Patent Application
20110138488
The present invention relates to animal models of metabolic disorders such as obesity, diabetes, metabolic syndrome, insulin resistance, hyperinsulinemia, glucose intolerance, hyperlipidemia, and the like. In particular, the invention relates to transgenic non-human animals having a reduction in functional Jhdm2a gene expression and use of such animals as well as cells having reduced Jhdm2a expression in drug discovery. The invention further relates to the identification of subjects having or at increased risk for developing a metabolic disorder based on a genetic marker in the Jhdm2a gene.
Histone methylation plays important roles in regulating diverse biological processes through controlling gene expression (Martin et al., Nat Rev Mol Cell Biol 6, 838 (November 2005). Recent studies indicate that the methylation state of histones can be dynamically regulated by histone methyltransferases and demethylases (Klose et al., Nat Rev Mol Cell Biol 8, 307 (2007)). The H3K9me1/2-specific demethylase JHDM2A/KDM3A plays an important role in nuclear hormone receptor mediated gene activation and male germ cell development (Okada et al., Nature 450, 119 (2007); Yamane et al., Cell 125, 483 (2006)).
In humans, excessive body fat deposition is closely linked to systemic insulin resistance, and diet-induced obesity is a primary risk factor for Type 2 diabetes. There is a need in the art for improved compositions and methods for treating and preventing metabolic disorders such as obesity, diabetes, metabolic syndrome and hyperlipidemia and methods of identifying those subjects that have or are at risk of developing such metabolic disorders.
The invention is based, in part, on the discovery that genetically modified non-human animals (e.g., avians, non-human mammals) having a reduction in functional Jhdm2a gene expression develop a phenotype that resembles metabolic disorders in humans such as obesity, diabetes, metabolic syndrome, insulin resistance, hyperinsulinemia, glucose intolerance, hyperlipidemia, and the like. Thus, as one aspect, the invention provides a genetically modified non-human animal having a reduction in functional Jhdm2a gene expression. As a further aspect, the invention provides methods of using the non-human animals of the invention in drug discovery to identify compounds for treating and/or preventing metabolic disorders. As still a further aspect, the invention provides methods for identifying subjects having or at increased risk for developing a metabolic disorder based on a genetic marker in the Jhdm2a gene.
Accordingly, as one aspect, the invention provides a transgenic non-human animal (e.g., mouse) whose genome comprises a homozygous disruption of the Jhdm2a gene, wherein the disruption results in a deficiency in functional Jhdm2a gene expression in the animal, and wherein the animal exhibits one or more of the following characteristics relative to a wild-type animal: (i) obesity, (ii) systemic insulin resistance, (iii) hyperinsulinemia, (iv) hyperlipidemia, (v) impaired energy expenditure, and (vi) reduced β-adrenergic receptor stimulated O2 consumption.
Also provided are isolated transgenic cells from the transgenic non-human animals of the invention and cell cultures comprising or produced by culturing the same.
As a further aspect, the invention provides a transgenic non-human animal (e.g., mouse) whose genome comprises a heterozygous disruption in the Jhdm2a gene, wherein the disruption results in a deficiency in functional Jhdm2a gene expression in the animal, and wherein the animal exhibits obesity on a high fat diet relative to a wild-type mouse. In particular embodiments, the animal is obese.
As another aspect, the invention provides a method of identifying a candidate compound for treating obesity, diabetes and/or metabolic syndrome, the method comprising:
administering a compound to an obese transgenic non-human animal (e.g., mouse) of the invention after the onset of obesity; and
determining the body weight and/or level of obesity in the transgenic animal,
wherein a reduction in body weight and/or obesity in the transgenic non-human animal administered the compound as compared with an untreated control animal indicates that the compound is a candidate for treating obesity, diabetes and/or metabolic syndrome.
As still a further aspect, the invention provides a method of identifying a candidate compound for preventing obesity, diabetes and/or metabolic syndrome, the method comprising:
administering a compound to a transgenic non-human animal (e.g., mouse) of the invention prior to the onset of obesity and for a time sufficient for the onset of obesity in an untreated control animal; and
determining the body weight and/or degree of obesity in the transgenic animal administered the compound,
wherein a reduction in body weight and/or obesity in the transgenic animal administered the compound relative to an untreated control animal indicates that the compound is a candidate for preventing obesity, diabetes and/or metabolic syndrome.
As yet a further aspect, the invention provides a method of identifying a candidate compound for treating obesity, diabetes and/or metabolic syndrome, the method comprising:
administering a compound to a transgenic non-human animal (e.g., mouse) of the invention after the onset of impaired energy expenditure; and
determining energy expenditure in the transgenic animal,
wherein an increase in energy expenditure in the transgenic animal administered the compound as compared with an untreated control animal indicates that the compound is a candidate for treating obesity, diabetes and/or metabolic syndrome.
