine ChREBP β.
SEQ ID NO: 7 is a murine ChREBP β promoter nucleic acid sequence. This is the genomic sequence 500 base pairs 5′ and 3′ to murine exon 1b. The upstream e-box is nucleotides 403 to 408. The ChoRE is nucleotides 658 to 674. The exon 1b sequence is nucleotides 501 to 671.
SEQ ID NO: 8 is a full-length murine ChREBP β mRNA nucleic acid sequence. The Exon 1b sequence is nucleotides 1 to 171.
The invention provides a variety of diagnostic, prognostic and monitoring (e.g. disease progression and/or treatment efficacy) by determining the level of ChREBP β expression products in a biological sample from a subject and, e.g., comparing them to suitable controls, such as annotated reference values for a particular disease or disorder, as well as monitoring them over time in a subject. Accordingly, the invention also provides kits, nucleic acids (e.g. oligonucleotides, including pairs of oligonucleotides for amplification) and antibodies for performing these methods.
Based on the prominent role of ChREBP in metabolism—including glucose flux and lipid metabolism—and the central role of these metabolic pathways in obesity, diabetes-related disorders, cancer and vascular disease, and further in view of the dramatic activity of ChREBP β expression products, the level of ChREBP β expression products is a powerful monitor of disease state and, accordingly, can elucidate the need, efficacy and/or progress of any treatments for these disorders.
For the methods provided by the invention, fragments of ChREBP β expression products (nucleic acid or protein) detected need not be functional per se, as these molecules are being used in such applications as biomarkers only. Thus, fragments of ChREBP β expression products detected by the methods provided by the invention described under this subheading, in general, need only be of a size and structure so as to be distinguishable from other molecules in a given biological sample (or derived fraction thereof), such as total protein or nucleic acid, or ChREBP α expression products.
As defined above, any mammalian subject can be evaluated by the methods of the invention, while human subjects are one particular exemplification. Also, the subject may be of any age, with adult human subjects serving as particular exemplifications. However veterinary applications, particularly in a research context to develop treatments for human subjects are clearly encompassed by the invention as well.
Samples for use in the methods provided by the invention include any suitable biological sample or fraction thereof (e.g., extracted total nucleic acids, mRNAs, cDNAs, total protein, or mixture or further subfractions thereof). In particular embodiments, the biological sample may be isolated from blood, liver, adipose tissue, brown adipose tissue, muscle, pancreas, islet cells, kidney, breast, small intestine, bone marrow, nervous tissue (central, including brain or spine, and/or periperheral), ovary, or prostate. In some embodiments, the biological sample includes cancerous, precancerous, or neoplastic tissue that may include tissue obtained from any of the foregoing tissues. In more particular embodiments, the biological sample is obtained from liver or adipose tissue. Biological samples may be assayed from fresh or fixed samples.
The classification of a sample as normal (or well-controlled) or associated with a disease state (and/or requiring a modified treatment protocol) depends on the particular indication being assayed for (e.g., cancer versus obesity and diabetes-related disorders, including, in some embodiments, associated vascular disease). For example, elevated levels of ChREBP β expression products in liver or in cancerous, precancerous, or neoplastic samples are associated with disease states and/or indicate a need for modified treatment regimens. With regard to cancer, this may be based, at least in part on what is termed the Warburg effect—where cancer cells shift their metabolic profiles towards glycolysis and lactic acid fermentation. See, e.g., Christofk et al., Nature 452(7184):230-33 (2008); see also Tong et al. (2009). Furthermore, the activity of ChREBP to promote lipogenesis may be important for cancer progression. Tong et al., (2009). Expression of lipogenic enzymes positively correlated with ChREBP activity such as ATP-citrate lyase, acetyl-CoA carboxylase, and fatty acid synthase are increased in many cancers and inhibiting the activity of these enzymes may be therapeutic or preventative for numerous different cancers. See, e.g., Chajès et al., Cancer Research 66, no. 10: 5287-5294(May 15, 2006); Hatzivassiliou et al., Cancer Cell 8, no. 4: 311-321(October 2005); Swinnen et al., Current Opinion in Clinical Nutrition and Metabolic Care 9, no. 4: 358-365 (July 2006); Alli et al., Oncogene 24, no. 1:39-46 (Oct. 18, 2004). In adipose tissue, in contrast, levels of ChREBP β expression products are negatively correlated with disease state—i.e. reduced levels ChREBP β expression products in adipose tissue are associated with disease states such as diabetes-related disorders.
Levels of nucleic acid ChREBP β expression products can be determined by any means known in the art, including polymerase chain reaction (PCR), including reverse transcriptase (rt) PCR, real-time and quantitative PCR methods (including, e.g., TAQMAN®, molecular beacon, LIGHTUP™, SCORPION™, SIMPLEPROBES®; see, e.g., U.S. Pat. Nos. 5,538,848; 5,925,517; 6,174,670; 6,329,144; 6,326,145 and 6,635,427)); Northern blotting; Southern blotting of reverse transcription products and derivatives; array based methods, including blotted arrays or in situ-synthesized arrays; and sequencing, e.g., sequencing by synthesis, pyrosequencing, dideoxy sequencing, and sequencing by ligation, or any other methods known in the art, such as discussed in Shendure et al., Nat. Rev. Genet. 5:335-44 (2004) or Nowrousian Euk. Cell, 9(9): 1300-1310 (2010), including such specific platforms as HELICOS®, ROCHE® 454, ILLUMINA®/SOLEXA®, ABI SOLiD®, and POLONATOR® sequencing.
In particular embodiments, the invention provides oligonucleotides for detecting nucleic acid ChREBP β expression products, as well as additional tools for evaluating ChREBP β expression products—e.g., oligonucleotides to detect total (i.e. α and β) ChREBP expression products, as well as ChREBP α expression products. Based on the transcription mechanism driving nucleic acid ChREBP β expression products, in certain embodiments, the oligonucleotides provided by the invention enable detection, by any means, of exon 1b—the novel first exon of ChREBP β. The skilled artisan can readily develop a wide array of oligonucleotides to detect nucleic acid ChREBP β expression products and particular oligonucleotide pairs for detection (by amplification) of mouse (e.g. TCTGCAGATCGCGTGGAG and CTTGTCCCGGCATAGCAAC; SEQ ID NOs: 39 and 39, respectively) and human (e.g. AGCGGATTCCAGGTGAGG and TTGTTCAGGCGGATCTTGTC; SEQ ID NOs: 64 and 65, respectively) nucleic acid ChREBP β expression products are shown in TABLE 6 in the examples. Other suitable oligonucleotides for detecting nucleic acid ChREBP β expression products from human, mouse, or any other organism can readily be reduced to practice without undue experimentation. One non-limiting example of an additional modality to detect nucleic acid ChREBP β expression products includes subtractive analysis, e.g. by measuring total nucleic acid ChREBP expression product levels (α and β) and detecting nucleic acid ChREBP α expression products (e.g. by using sequence directed to the first exon of ChREBP α expression products, which is not present in nucleic acid ChREBP β expression products).