As another aspect, the invention provides a method of identifying a candidate compound for preventing obesity, diabetes and/or metabolic syndrome, the method comprising:
administering a compound to a transgenic non-human animal (e.g., mouse) of the invention prior to the onset of impaired energy expenditure and for a time sufficient for the onset of impairments in energy expenditure in an untreated control animal; and
determining energy expenditure in the transgenic animal,
wherein an increase in energy expenditure in the transgenic animal administered the compound as compared with an untreated control animal indicates that the compound is a candidate for preventing obesity, diabetes and/or metabolic syndrome.
As a further aspect, the invention provides a method of identifying a candidate compound for treating obesity, diabetes and/or metabolic syndrome, the method comprising:
administering a compound to a transgenic non-human animal of the invention after the onset of reduced β-adrenergic receptor stimulated O2 consumption; and
determining β-adrenergic receptor stimulated O2 consumption by the transgenic animal,
wherein an increase in β-adrenergic receptor stimulated O2 consumption in the transgenic animal administered the compound as compared with an untreated control animal indicates that the compound is a candidate for treating obesity, diabetes and/or metabolic syndrome.
As still a further aspect, the invention provides a method of identifying a candidate compound for preventing obesity, diabetes and/or metabolic syndrome, the method comprising:
administering a compound to a transgenic non-human animal (e.g., mouse) of the invention prior to the onset of reduced β-adrenergic receptor stimulated O2 consumption and for a time sufficient for the onset of reduced β-adrenergic receptor stimulated O2 consumption in an untreated control animal; and
determining β-adrenergic receptor stimulated O2 consumption by the transgenic animal,
wherein an increase in β-adrenergic receptor stimulated O2 consumption in the transgenic animal administered the compound relative to an untreated control animal indicates that the compound is a candidate for preventing obesity, diabetes and/or metabolic syndrome.
Still further, the invention provides a method of identifying a candidate compound for preventing and/or treating obesity, diabetes and/or metabolic syndrome, the method comprising:
contacting a cell (e.g., a genetically modified cell) having reduced expression of Jhdm2a with a compound,
determining energy expenditure and/or the activity of the AMPK-PGC-1α axis in the cell,
wherein increased energy expenditure and/or activity of the AMPK-PGC-1α axis in the cell contacted with the compound relative to an untreated control cell indicates that the compound is a candidate for preventing and/or treating obesity, diabetes and/or metabolic syndrome.
As yet another aspect, the invention provides a method of identifying a candidate compound for preventing and/or treating obesity, diabetes and/or metabolic syndrome, the method comprising:
contacting a cell (e.g., a genetically modified cell) having reduced expression of Jhdm2a with a compound,
determining PPARα signaling pathway activity and/or β-adrenergic receptor signaling pathway activity in the cell,
wherein an increase in PPARα signaling pathway activity and/or an increase in β-adrenergic receptor signaling pathway activity in the cell contacted with the compound relative to an untreated control cell indicates that the compound is a candidate for preventing and/or treating obesity, diabetes and/or metabolic syndrome.
The invention also provides methods of identifying a mammalian subject at increased risk for developing obesity, diabetes and/or metabolic syndrome, the method comprising detecting in the subject the presence of a genetic marker in the Jhmda2a gene, wherein the genetic marker is correlated with an increased risk of developing obesity, diabetes and/or metabolic syndrome, thereby identifying a mammalian subject having increased risk of developing obesity, diabetes and/or metabolic syndrome.
Also provided is a method of identifying a mammalian subject at increased risk for developing obesity, diabetes and/or metabolic syndrome, the method comprising:
(a) correlating the presence of a genetic marker in the Jhdm2a gene with an increased risk of developing obesity, diabetes and/or metabolic syndrome; and
(b) detecting the presence of the genetic marker of (a) in a mammalian subject, thereby identifying a mammalian subject at increased risk for developing obesity, diabetes and/or metabolic syndrome.
As yet another aspect, the invention provides a method of identifying a genetic marker in the Jhdma2 gene correlated with increased risk of a mammalian subject developing obesity, diabetes and/or metabolic syndrome, the method comprising:
(a) identifying a mammalian subject that has developed obesity, diabetes and/or metabolic syndrome;
(b) detecting in the mammalian subject the presence of a genetic marker in the Jhdm2a gene; and
(c) correlating the presence of the genetic marker of step (b) with the development of obesity, diabetes and/or metabolic syndrome, thereby identifying a genetic marker in the Jhdm2a gene correlated with increased risk of a mammalian subject developing obesity, diabetes and/or metabolic syndrome.
Also provided is a method of identifying a genetic marker in the Jhdma2 gene correlated with increased risk of a mammalian subject developing obesity, diabetes and/or metabolic syndrome, the method comprising:
(a) identifying a mammalian subject that has developed obesity, diabetes and/or metabolic syndrome;
(b) determining the nucleotide sequence of the Jhdm2a gene of the mammalian subject of (a);
(c) comparing the nucleotide sequence of (b) with the wild-type nucleotide sequence of the Jhdm2a gene;
(d) detecting a genetic marker in the nucleotide sequence of (b); and
(e) correlating the presence of the genetic marker of step (b) with the development of obesity, diabetes and/or metabolic syndrome in the mammalian subject of (a).
These and other aspects of the invention are described in more detail in the following description of the invention.
in Ctrl cells without AICAR treatment is defined as 1. *p<0.05.
The present invention will now be described with reference to the accompanying drawings, in which preferred embodiments of the invention are shown. This invention can be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. For example, features illustrated with respect to one embodiment can be incorporated into other embodiments, and features illustrated with respect to a particular embodiment can be deleted from that embodiment. In addition, numerous variations and additions to the embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure, which do not depart from the instant invention.
Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination.
Any feature of the invention that is specifically described herein can be included or omitted from the invention (e.g., can be disclaimed).
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
All publications, patent applications, patents, and other references mentioned herein are incorporated by reference herein in their entirety.
As used in the description of the invention and the appended claims, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.
As used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).
As used herein the term “consisting essentially of” (and grammatical variations) means that the composition, article, product or method does not comprise any elements that materially change the functioning of the composition, article, product or method other than those elements specifically recited.
The invention provides transgenic non-human animals that have been genetically modified to reduce functional Jhdm2a gene expression. Animals of the present invention are, in general, non-human mammals and/or avians including but not limited to monkeys, chimps, dogs, cats, sheep, goats, cattle, rabbits, pigs, rodents (e.g., hamsters, gerbils, rats, guinea pigs and/or mice), chickens, turkeys, ducks and/or quail. In some embodiments, the non-human animal is a laboratory animal (e.g., monkey, rodent, dog, pig, bird, etc.). In some embodiments, the animal is a non-human primate, a species of domestic livestock (e.g., horse, cattle, sheep, pig, goat, chicken and the like) and/or a companion animal (e.g., cat, dog, guinea pig, gerbil, hamster, and the like).
Animals may be male and/or female and may be of any age, including neonatal, immature, adult and/or senescent animals. In particular embodiments, the animal is an immature animal (e.g., before sexual maturity; sexual maturity is at about four to eight weeks in the mouse). In embodiments of the invention, the animal is an adult animal (e.g., after sexual maturity), optionally after the onset of metabolic defects in energy expenditure and/or obesity due to the reduction in functional Jhmd2a gene expression.
A “metabolic disorder” as used herein includes but is not limited to any disorder or condition associated with an impairment in energy metabolism including but not limited to obesity, diabetes (e.g., type II/non-insulin dependent diabetes), metabolic syndrome, insulin resistance, hyperinsulinemia, glucose intolerance, hyperglycemia (e.g., fasting hyperglycemia), hyperlipidemia, and the like.
In general, the non-human animals of the invention deposit excess fat as compared with wild-type animals (e.g., wild-type littermates) after reaching sexual maturity. For example, a transgenic mouse having reduced functional Jhmd2a expression may begin to deposit excess fat as compared with wild-type littermates at about 5 to 6 months of age, and will generally be clearly obese by 7 months, and fat deposition will peak at about one year of age. Metabolic impairments in energy expenditure (e.g., reductions in basal metabolic rate, physical activity, and/or adaptive thermogenesis) will generally precede the development of obesity in the animal.
A “genetically modified,” “recombinant” or “transgenic” non-human animal (and like terms) of the invention comprises modifications to its genome such that the animal has a reduction in functional Jhdm2a gene expression. The genomic modification can be present in the somatic and/or germ cells of the animal, optionally, in all of the somatic cells and/or all of the germ cells of the animal.
“Congenic” strains may be created, which are useful for generating non-human animals (e.g., mice) that are nearly identical except for a selected genotype/phenotype (see, e.g., U.S. Pat. No. 7,202,393 to Matsushima). Congenic animals can be generated by mating two genetically distinct inbred strains and then backcrossing the descendants with one of the parental or ancestral strains (the “recipient” strain), e.g., for two generations, followed by inbreeding sister and brother, with or without selecting for particular markers or phenotypes. Using this method, the recipient on average contributes the greater proportion of the genome to each congenic strain. Backcrossing generally increases homozygosity twice as fast as sibling mating. Other methods of creating congenic strains may also be used, and alternative methods may be used, as will be appreciated by those of skill in the art. For example, the number of backcrosses may vary, resulting in different genomic proportions from the recipient. Selection for the genotype/phenotype of interest may also be performed at certain steps as desired.