The oligonucleotides provided by the invention can readily be designed using ordinary skill in the art of molecular biology to arrive at oligonucleotides that are specific for a nucleic acid ChREBP β expression product, including, in certain embodiments, exon 1b (as well as fragments and similar nucleic acid sequences, as described above)—i.e., so that the oligonucleotides can discriminate the target nucleic acid from other nucleic acids present (or expected to be present) in a sample, including ChREBP α, entire transcriptomes and/or oligonucleotides directed to other genes.
The size of the nucleic acid ChREBP β expression product, e.g. the size of an amplified region to be detected, can be adjusted so as to provide the necessary resolution, based on the biological sample provided, e.g. the relative heterogeneity of nucleic acids in the sample or a fraction thereof. For example the nucleic acid ChREBP β expression product, or fragment thereof, detected may be at least about 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3050, 3100 nucleotides, or more, in length and, in particular embodiments, comprises a nucleic acid that has at least 60% identity to SEQ ID NO: 1 or 5, e.g. at least 60, 65, 70, 80, 85, 90, 95, 96, 97, 98, 99 or 100% identity or which hybridizes to SEQ ID NO: 1 or 5 under highly stringent conditions.
Similarly, the length (e.g., about 10-100 nucleotides, e.g., about 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100 nucleotides, or more) and sequence (i.e., the particular portion of a nucleic acid ChREBP β expression product to which the oligonucleotide hybridizes) of the oligonucleotides can readily be adjusted to achieve, e.g., a desired melting temperature (Tm; e.g., about 45-72° C., e.g., about 45, 50, 55, 60, 65, 70, 72° C. or more) and specificity, e.g., depending on the other materials present in the biological sample or fraction thereof. The skilled artisan will readily account for factors such as secondary structures, primer dimers, salt concentrations, nucleic acid concentrations, et cetera. Oligonucleotide primers provided by the invention may consist of (or consist essentially of) naturally occurring deoxribonucleotides or, optionally, may include modifications such as non-natural nucleotides, artificial backbones (such as PNAs), and detectable labels, such as florescent labels, biotinylation, et cetera.
Levels of protein ChREBP β expression products may be detected by any means of protein detection known in the art, including, for example, ELISA, Western Blotting, RIA (radioimmunoassay), nucleic acid-based or protein-based aptamer techniques, HPLC (high precision liquid chromatography), SPR (surface plasmon resonance), SAT (suspension array technology—including both immune-based, aptamer-based, or combination methods), direct peptide sequencing (such as Edman degradation sequencing), or mass spectrometry (such as MS/MS, optionally coupled to HPLC).
In particular embodiments, the methods provided by the invention that encompass detecting protein ChREBP β expression products employ antibodies directed to ChREBP, but which do not bind to the N-terminus of ChREBP—i.e., the portion of ChREBP that is absent in ChREBP β expression products. For example, antibodies suitable for use in detecting protein ChREBP β expression products include those that specifically bind a polypeptide having an amino acid sequence with at least 60% identity to SEQ ID NO: 2 or 6, e.g., bind a polypeptide having an amino acid sequence with at least 60, 65, 70, 80, 85, 90, 95, 96, 97, 98, 99 or 100% identity to SEQ ID NO: 2 or 6, or a fragment thereof, wherein the fragment is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 amino acids in length. In certain embodiments, subtractive or comparative methods employing multiple antibodies may be used. For example, total protein ChREBP expression products (i.e. α and β) can be measured using an antibody that binds to regions common to the α and β forms, while a second antibody that specifically binds a portion of the N-terminus of ChREBP α (e.g. within the first approximately 170-180, e.g. the first 178 amino acids of ChREBP α) not present in protein ChREBP β expression products is used to determine the proportion of total ChREBP protein that is ChREBP α, thereby providing the level of ChREBP β expression product). Similar approaches will be readily recognized by the skilled artisan based on the teachings of the application.
Levels of ChREBP β expression products can be evaluated and classified by a variety of means such as general linear model (GLM), ANOVA, regression (including logistic regression), support vector machines (SVM), linear discriminant analysis (LDA), principal component analysis (PCA), k-nearest neighbor (kNN), neural network (NN), nearest mean/centroid (NM), and baysian covariate predictor (BCP). Suitable cutoffs for evaluating levels of ChREBP β expression products (e.g., for classification as abnormal (obsese; positive or at risks for a diabetes-related disorder, cancer or vascular disease; requiring modification of a treatment regime) or normal (or low risk, or positive response to treatment) can be determined using routine methods, such as ROC (receiver operating characteristic) analysis, and may be adjusted to achieve the desired sensitivity (e.g., at least about 50, 52, 55, 57, 60, 62, 65, 67, 70, 72, 75, 77, 80, 82, 85, 87, 90, 92, 95, 97, or 99% sensitivity) and specificity (e.g., at least about 50, 52, 55, 57, 60, 62, 65, 67, 70, 72, 75, 77, 80, 82, 85, 87, 90, 92, 95, 97, or 99% specificity).
For example, in particular embodiments, expression levels are converted to a disease index. In particular embodiments, a disease index can use raw or transformed (e.g. normalized to any suitable metabolite, log-normalized, expressed as a ratio of ChREBP β and ChREBP α levels, percentile ranked, ranked as quartiles, et cetera) levels of ChREBP β expression products. A disease index for a particular individual can be compared to reference values as, for example, a percentile rank. Using percentile ranks the skilled artisan can then diagnose, prognose, or otherwise clinically stratify a subject by comparing the subject's disease index to these reference values. For example, in certain embodiments, a subject with a disease index percentile rank of at least 40, 45, 50, 60, 65, 70, 75, 80, 85, 90, or 95 may be classified as having, being at an increased risk for developing, or needing additional/alternative treatment for obesity, a diabetes-related disorder, cancer or vascular disease. In more particular embodiments, a subject exhibiting a disease index in at least the 60th, e.g., at least 70th or at least 75th percentile is classified as having, being at an increased risk for developing, or needing additional/alternative treatment for obesity, a diabetes-related disorder, cancer or vascular disease. A selected threshold for a disease index can be set to achieve a desired sensitivity or specificity, as described above, and/or to stratify subjects based on a relative hazard ratio between stratification groups. For example, in some embodiments, a disease index threshold is set to achieve a “hazard ratio” (ratio of frequency of a disorder between two stratification groups, e.g., high and low risk of disease or complication) of about 1.1, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.5, 2.6, 2.8, 3.0, 3.5, 4.0, or more. In more particular embodiments, the index threshold is set to achieve a hazard ratio of at least about 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, or more, e.g., 5, 6, 7, 8, 9, or 10. “Stratification groups” comprises the member of a data set satisfying one or more stratification criteria—for example, a percentile rank of disease index, such as all group members with a disease index greater than or equal to the 60th percentile. Stratification groups may be compared by any means by any statistic, such as, mean, median, mode, and/or standard deviation of any clinical parameter, such as age, duration of disease, frequency of death, et cetera.