In representative embodiments, the non-human animal of the invention is obese. Typically, the animal exhibits the onset of obesity in adulthood (e.g., after reaching sexual maturity). For example, in mice, the onset of obesity may be at about 1, 2, 3, 4 or 5 months. Generally, obesity is understood to be an increase in body mass of 20% or more over the ideal body mass. In particular embodiments, the animal has an increase in body mass of at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200% or more and/or an increase in the mass and/or proportion of body fat by weight of at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200% or more relative to a suitable control (e.g., a wild-type animal of the same species, strain, general age and/or gender, for example, a wild-type littermate). Methods of determining normal body weight are known in the art. For example, in humans, normal body weight can be defined as a BMI index of 18.5-24.9 kg/meter2 (NHLBI (National Heart Lung and Blood Institute) Obesity Education Initiative. The Practical Guide-Identification, Evaluation and Treatment of Overweight and Obesity in Adults. NIH Publication No. 00-4084 (2000); obtainable at http://www.nhlbi.nih.gov/guidelines/obesity/prctad_b.pdf). In particular embodiments, the non-human animal has a BMI index of about 24.9 kg/meter2 or greater.
In exemplary embodiments, the non-human animal exhibits obesity when fed a high fat diet (e.g., adult-onset obesity on a high fat diet). The term “high fat diet” is understood in the art and includes but is not limited to diets in which at least about 50%, 60%, 70% or more of the calories are derived from fat. In a representative example of this embodiment, the non-human animal is heterozygous for the genetic modification resulting in a reduction in functional Jhdm2a gene expression, e.g., an obese heterozygous animal.
Obesity can be determined by any suitable method known in the art, such as by determining body weight, BMI, mass of body fat, body fat as a percentage of body weight, and the like.
In embodiments of the invention, the animal exhibits glucose intolerance, hyperglycemia (e.g., fasting hyperglycemia), insulin resistance, hyperinsulinemia and/or hyperlipidemia relative to a wild-type animal. In embodiments of the invention, the onset of these characteristics is in the adult animal (e.g., after sexual maturity). Hyperglycemia is characterized by excessive blood (or plasma) glucose levels. Methods of diagnosing and evaluating hyperglycemia are known in the art. In general, fasting hyperglycemia is characterized by blood or plasma glucose concentration above the normal range after a subject has fasted for at least eight hours (e.g., the normal range is about 70-120 mg/dL). Postprandial hyperglycemia is generally characterized by blood or plasma glucose concentration above the normal range one to two hours after food intake by a subject.
By “insulin resistance” or “insulin insensitivity” it is meant a state in which a given level of insulin produces a less than normal biological effect (e.g., uptake of glucose). Insulin resistance is particularly prevalent in obese individuals or those with type-2 diabetes or metabolic syndrome. In type-2 diabetics, the pancreas is generally able to produce insulin, but there is an impairment in insulin action. As a result, hyperinsulinemia is commonly observed in insulin-resistant subjects. Insulin resistance is less common in type-I diabetics; although in some subjects, higher dosages of insulin have to be administered over time indicating the development of insulin resistance/insensitivity. The term “insulin resistance” or “insulin insensitivity” refers to systemic insulin resistance/insensitivity unless specifically indicated otherwise. Methods of evaluating insulin resistance/insensitivity are known in the art, for example, hyperinsulinemic/euglycemic clamp studies, insulin tolerance tests, uptake of labeled glucose and/or incorporation into glycogen in response to insulin stimulation, and measurement of known components of the insulin signaling pathway.
“Glucose intolerance” is characterized by an impaired ability to maintain blood (or plasma) glucose concentrations following a glucose load (e.g., by ingestion or infusion) resulting in hyperglycemia. Glucose intolerance is generally indicative of an insulin deficiency or insulin resistance. Methods of evaluating glucose tolerance/intolerance are known in the art, e.g., the oral glucose tolerance test.
“Hyperlipidemia” can include elevations in serum free fatty acids, triglycerides, LDL, HDL, VLDL and/or total cholesterol. Methods of measuring serum lipids are standard in the art.
In representative embodiments, the non-human transgenic animals of the invention display metabolic impairments in energy metabolism and/or impairments in the AMPK-PGC-1α axis.
Accordingly, in some embodiments, the animal has a reduction in energy expenditure (e.g., basal metabolic rate, physical activity and/or adaptive thermogenesis) relative to a wild-type animal. Methods of determining basal metabolic rate are known in the art, for example, by measuring oxygen consumption and/or heat generation by the animal. Methods of evaluating physical activity are known in the art, e.g., by measuring horizontal and/or vertical movement. Methods of measuring adaptive thermogenesis are also known in the art, for example, the ability of the animal to maintain body weight in response to cold stress.
In representative embodiments, there are defects in brown adipose tissue function and/or cold intolerance in the non-human animals of the invention relative to a wild-type animal.
In embodiments of the invention, there is a reduction in cold stress induced PPARα, UCP1 and/or PGC-1α upregulation in the animal (e.g., in brown adipose tissue, white adipose tissue, liver, skeletal muscle and/or testis) relative to the level of induction observed in a wild-type animal.
In embodiments of the invention, expression of PGC-1α and/or its downstream targets (including without limitation MCAD, ATP5J, UCP2, COX7A and other downstream targets of PGC-1α), PPARα, and/or PGC-1β is reduced in the animal (e.g., brown adipose tissue, white adipose tissue, liver, skeletal muscle and/or testis) relative to a wild-type animal.
As used herein, the terms “express,” “expressing,” or “expression” (or grammatical variants thereof) in reference to a gene or coding sequence can refer to transcription to produce an RNA and, optionally translation to produce a functional protein or peptide. Thus, the terms “express,” “expressing,” “expression” and the like can refer to events at the transcriptional, post-transcriptional, translational and/or post-translational level.
In embodiments of the invention, serum leptin levels are increased and/or there is increased AMPK phosphorylation in the animal (e.g., in brown adipose tissue, white adipose tissue, liver, skeletal muscle and/or testis) relative to a wild-type animal. According to this embodiment of the invention, the animal can be an adult animal (e.g., after sexual maturity) after the onset of excess fat accumulation and is optionally an obese animal.
In embodiments of the invention, there is a reduction in AICAR-induced AMPK activation of cellular respiration (e.g., in brown adipose tissue, white adipose tissue, liver, skeletal muscle and/or testis) by the non-human animals of the invention relative to a wild-type animal.
In embodiments of the invention, there is a reduction in AMPK-mediated PGC-1α induction (e.g., in brown adipose tissue, white adipose tissue, liver, skeletal muscle and/or testis) in the non-human animals of the invention relative to a wild-type animal.
In embodiments of the invention, there is a reduction in β-oxidation (e.g., in brown adipose tissue, white adipose tissue, liver, skeletal muscle and/or testis) in the non-human animals of the invention relative to a wild-type animal.
In embodiments of the invention, there is a reduction in mono- and/or di-methylation at lysine 9 of histone H3 (H3K9me1 and H3K9me2) in response to AICAR induced H3K9 demethylation at the PGC-1α promoter (e.g., in brown adipose tissue, white adipose tissue, liver, skeletal muscle and/or testis) in the non-human animals of the invention relative to a wild-type animal.
In other representative embodiments of the invention, there is an impairment in the PPARα signaling pathway (e.g., in brown adipose tissue, white adipose tissue, liver, skeletal muscle and/or testis) in the non-human animals of the invention relative to a wild-type animal. As non-limiting examples, there can be impaired expression and/or activity of PPARα and/or one or more of its downstream targets (including without limitation UCP2, MCAD, LCAD, VLCAD, AQP7).
In representative embodiments of the invention, H3K9me2 levels at the PPAR responsive element (PPRE) of the PPARα gene are increased (e.g., in brown adipose tissue, white adipose tissue, liver, skeletal muscle and/or testis) in the non-human animals of the invention relative to a wild-type animal.
In representative embodiments, there is an impairment in β-adrenergic signaling pathways (e.g., in brown adipose tissue, white adipose tissue, liver, skeletal muscle and/or testis) in the non-human animals of the invention relative to a wild-type animal. As a non-limiting illustration, β-adrenergic receptor stimulated (e.g., in the presence of β-adrenergic receptor agonists such as isoproterenol) O2 consumption and/or glycerol release is reduced (e.g., in brown adipose tissue, white adipose tissue, liver, skeletal muscle and/or testis) in the non-human animals of the invention relative to a wild-type animal. As a further example, β-adrenergic receptor stimulated (e.g., in the presence of β-adrenergic receptor agonists such as isoproterenol) expression and/or activity of one or more downstream targets involved in mitochondrial function (including without limitation PPARα, UCP3, CPT2 and/or LCAD) is reduced (e.g., in brown adipose tissue, white adipose tissue, liver, skeletal muscle and/or testis) in the non-human animals of the invention relative to a wild-type animal.
In representative embodiments of the invention, β-adrenergic receptor mediated reductions in H3K9me2 levels at the Ucp1 enhancer region are reduced (e.g., in brown adipose tissue, white adipose tissue, liver, skeletal muscle and/or testis) in the non-human animals of the invention relative to a wild-type animal.
In representative embodiments of the invention, β-adrenergic receptor mediated increase in recruitment of transcription factors and/or co-activators to the Ucp1 gene is reduced (e.g., in brown adipose tissue, white adipose tissue, liver, skeletal muscle and/or testis) in the non-human animals of the invention relative to a wild-type animal. For example, β-adrenergic receptor mediated increase in PPARγ, RXRα, PGC1α, CBP/p300 and/or SRC1 binding to the UCP1 enhancer is reduced (e.g., in brown adipose tissue, white adipose tissue, liver, skeletal muscle and/or testis) in the non-human animals of the invention relative to a wild-type animal.