Kits provided by the invention contain reagents to perform any of the methods provided by the invention, e.g. oligonucleotides and/or antibodies as described above. In particular embodiments, the kits include instructions for use. Optionally, the kits may include “suitable positive controls,” which are compositions comprising (consisting essentially of, or consisting of) nucleic acids, proteins, or nucleic acids and proteins that contain known concentrations of ChREBP β expression products and, in certain embodiments, ChREBP α expression products. For example, suitable controls may be from a clinical source known to have obesity, a diabetes-related disorder, cancer or vascular disease and may include either fixed or preserved but otherwise unprocessed biological sample, or, alternatively, isolated fractions from such samples, including fractions comprising (consisting of or consisting essentially of) nucleic acids (e.g., mRNA or cDNAs thereof), protein, and combinations thereof (e.g., at least 20, 40, 50, 60, 70, 80, 90, 95, 97, 99% by dry weight, or more, nucleic acid and/or protein). Alternatively, in certain embodiments, the suitable positive controls may comprise artificial mixtures of nucleic acids and/or proteins, e.g., combined in proportions characteristic of an abnormal levels of ChREBP β expression products for a particular disorder and/or particular ranges of concentrations.
In another aspect, the invention provides methods of treatment comprising administering a suitable prophylaxis or treatment to a subject in need thereof, as determined by the methods provided by the invention—e.g., according to any of the methods described under the previous subheading. Specifically, in certain embodiments, the subject is administered any clinically acceptable prophylaxis or treatment (including new regimens or modifications of existing regimens) for their given indication, based on the determination that the subject exhibits an abnormal level of ChREBP β expression products according to the methods provided by the invention. In more particular embodiments, any of the methods described in the claims or under the previous subheading further comprise the steps of follow-on diagnosis, prognosis or treatment. In other embodiments, the follow-on diagnosis, prognosis or treatment is performed by a provider advised of the presence of an abnormal level of a ChREBP β expression product, but who did not necessarily make the determination. For example, where the subject has been identified as having, being at an increased risk for, or needing further treatment for obesity, a diabetes-related disorder, cancer or vascular disease, at least in part, on the basis of abnormal levels of ChREBP β expression products, a provider administers or directs the subject to undergo a prophylactic and/or treatment regime suitable for the indication.
Suitable prophylaxes or treatments for diabetes-related disorders include, for example, weight loss programs, increased exercise, modified diet (e.g., reduced glycemic index), GLP-1R (human GeneID 2740) agonists (such as exenatide and liraglutide); DPP-4 antagonists (e.g., saxagliptin, vildagliptin); pramlintide; insulins (e.g., glulisine, detemir, glargine, lispro, aspart); SGLT2 (human GeneID No. 6524) inhibitors; inhibitors of glucose synthesis or release (FR-225654, CS-917 and MB07803); inhibitors of pyruvate kinase M2 (human GeneID No. 5315) (including agents described in U.S. Patent Application Publication No. 20100099726 A1, incorporated by reference in its entirety); insulin sensitizers (such as biguanidines, including metformin); adiponectin receptor 1 (human GeneID No. 51094) and adiponectin receptor 2 (human GeneID No. 79602) agonists; leptin receptor (human GeneID No. 3953) agonists; anoretics (e.g., sibutramine, rimonabant, bupropion); and the like, including combinations of the foregoing.
Suitable prophylaxes or treatments for obesity include weight loss programs, increased exercise, modified diet (e.g. reduced caloric, carbohydrate, or fat diets), gastric bypass or laproscopic banding, SGLT2 (human GeneID No. 6524) inhibitors; inhibitors of glucose synthesis or release (SB-204990, 2-deoxy-D-glucose (2DG), 3-bromopyruvate (3-BrPA, Bromopyruvic acid, or bromopyruvate), 3-BrOP, 5-thioglucose and dichloroacetic acid (DCA), FR-225654, CS-917 and MB07803); inhibitors of pyruvate kinase M2 (human GeneID No. 5315) (including agents described in U.S. Patent Application Publication No. 20100099726 A1, incorporated by reference in its entirety); leptin receptor (human GeneID No. 3953) agonists; anoretics (e.g., sibutramine, rimonabant, bupropion); and the like, including combinations of the foregoing.
Suitable prophylaxes or treatments for cancer include chemotherapy, hormonal therapy, immunotherapy, radiotherapy, surgery, targeted gene therapies (e.g., epidermal growth factor receptor-tyrosine kinase inhibitors, such as gefitinib; and agents targeting ALK mutations and rearrangements, such as crizotinib, et cetera), glycolytic inhibitors (e.g., SB-204990, 2DG, 3-BrOP, 5-thioglucose, DCA, as well as those agents described in U.S. Patent Application Publication No. 20100099726 A1), inhibitors of ATP-citrate lyase, inhibitors acetyl-CoA carboxylase, inhibitors fatty acid synthase, and combinations of the foregoing.
Suitable prophylaxes or treatments for vascular disease include weight loss programs (such as the pharmaceutical obesity treatments, above), increased exercise, modified diet (e.g. reduced salt), statin treatment (e.g. atorvastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin and simvastatin), bypass surgery and stenting.
It is, of course, encompassed by the methods provided by the invention that a subject determined to have obesity, a diabetes-related disorder, cancer or vascular disease may have one or more of these disorders, e.g. 2, 3 or 4 of these disorders, and may therefore provide an indication for treatment with combinations of prophylaxes or treatments for the different disorders, i.e. combinations of prophylaxes or treatments for obesity, a diabetes-related disorder, cancer or vascular disease as described above.
In another aspect, the invention provides methods of modulating the activity of ChREBP β expression products, i.e. increasing or decreasing their activity. Such methods can be performed in vitro or in vivo, e.g. in methods of treatment. For example, to increase the activity of ChREBP β expression products, an agonists of ChREBP β expression products can be contacted with a target cell, e.g., within a subject. This contact can be any means including by, e.g., intra venous, or intra peritoneal, rectal, oral or nasal administration of a recombinant polypeptide corresponding to a protein ChREBP β expression product, but which does not comprise a functional “LID” domain, as depicted in
Similarly, antagonists of ChREBP β expression products may be used in methods of treatment where reduced levels of ChREBP β expression products are desireable, e.g., in cancer, or, for diabetes-related disorders, elevated ChREBP β expression products in the liver. Suitable antagonists of ChREBP β expression products include siRNA directed to a nucleic acid ChREBP β expression products, e.g., in certain embodiments the siRNA is directed to exon 1b of a nucleic acid ChREBP β expression product. In other embodiments, the antagonist may be a dominant negative recombinant polypeptide corresponding to a ChREBP β expression product—i.e., that does not include a functional “LID” domain, as depicted in
In another aspect, the invention provides screening methods for identifying an agent that modulates the expression of a ChREBP β target gene, as defined above. The methods comprise determining the level of a ChREBP β expression product reporter in a cell contacted with a candidate agent where a change in the level of the ChREBP β expression product reporter in the cell relative to a control cell not contacted with the agent indicates that the agent modulates the expression of a ChREBP β target gene.
Any cell type may be useful in such methods including, yeast or insect cells, or a mammalian cell, e.g. a primate, murine, bovine, ovine, leporine, or porcine cell. The cell may be isolated, e.g., the method is performed in vitro, in a suitable culture, e.g., with either primary or established cell lines. In some embodiments, the cells are recombinant while in other embodiments the cells are non-recombinant. In other embodiments, the cell is in situ, i.e., the method is performed in vivo in a non-human animal, and in more particular embodiments in a non-human mammal, such as a non-human primate, a leporine or murine animal. The non-human animal may be transgenic or non-transgenic and such animals may also serve as the source of cells for the in vitro screening methods provided by the invention. The cell, in certain embodiments, further expresses Mlx. In other embodiments, the cell further express GLUT4 (SLC2A4, human GeneID No. 6517) and in more particular embodiments, the cell further expresses both Mlx and GLUT4. Although not essential to all aspects of these screening methods, in some embodiments, the methods include incubating the cell in the presence of glucose or fructose or their metabolites or analogs such as glucose-6-phosphate, xyulose-5-phosphate, fructose-2,6-bisphosphate, mannoheptulose, 2-deoxyglucose (2DG), or fluorodeoxyglucose.
The screening methods provided by the invention can, in some embodiments, include screening the candidate agent in the presence of additional agents, e.g., a second (or more) candidate therapeutic. For example, second therapeutic agents can include one or more of any of the therapeutics described under the previous subheading in order to identify combinations of agents with synergistic interactions.
In some embodiments, the ChREBP β expression product reporter is a ChREBP β target gene (i.e., a non-recombinant, endogenous product). In other embodiments, the ChREBP β expression product reporter is a synthetic (e.g. recombinant) ChREBP β expression product reporter and in more particular embodiments the synthetic ChREBP β expression product reporter is an expression product of a nucleic acid construct comprising a ChREBP β promoter, e.g., comprises a sequence having at least 60% identity to SEQ ID NO: 1 or 5. In still more particular embodiments, the construct comprises a nucleic acid sequence that has at least 60% identity to SEQ ID NO: 3 in operative association with a heterologous sequence. Exemplary heterologous sequences useful in these methods include, for example, a fluorescent protein, luciferase, aequorin, a peptide epitope, or an enzyme. Accordingly, in another aspect, the invention provides isolated nucleic acids encoding any of the foregoing reporter constructs.
Agents identified by these screening methods may either increase or decrease the expression of a ChREBP β target gene and/or increase or decrease the activity of a ChREBP β expression product. Methods of treating obesity, a diabetes-related disorder, cancer or vascular disease by administering an effective amount of one or more of the agents identified by these screening methods are contemplated and encompassed by the present invention.
The following examples serve to illustrate, and in no way to limit, the present invention.
Insulin resistance is a common complication of obesity and is a major factor in the pathogenesis of type 2 diabetes and cardiovascular disease1. Adipose tissue contributes to the development of obesity-related insulin resistance through increased release of fatty acids, altered adipokine secretion, and/or macrophage infiltration and cytokine release2,3. Altered adipose tissue glucose metabolism is also an important cause of insulin resistance, and adipose tissue Glut4 (Slc2a4), the major insulin-responsive glucose transporter, plays a more central role in systemic glucose metabolism than previously appreciated1,4,5. In insulin-resistant states, Glut4 is down-regulated in adipose tissue, but not in muscle1, the major site of insulin-stimulated glucose uptake. In addition, mice with adipose-specific Glut4 overexpression (AG4OX) have improved glucose homeostasis5 while adipose-specific Glut4 knockout mice (AG4KO) have insulin resistance and type 2 diabetes4. We investigated how altering adipose tissue glucose flux regulates glucose homeostasis. Here we show that carbohydrate responsive-element binding protein (ChREBP, also known as Mlxipl), which is a glucose-responsive transcription factor that regulates fatty acid synthesis and glycolysis,6 is highly regulated by Glut4 in adipose tissue and is a key determinant of systemic insulin sensitivity and glucose homeostasis. We also demonstrate that ChREBP in adipose tissue is required for the improved glucose homeostasis resulting from increased adipose-Glut4 expression. Glut4-mediated glucose uptake induces ChREBP which activates adipose tissue de novo lipogenesis. The latter is associated with enhanced insulin sensitivity7-10. We also show in obese humans that adipose-ChREBP gene expression correlates with insulin sensitivity, suggesting that ChREBP protects against obesity-associated insulin resistance. In addition, we discovered a novel mechanism for glucose-regulated ChREBP expression which involves a new ChREBP isoform, ChREBPβ. ChREBPβ is expressed from an alternative promoter in a glucose- and ChREBP-dependent manner. In contrast, the expression of the canonical ChREBPα isoform is not regulated by glucose flux. However, glucose-induced ChREBPα transcriptional activity increases ChREBPβ expression. Furthermore, expression of ChREBPβ is more highly regulated than ChREBα in adipose tissue in insulin resistant states. Thus, activation of adipose tissue ChREBP, and particularly ChREBPβ, may be a novel strategy for preventing and treating obesity-related metabolic dysfunction and type 2 diabetes.