In reference to the metabolic impairments and dysregulation of gene expression discussed herein, the terms “reduce,” “reduced,” “reducing” and the like refer to a decrease of at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98% or more in the relevant parameter.
In reference to the metabolic impairments and dysregulation of gene expression discussed herein, the terms “increase,” “increases,” “increasing” and like terms refer to an elevation of at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200% or more in the relevant parameter.
In embodiments of the invention, the impairments in energy expenditure, gene regulation and/or plasma leptin concentrations described herein commence in the adult animal (e.g., after the animal reaches sexual maturity). Alternatively, in embodiments of the invention, the impairments in energy expenditure, gene regulation, and plasma leptin concentrations described herein commence in the immature animal (e.g., prior to sexual maturation, for example, an animal that is younger than about 8, 7, 6, 5, 4, 3, 2 or 1 week(s) of age).
In some embodiments, the animal does not have a significant change in plasma corticosterone, triiodothyronine (T3) and/or norepinephrine as compared with a suitable control.
The transgenic non-human animals of the invention have been genetically modified to result in a reduction in functional Jhdm2a gene expression. “Functional Jhdm2a gene expression” and like terms refers to expression at the mRNA and/or protein level, e.g., transcription to produce an mRNA encoding a functional JHDM2A protein (for example, having H3K9 demethylase activity). Thus, the genetic modification can result in an impairment at the transcriptional, post-transcriptional, translational and/or post-translational level and/or can produce a protein that has substantially reduced or no biologic activity (e.g., histone demethylase activity).
A variety of methods are known in the art for reducing functional gene expression including methods for producing “knockout,” “knockin,” and “knockdown” animals, all of which may be used to produce the transgenic non-human animals of the present invention. Thus, in some embodiments, the transgenic non-human animal comprises a heterozygous and/or homozygous disruption in the Jhdm2a gene, e.g., as a knockout or knockin mutation. The disruption can encompass any portion of the gene that results in reduced functional Jhdm2A protein including the 5′ untranslated region, one or more exons and/or one or more introns. In particular embodiments, the disruption comprises the catalytic JmjC domain. For example, in a transgenic mouse of the invention, the disruption can comprise the catalytic JmjC domain in exons 22, 23 and 24, and can optionally comprise all of exons 22, 23 and 24. In other embodiments, the disruption can comprise a “knockin” upstream of the JmjC catalytic domain.
In particular embodiments, the non-human animal of the invention is homozygous for the genetic modification (e.g., disruption to the Jhdm2a gene) that results in reduced functional Jhdm2a gene expression. According to this embodiment, production of functional JHDM2a protein can be reduced by 75%, 85%, 90%, 95%, 99% or more relative to a suitable control in one or more, or even all, tissues.
In other embodiments, the non-human animal of the invention is heterozygous for the genetic modification that results in reduced functional Jhdm2a gene expression. According to this embodiment, production of functional JHDM2a protein can be reduced about one-half the level observed in homozygous animals (e.g., by about 40% or 45% to about 50%, 55% or 60% or about 50% to about 55% or 60%).
In exemplary embodiments, the invention provides a transgenic non-human animal (e.g., mouse) whose genome comprises a homozygous disruption of the Jhdm2a gene, wherein the disruption results in a deficiency in functional Jhdm2a gene expression in the animal, and wherein the animal exhibits one or more of the following characteristics relative to a wild-type animal: (i) obesity, (ii) systemic insulin resistance, (iii) hyperinsulinemia, (iv) hyperlipidemia, (v) reduction in energy expenditure, (vi) reduction in cold stress induced PPARα, UCP1 and/or PGC-1α upregulation; (vii) reduction in expression of PGC-1α and/or its down-stream targets (including UCP2, MCAD, ATP5J, COX7A), PPARα and/or PGC-16; (viii) increase in leptin concentrations in blood, plasma or serum; (ix) increased AMPK phosphorylation; (x) reduction in AICAR-induced AMPK activation of cellular respiration; (xi) reduction in AMPK-mediated PGC-1α induction; (xii) reduction in β-oxidation; (xiii) reduction in AICAR induced H3K9me1 and H3K9me2 demethylation at the PGC-1α promoter; (xiv) reduction in PPARα signaling pathway activity (described herein); (xv) increase in H3K9me2 levels at the PPRE of the PPARα gene; (xvi) reduction in β-adrenergic receptor signaling pathway activity (described herein); and/or (xvii) reduction in β-adrenergic receptor mediated H3K9 demethylation at the Ucp1 enhancer region. In particular embodiments, one or more of the foregoing characteristics exhibits a pattern of adult-onset.