AG4OX, AG4KO, and ChREBP KO mice were described previously4-6 except that AG4KO mice were generated using adiponectin-Cre expressing mice43 rather than aP2-Cre expressing mice. Phenotypic analyses were performed as previously described4,5,44. All mouse studies were conducted in accordance with federal guidelines and were approved by the BIDMC Institutional Animal Care and Use Committee. For microarray analysis, RNA was isolated from epididymal adipose tissue and the cDNA was analyzed on the Affymetrix MG-U74-A.v2 Genechip. Quantitative real-time PCR was performed on the Applied Biosystems 7900 HT using SYBR Green PCR Master Mix. Cloning, mutagenesis, and luciferase assays were performed by standard methods. Human subjects were recruited as previously described41,45. Human studies were approved by the Human Studies Committee of Washington University School of Medicine or the Ethics Committee of the University of Leipzig.
Generation and initial metabolic characterization of the adipose-specific Glut4-overexpressing mice (AG4OX), adipose-specific Glut4 knock-out mice (AG4KO), and ChREBP KO mice were previously described4-6 except that AG4KO mice were generated using adiponectin-Cre expressing mice43 rather than aP2-Cre expressing mice. WT and adiponectin-Cre littermates were used as controls for AG4KO mice. Mice were housed at Beth Israel Deaconess Medical Center with a 14/10 light-dark cycle and were fed standard chow (Formulab 5008) or HFD (Harlan-Teklad TD.93075) ad libitum. All studies were performed on age- and sex-matched littermates. Blood collections were performed by submandibular vein or tail vein bleeding. Body composition was measured by NMR (Echo Medical Systems). Glucose tolerance tests were performed by injection of 1 mg glucose per kg body weight i.p. after 5 hours food removal. For the fasting glycemic time course experiment, food was removed at 8 AM. Mice were sacrificed by CO2 euthanasia or decapitation and serum was collected, and tissues were harvested, snap frozen in liquid nitrogen, and stored at −80 C for processing. Mouse studies were conducted in accordance with federal guidelines and were approved by the BIDMC Institutional Animal Care and Use Committee.
For the cross-sectional cohort, adipose tissue samples were obtained from 123 men (n=64) and women (n=59) who underwent open abdominal surgery for gastric banding, cholecystectomy, appendectomy, weight reduction surgery, abdominal injuries or explorative laparotomy. These patients represent a normal glucose tolerant subset of patients previously described45. All subjects had a stable weight, defined as the absence of fluctuations of >2% of body weight for at least 3 months before surgery. Patients with malignant diseases or any acute or chronic inflammatory disease, as determined by a leukocyte count of >7,000 Gpt/l, C-reactive protein levels of >50 mg/l or clinical signs of infection, were excluded from the study. After resection, samples of visceral and subcutaneous adipose tissue were immediately frozen in liquid nitrogen. Euglycemic-hyperinsulinemic clamp studies were performed prior to surgery in subjects undergoing bariatric surgery or 3 months after surgery in subjects who underwent non-elective surgeries. The clamp study was performed with an insulin infusion rate of 20 mU/kg/min, as described and the glucose disposal rate was defined as the glucose infusion rate during the last 30 min of the study45. The study was approved by the ethics committee of the University of Leipzig. All participants gave written informed consent before taking part in the study.
For the obese cohort, 38 obese subjects (10 men, 28 women; 41±11 years old) who had normal oral glucose tolerance were studied. Subjects were sedentary (i.e., participated in regular exercise <1 hour/wk and ≦1 time/wk) and weight stable. All subjects provided written, informed consent before participating in the study, which was approved by the Human Studies Committee of Washington University School of Medicine in St. Louis.
Subjects were admitted to the Clinical Research Unit at Washington University School of Medicine in the evening before the clamp procedure. The next morning, after subjects fasted for 12 h overnight, a hyperinsulinemic-euglycemic clamp procedure in conjunction with [6,6-2H2]glucose tracer infusion was performed, as described previously41. Euglycemia was maintained at a blood glucose concentration of approximately 5.6 mmol/L (100 mg/dL) by infusing 20% dextrose enriched to 2.5% with [6,6-2H2]glucose. Skeletal muscle insulin sensitivity was determined as the relative increase in the rate of glucose uptake during insulin infusion (50 mU/m2/min).
Total RNA from epididymal adipose tissue was extracted using the RNeasy Mini Kit from Qiagen from three mice from each of four genotypes: aP2-Cre transgenic littermates (controls for AG4KO mice), AG4KO mice; FVB littermates (controls for AG4OX) and AG4OX. RNA from each mouse was hybridized on an Affymetrix MG-U74-A.v2 Genechip microarray. Affymetrix gene chip hybridization and analysis were performed at the Genomics Core Facility of the Beth Israel Deaconess Medical Center. Genome-wide expression analysis of the microarray data were performed using gene set enrichment analysis (GSEA)11 and the s2.mgu74av2.gmt gene set database available through the Broad Institute.
Mouse tissues were harvested following CO2 euthanasia or decapitation, snap frozen in liquid nitrogen, and stored at −80 C for processing. Total RNA was extracted from frozen tissue with TRI Reagent (Molecular Research Center, Inc.). Reverse transcription was performed using the Advantage RT-for-PCR kit (Clontech). Quantitative PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems) in a 7900 HT thermocycler (Applied Biosystems). SDS 2.3 (Applied Biosystems) was used for calculation of cycle thresholds. Relative expression levels were determined using the standard curve method with normalization of target gene expression levels to 18 s or Tbp. See Table 6 for primer sequences.
For the cross-sectional cohort, total RNA was isolated from subcutaneous adipose tissue samples using TRIzol (Life Technologies) and cDNA was synthesized with standard reagents (Life Technologies). Quantitative PCR was performed using Brilliant SYBR Green QPCR Core Reagent Kit (Stratagene) in a PRISM 7900 thermocycler (Applied Biosystems). Relative expression levels were determined using the standard curve method with normalization of target gene expression levels to 18 s.
For the obese cohort, total RNA was isolated from subcutaneous adipose tissue samples using TRIzol (Invitrogen) and cDNA was synthesized using Taqman Reverse Transcription (Applied Biosystems). Quantitative PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems) on the ABI 7500 Real-Time PCR System (Applied Biosystems). Relative expression levels were determined using the 2−ΔCt method with normalization of target gene expression levels to the 36B4 gene.
For adipose tissue lysates, aliquots of frozen adipose tissue were homogenized on ice in RIPA buffer supplemented with phospho-preserving and anti-protease agents (sodium fluoride, sodium pyrophosphate, sodium orthovanadate, PMSF, aprotinin, and leupeptin). For adipose tissue nuclear lysates, an adipose tissue sample was ground under liquid nitrogen, then dounce homogenized in low salt buffer [potassium chloride, 10 mM; Hepes pH 7.9, 10 mM; EDTA 0.1 mM; EGTA 0.1 mM; DTT 1 mM] with protease inhibitors. Homogenates were centrifuged at 1000 RCF for 10 minutes at 4 C and nuclear pellets were collected and washed with low salt buffer. The nuclear pellets were resuspended in RIPA buffer and vortexed vigorously. Nuclear lysates from 3 to 4 animals from the same genotype were pooled for further processing. Protein concentrations were assayed using the BCA assay. Equal amounts of protein were loaded and transferred to nitrocellulose membranes. Membranes were probed with antibodies against Acetyl-CoA Carboxylase (Cell Signaling), Fatty Acid Synthase (Cell Signaling), Glut4 (provided by H. Haspel), SREBP1 (Novus Biologicals), and Lamin B (Santa Cruz) and PI3 kinase p85 (Upstate). Film images were scanned (Epson Expression 10000 XL) and results were quantified with ImageQuant TL software (GE).
Conscious, freely moving mice in the fed state were injected intraperitoneally with 5 mCi of 3H2O (MP Biomedicals) and 10 μCi of [U-14C]-glucose (Perkin Elmer) and sacrificed 1 hour later using an overdose of ketamine and xylazine (160 and 24 mg/kg i.p., respectively). Samples of plasma were obtained serially at 5, 10, 30, and 60 minutes after injection for measurement of the plasma glucose levels and the specific activity of plasma water and glucose. At sacrifice, tissues were rapidly removed, frozen in liquid N2 and stored at −80° C. for processing. Lipids were extracted by the Folch method with chloroform/methanol (2:1). Fatty acids were obtained by saponification and extraction with petroleum ether. Incorporation of 3H and 14C into saponified fatty acids were measured. The rate of synthesis was calculated as a molar rate based on the estimate that 13.3 mol of H2O are incorporated into each newly synthesized C16 fatty acid46. The rate of glucose incorporation into newly synthesized fatty acids was calculated as nanomoles of 14C-glucose incorporated into fatty acids/gram tissue per minute, using 14C counts in saponified fatty acids extracted from tissue normalized by the time averaged specific activity of glucose in plasma over the course of the hour-long experiment.
Adipocytes were isolated from perigonadal fat pads and glucose uptake was measured as previously described5. Briefly, perigonadal fat pads were digested with collagenase (1 mg/ml) and cells were incubated at 37 “C with constant shaking in Krebs-Ringer-Hepes (30 mM) buffer (pH 7.4) with 2% bovine serum albumin, 200 nM adenosine, and without (basal) or with (insulin-stimulated) 80 nM insulin. Following a 30-min incubation with or without insulin, U-14C glucose (3 μM) was added for 60 min and the reaction was terminated by separating cells from media by spinning the suspension through dinonyl-phthalate oil. A portion of isolated adipocytes from each sample were fixed with osmic acid and counted in a Coulter counter to normalize glucose uptake per cell.
A position weighted matrix representative of a consensus ChoRE was generated from sequences for 16 mouse, human, or rat ChoREs previously identified and experimentally validated (
PCR of bacterial artificial chromosome containing the mouse ChREBP gene (CH29-535J6, Children's Hospital Oakland Research Institute) was used to generate the mouse ChREBPβ promoter sequence (−631 to +224, in reference to exon 1b transcriptional start site) which was cloned into the pGL3_basic reporter vector. The putative ChoRE sequence (+157 to +174) and E-box (−98 to −92) were deleted by site-directed mutagenesis (QuickChange II Site-Directed Mutagenesis Kit, Agilent). The exon 1b promoter_pGL3, mutant pGL3 constructs, or an empty pGL3_basic control were co-transfected using Lipofectamine LTX (Invitrogen) in HEK293T cells with Flag-tagged ChREBPα (ζ-isoform) and HA-tagged Mlx, both in the CMV4 vector33, along with a renilla luciferase control. After 24 hours of transfection, cells were cultured in DMEM containing low (2.5 mM) versus high (25 mM) glucose. Cells were collected and firefly luciferase activity was measured 24 hours after the media change and normalized to renilla luciferase activity (Dual Luciferase Assay System, Promega). All experiments were repeated at least three times with comparable results.
Flag-tagged ChREBPβ was generated by deletion of N-terminal sequence in the mouse Flag-tagged ChREBPα (GenBank: AF245475) in the CMV4 vector33 by site-directed mutagenesis (QuickChange II Site-Directed Mutagenesis Kit, Agilent). ChREBPβ sequence was confirmed by sequencing. Flag-tagged ChREBPα, Flag-tagged ChREBPβ, or empty pGL3_basic control were co-transfected with HA-tagged Mlx and a reporter plasmid containing a promoter region consisting of two copies of the ACC ChoRE fused to the firefly luciferase gene40. 24 hours after transfection, cells were cultured in DMEM containing low (2.5 mM) or high (25 mM) glucose. Cells were collected and firefly luciferase activity was measured 24 hours after the media change and normalized to renilla luciferase activity (Dual Luciferase Assay System, Promega). All experiments were repeated at least three times with comparable results.
All values are given as means±S.E. Differences between two groups were assessed using unpaired two-tailed Student's t-tests unless otherwise specified in figure legends. Comparisons between multiple groups were performed by ANOVA with pairwise comparisons and adjustment for multiple comparisons by the Tukey test unless otherwise specified in figure legends. For analyses with non-normal distributions or unequal variances, ANOVA and pairwise comparisons were performed on log-transformed values. All data presented in the figures are of non-transformed values. Statistical Analyses including linear regressions were performed with SPSS 8.0.