“Wild-type” gene sequences of a given species are those DNA or protein sequences that are most highly conserved within or across species and/or which are generally accepted as the wild-type gene in the art. For example, the genomic sequences of the mouse and human Jhdm2a genes can be found at GenBank Accession Nos. NC—000072.5. and NC—000002.10, respectively.
As used here, a “wild type” non-human animal is one that does not contain the genetic manipulations of the invention to reduce functional Jhdm2a gene expression. In representative embodiments, the genome of the wild-type non-human animal is otherwise significantly or substantially identical to the transgenic and/or congenic non-human animal of the invention (e.g., littermates).
A “knockout” of a target gene means an alteration in a host cell genome that results in reduced expression of the target gene, e.g., by introduction of a mutation (e.g., insertion) into a coding or noncoding region of the target gene, which mutation reduces expression of the target gene. Mammals containing a knockout of Jhdm2a may be heterozygous or homozygous with respect to the mutation that causes the knockout.
The term “conditional knockout” as used herein refers to an animal in which the knocked out gene is selectively or preferentially knocked out in a particular tissue (e.g., in brown adipose tissue, white adipose tissue, skeletal muscle, testis and/or liver) and/or at a particular time of development. Such conditional knockout animals can be produced by a variety of techniques, such as with site-specific recombinases such as Cre/lox (to create “floxed” mice or mice having a “floxed” gene) or Tnpl/TRT (see, e.g., U.S. Pat. No. 7,083,976), and/or with a tetracycline-controllable transactivator (see, e.g., U.S. Pat. Nos. 6,783,757 and 6,252,136).
“Floxed” mice or “Cre/lox conditional knockout” animals are known. The Cre recombinase catalyzes recombination between 34 by loxP recognition sequences (Sauer, B. and Henderson, N., Proc. Natl. Acad. Sci. USA 85:5166-5170, 1988). The loxP sequences can be inserted into the genome of embryonic stem cells by homologous recombination such that they flank one or more exons of a gene of interest (making a “floxed” gene). Animals homozygous for the floxed gene are generated from these embryonic stem cells by conventional techniques and are crossed to a second animal that harbors a Cre transgene under the control of a tissue type- or cell type-specific transcriptional promoter. In progeny that are homozygous for the floxed gene and that carry the Cre transgene, the floxed gene will be deleted by Cre/loxP recombination, but only in those cell types in which the Cre gene-associated promoter is active. See U.S. Pat. No. 6,583,333; see also U.S. Pat. No. 6,946,244.
The recombinase enzyme can be provided by any suitable method. For example, purified recombinase enzyme can be provided by direct microinjection. In another embodiment, recombinase is expressed from a co-transfected construct or vector in which the recombinase gene is operably linked to a functional promoter. Further, tissue-specific or inducible recombinase constructs can be employed which allow the choice of when and where recombination occurs. One method for practicing the inducible forms of recombinase-mediated recombination involves the use of vectors that use inducible or tissue-specific promoters or other gene regulatory elements to express the desired recombinase activity. The inducible expression elements can optionally be positioned to allow the inducible control or activation of expression of the desired recombinase activity. Examples of such inducible promoters or other gene regulatory elements include, but are not limited to, tetracycline, metallothionein, ecdysone, and other steroid-responsive promoters, rapamycin responsive promoters, and the like (No, et al. Proc. Natl. Acad. Sci. USA, 93:3346-51 (1996); Furth, et al. Proc. Natl. Acad. Sci. USA, 91:9302-6 (1994)). Additional control elements that can be used include promoters requiring specific transcription factors such as viral promoters. Vectors incorporating such promoters only express recombinase activity in cells that express the necessary transcription factors. In particular embodiments, the promoters or regulatory elements is preferentially or specifically active or inducible in brown adipose tissue, white adipose tissue, liver, skeletal muscle and/or testis.
Another example of a conditional knockout can be made using FRT-Flpase technology, also well know to those skilled in the art of gene-targeting mice.