To understand the cellular mechanisms by which adipocytes respond to changes in glucose flux, we undertook global gene expression analyses of adipose tissue from AG4OX and AG4KO mice. Gene-set enrichment analysis11 demonstrated coordinate up-regulation of enzymes of fatty acid synthesis in AG4OX mice (Table 1). We confirmed that in adipose tissue, the expression of the enzymes of fatty acid synthesis are coordinately up-regulated in AG4OX mice and down-regulated in AG4KO mice (
In AG4KO and control mice, adipose-ChREBP expression strongly correlated with Glut4 expression (
Increased fatty-acid synthesis in rodent adipose tissue is associated with enhanced glucose tolerance and insulin sensitivity24,25. Consistent with the increases in ChREBP and lipogenic enzyme expression (
To determine whether adipose tissue ChREBP was required for the increased adipose tissue fatty-acid synthesis resulting from Glut4 overexpression, we crossbred AG4OX mice with whole-body ChREBP KO mice6. Fatty acid synthesis was normalized in perigonadal and subcutaneous adipose tissue after deletion of ChREBP in AG4OX mice (
In parallel with the changes in lipogenesis, the marked increases in the expression of fatty acid synthetic enzymes in AG4OX adipose tissue normalize following deletion of ChREBP (
AG4OX mice were obese compared to wild-type mice (
High-fat feeding causes obesity and insulin resistance in humans and rodents and down-regulates Glut4 expression selectively in adipose tissue1. To investigate whether adipose ChREBP expression may play a role in the insulin resistance that results from down-regulation of adipose-Glut4 expression with high-fat feeding, we subjected wild-type and AG4OX mice to high-fat diet (HFD). On chow, AG4OX mice were obese compared to chow-fed wild-type mice (
HFD induced insulin resistance in both genotypes reflected by increased glycemia, insulinemia, and glucose-insulin product (Table 3). In wild-type mice, HFD caused a diabetic GTT (
HFD reduces the expression of adipose tissue fatty acid synthetic enzymes in rodents28,29 and recent data from studies in humans show that downregulation of fatty-acid synthetic enzymes in adipose tissue correlates with insulin resistance7-10. Therefore, we examined whether downregulation of adipose tissue ChREBP and fatty acid synthesis may contribute to the insulin resistance resulting from HFD. HFD in wild-type mice reduced fatty acid synthetic rates in subcutaneous (78%) adipose tissue, but not perigonadal fat (
To investigate the molecular mechanisms responsible for the HFD-induced changes in adipose tissue fatty acid synthesis, we examined the expression of lipogenic transcription factors and fatty acid synthetic enzymes. In wild-type mice, HFD diminished ChREBP but not SREBP-1c expression in white adipose tissue (WAT) (
Many of the fatty acid synthetic genes that were up-regulated in WAT of AG4OX on chow are down-regulated in HFD WT and AG4OX mice (
In contrast, both ChREBP and SREBP-1c expression were down-regulated in WAT in AG4OX mice on HFD compared to AG4OX on chow (
LXRs were unlikely to contribute to the down-regulation of ChREBP expression, fatty-acid synthesis, or lipogenic enzyme expression in WT mice on HFD because expression of LXRs (
In the liver, neither adipose-specific Glut4 overexpression nor HFD affected fatty acid synthesis compared to WT-chow (
We next sought to determine whether adipose tissue ChREBP might contribute to regulating insulin sensitivity and glucose homeostasis in humans. In 123 non-diabetic individuals with normal glucose tolerance and widely ranging body mass index (20.8-50.9 kg/m2; Table 4), ChREBP expression in subcutaneous adipose tissue correlated strongly with insulin sensitivity measured by glucose infusion rate during a euglycemic-hyperinsulinemic clamp procedure (R=0.539, P<0.001;
Most, but not all, obese people are insulin resistant. To determine whether adipose tissue-ChREBP expression could have a role in regulating insulin sensitivity in obese people, we also investigated adipose tissue-ChREBP expression in a group of non-diabetic obese individuals (BMI=36.6±4.6 kg/m2) with widely ranging insulin-sensitivity (Table 4). Adipose tissue-ChREBP expression was directly associated with insulin-stimulated glucose uptake during a hyperinsulinemic-euglycemic clamp procedure, independent of BMI (R=0.605; P<0.001;
The mechanism by which glucose activates ChREBP transcriptional activity is complex and controversial. It has been attributed to xylulose-5-phosphate induced dephosphorylation of specific ChREBP residues34. Alternatively, glucose or a glucose metabolite may prevent the interaction of an N-terminal inhibitory domain with a glucose-activating domain within the ChREBP protein35. Glucose and other carbohydrates can also induce ChREBP expression31 but whether this contributes to ChREBP activity is unknown. The mechanism for carbohydrate-mediated ChREBP induction may involve “feed-forward” auto-regulation since a dominant negative mutant of Mlx which inhibits ChREBP activity reduces endogenous ChREBP expression21,36. To determine whether this occurs in vivo, we used qPCR primers proximal to the deleted exons to detect ChREBP mRNA. ChREBP was downregulated in adipose tissue (
To determine the molecular mechanisms by which ChREBP transactivates its own expression, we searched the genomic sequence up- and downstream of the ChREBP transcriptional start site for a carbohydrate response element (ChoRE). The ChoRE is defined by two E-boxes (CACGTG) separated by 5 nucleotides37. Using the Transcription Element Search System and a positional weight matrix defined by known ChoREs (
To determine whether the two distinct ChREBP mRNA species are physiologically regulated, we performed qPCR with primers specific for ChREBPα defined by the presence of exon 1a and ChREBPβ defined by the presence of exon 1b. In adipose tissue, ChREBPα expression declined modestly with overnight fasting, and increased 64% after 3 hours of refeeding a chow diet compared to ad libitum fed mice (
To determine whether alterations in glucose flux selectively regulate ChREBPα or β, we examined the expression of the two isoforms in AG4OX and AG4KO adipose tissue. Total ChREBP expression increased 50% in AG4OX and decreased 50% in AG4KO adipose tissue (
To investigate the molecular mechanism by which ChREBPβ expression is regulated in response to changes in adipose tissue Glut4 expression, we cloned the ChREBPβ promoter into a luciferase reporter plasmid. Expression of the ChREBPβ-promoter-luciferase construct was markedly increased in a glucose-dependent manner with co-transfection of ChREBPα and Mlx (
The translational start site for ChREBPα resides in exon 1a and translation from this site produces an 864 amino acid protein. No translational start site was present in exon 1b and translation beginning at the next start site, in exon 4, produces a 687 amino acid protein in which two nuclear export signals, a nuclear localization signal, and a domain that inhibits ChREBP transcriptional activity in low glucose conditions are deleted (
To determine whether regulation of distinct ChREBP isoforms might contribute to pathophysiology, we examined the regulation of ChREBPα and β in mice subjected to HFD. On HFD, total ChREBP and ChREBPβ expression declined in adipose tissue while ChREBPα expression remained unchanged (
To determine whether adipose tissue ChREBPα or β contributed more to insulin sensitivity in humans, we re-examined isoform-specific ChREBP expression in non-diabetic obese subjects. Expression of both isoforms correlated with insulin sensitivity (
In the past decade, the adipocyte has been recognized as a key regulator of whole-body energy homeostasis and metabolic function2,4. Our prior work demonstrating that adipose tissue Glut4 expression regulates systemic insulin sensitivity4,5 indicated that adipocytes are capable of sensing and coordinating responses to changes in glucose availability. The elucidation here of the molecular mechanism by which glucose-mediated ChREBP activation transactivates expression of a novel, potent ChREBPβ (
The beneficial effects of adipose tissue-ChREBP on glucose homeostasis may result from upregulation of adipose tissue-fatty acid synthesis and/or other ChREBP-dependent actions such as changing the metabolic fate of glucose after it enters adipocytes and/or regulating a novel adipokine. The strong, direct correlation between adipose-ChREBP expression and insulin sensitivity in human subjects suggests that adipose tissue ChREBP may be involved in regulating whole-body insulin action in people. The beneficial metabolic effects of increased fatty acid synthesis in adipose tissue contrast with the adverse effects of increased fatty acid synthesis in the liver. The latter increases intrahepatic triglyceride content and may contribute to insulin resistance and other features of the metabolic syndrome41,42. In summary, our data support the importance of adipose-ChREBP in regulating insulin action and glucose homeostasis in human obesity and diabetes. These data suggest that selective activation of adipose tissue ChREBP, and specifically ChREBPβ, could be an effective therapeutic strategy for preventing and treating type 2 diabetes and obesity-related metabolic diseases.
Total RNA from epididymal adipose tissue was extracted using the RNeasy Mini Kit from Qiagen from three mice from each of two genotypes: AG4OX and FVB littermate controls. RNA from each mouse was hybridized on an Affymetrix MG-U74-A.v2 Genechip microarray containing (12,421 probes). Genome-wide expression analysis of the microarray data was performed using gene set enrichment analysis (GSEA) software version 2.05 available from the Broad Institute (http://www.broadinstitute.org/gsea/downloads.jsp)11 and gene set database version s2.mgu74av2.gmt containing 522 distinct gene sets.
In Table 1A, the top ten up-regulated gene sets ranked by “NES”—normalized enrichment score—in AG4OX adipose tissue compared to controls are listed. “Name” indicates the name of the gene set as listed in the s2.mgu74av2.gmt database. “Size” refers to the number of genes in the gene set. “Nom p-val” and “FDR q-val” indicate the nominal p-values and false discovery rates for the individual sets, respectively, as calculated by the GSEA analysis software.
In Table 1B, the Affymetrix Probe number, Gene Symbol, and Gene Name are listed for each gene included in the fatty acid synthesis gene set according to the s2.mgu74av2.gmt database. ‘Rank in Gene List’ refers to the position of the gene in the ranked list of all genes present in our expression dataset and ranked by signal to noise ratio. The GSEA algorithm calculates an enrichment score reflecting the degree to which the genes included in a gene set are overrepresented at the top or bottom of the ranked list of all genes present in the expression dataset.
aPredictors: (Constant), Log AT ChREBP Beta (AU), Log AT ChReBP Alpha (AU)
bDependent Variable: Glucose Rd % stim
aPredictors: (Constant), Log AT ChREBP Beta (AU), Log AT ChREBP Alpha (AU)
bDependent Variable: Glucose Rd % stim
aDependent variable: Glucose Rd % stim
It should be understood that for all numerical bounds describing some parameter in this application, such as “about,” “at least,” “less than,” and “more than,” the description also necessarily encompasses any range bounded by the recited values. Accordingly, for example, the description at least 1, 2, 3, 4, or 5 also describes, inter alia, the ranges 1-2, 1-3, 1-4, 1-5, 2-3, 2-4, 2-5, 3-4, 3-5, and 4-5, et cetera.
For all patents, applications, or other reference cited herein, such as non-patent literature and reference sequence information, it should be understood that it is incorporated by reference in its entirety for all purposes as well as for the proposition that is recited. Where any conflict exits between a document incorporated by reference and the present application, this application will control. All information associated with reference gene sequences disclosed in this application, such as GeneIDs or accession numbers (typically referencing NCBI accession numbers), including, for example, genomic loci, genomic sequences, functional annotations, allelic variants, and reference mRNA (including, e.g., exon boundaries or response elements) and protein sequences (such as conserved domain structures) are hereby incorporated by reference in their entirety.
Headings used in this application are for convenience only and do not affect the interpretation of this application.
Preferred features of each of the aspects provided by the invention are applicable to all of the other aspects of the invention mutatis mutandis and, without limitation, are exemplified by the dependent claims and also encompass combinations and permutations of individual features (e.g. elements, including numerical ranges and exemplary embodiments) of particular embodiments and aspects of the invention including the working examples. For example, particular experimental parameters exemplified in the working examples can be adapted for use in the claimed invention piecemeal without departing from the invention. For example, for materials that are disclosed, while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. Thus, if a class of elements A, B, and C are disclosed as well as a class of elements D, E, and F and an example of a combination of elements, A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, is this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. This concept applies to all aspects of this application including, elements of a composition of matter and steps of method of making or using the compositions.
The forgoing aspects of the invention, as recognized by the person having ordinary skill in the art following the teachings of the specification, can be claimed in any combination or permutation to the extent that they are novel and non-obvious over the prior art—thus to the extent an element is described in one or more references known to the person having ordinary skill in the art, they may be excluded from the claimed invention by, inter alia, a negative proviso or disclaimer of the feature or combination of features.
The described methods may be, at least in part, embodied as computer-readable implementations that can be implemented in software, hardware, or a combination of hardware and software. Examples of hardware include computing or processing systems, such as personal computers, servers, laptops, mainframes, and micro-processors. In addition, one of ordinary skill in the art will appreciate that the records and fields shown in the figures may have additional or fewer fields, and may arrange fields differently than the figures illustrate. Any of the computer-readable implementations provided by the invention may, optionally, further comprise a step of providing a visual output to a user, such as a visual representation of, for example, sequencing results, e.g., to a physician, optionally including suitable diagnostic summary and/or treatment options or recommendations.
While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.
This application claims the benefit of U.S. Provisional Application No. 61/590,012, filed on Jan. 24, 2012. The entire teachings of the above application are incorporated herein by reference.
This invention was made with government support under R37 DK43051 (B.B.K), K08 DK076726 (M.A.H), and BADERC DK057521 (B.B.K, M.A.H) awarded by the NIH/NIDDK. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2013/022521 | 1/22/2013 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61590012 | Jan 2012 | US |