A large number of suitable alternative site-specific recombinases have been described, and their genes can be used to produce the transgenic non-human animals of the invention. Such recombinases include the Int recombinase of bacteriophage lambda (with or without Xis) (Weisberg, R. et. al., in Lambda II, (Hendrix, R., et al., Eds.), Cold Spring Harbor Press, Cold Spring Harbor, N.Y., pp. 211-50 (1983)); Tpnl and the β-lactamase transposons (Mercier, et al., J. Bacteriol., 172:3745-57 (1990)); the Tn3 resolvase (Flanagan & Fennewald J. Molec. Biol., 206:295-304 (1989); Stark, et al., Cell, 58:779-90 (1989)); the yeast recombinases (Matsuzaki, et al., J. Bacteriol., 172:610-18 (1990)); the B. subtilis SpoIVC recombinase (Sato, et al., J. Bacteriol. 172:1092-98 (1990)); the Hin recombinase (Glasgow, et al., J. Biol. Chem., 264:10072-82 (1989)); immunoglobulin recombinases (Malynn, et al., Cell, 54:453-460 (1988)); and the Cin recombinase (Haffter & Bickle, EMBO J., 7:3991-3996 (1988); Hubner, et al., J. Molec. Biol., 205:493-500 (1989)). Such systems are discussed by Echols (J. Biol. Chem. 265:14697-14700 (1990)); de Villartay (Nature, 335:170-74 (1988)); Craig, (Ann. Rev. Genet., 22:77-105 (1988)); Poyart-Salmeron, et al., (EMBO J. 8:2425-33 (1989)); Hunger-Bertling, et al. (Mol Cell. Biochem., 92:107-16 (1990)); and Cregg & Madden (Mol. Gen. Genet., 219:320-23 (1989)).
Other transgenic non-human animals of the invention can comprise any mutation that impairs functional Jhdm2a gene expression, by reducing transcription, translation and/or post-translational processing and the like and/or by producing a JHDM2A protein with substantially reduced or no detectable histone demethylase activity. Any nucleotide in a 5′ regulatory region (e.g., the promoter), an exonic region and/or an intron region of the Jhdm2a gene can be mutated or deleted in order to produce a mouse that is defective for functional Jhdm2a gene expression. Such mice can be generated with a targeting construct containing a site-specific mutation, or as a “knock-in”, in which a mutated sequence is targeted to an acceptor site, such as one generated in a conventional gene-targeting strategy.
An “RNAi knockdown” of a target gene means an alteration in the animal's genome that results in altered expression of the target gene, e.g., by introduction of a expression cassette that encodes an RNAi (e.g., shRNA or siRNA) that binds to the target gene or its transcripts to decrease expression thereof. Mammals containing a knockdown of Jhdm2a may be heterozygous or homozygous with respect to the insert that expresses the sequence responsible for expressing RNAi targeted against Jhdm2a. See, e.g., D. Pawitt et al., RNAi-knock-down mice: an emerging technology for post-genomic functional genetics, Cytogenet. Genome Res. 105 (2-4): 412-21 (2004).
A “knock-in” of a target gene generally refers to the replacement of endogenous genetic material (e.g., a gene or a portion of a gene) with exogenous genetic material. The transgenic non-human animal may be heterozygous or homozygous with respect to the mutation (e.g., insertion) that causes the knock-in.
The production of transgenic animals, including “knockout,” “knockin,” and “knockdown” animals, is known and can be carried out in accordance with known techniques or variations thereof which will be apparent to those skilled in the art, for example, U.S. Pat. No. 7,115,795 to Forsberg et al. (University of Guelph), U.S. Pat. No. 6,888,047 to Wu et al. (New York University), and U.S. Pat. No. 6,872,868 to Wagner et al. (Ohio University) all disclose transgenic non-human mammals derived through microinjection of DNA into the pronucleus or cytoplasm of embryos at the single cell stage. See also U.S. Pat. No. 5,175,385 to Wagner et al. and U.S. Pat. No. 5,175,384 to Krimpenfort et al. More specifically, “knockin” and “knockout” transgenic rats derived through microinjection of fertilized ova are disclosed in U.S. Pat. No. 7,262,336 to Young et al. (Wyeth) and U.S. Pat. No. 6,372,956 to Goldsmith et al. (The J. David Gladstone Institutes) “Knockin” and “knockout” cows are also known: U.S. Pat. No. 7,074,983 to Robl et al.
See also U.S. Pat. No. 7,022,893 to Takeda et al. and U.S. Pat. No. 6,218,595 to Giros et al., as well as U.S. Pat. No. 6,344,596 to W. Velander et al. (American Red Cross); U.S. Pat. No. 6,339,183 to T. T. Sun (New York University); U.S. Pat. No. 6,331,658 to D. Cooper and E. Koren; U.S. Pat. No. 6,255,554 to H. Lubon et al. (American National Red Cross; Virginia Polytechnic Institute); U.S. Pat. No. 6,204,431 to P. Prieto et al. (Abbott Laboratories); U.S. Pat. No. 6,166,288 to L. Diamond et al. (Nextran Inc., Princeton, N.J.); U.S. Pat. No. 5,959,171 to J. M. Hyttinin et al. (Pharming BV); U.S. Pat. No. 5,880,327 to H. Lubon et al. (American Red Cross); U.S. Pat. No. 5,639,457 to G. Brem; U.S. Pat. No. 5,639,940 to I. Garner et al. (Pharmaceutical Proteins Ltd.; Zymogenetics Inc); U.S. Pat. No. 5,589,604 to W. Drohan et al. (American Red Cross); U.S. Pat. No. 5,602,306 to Townes et al. (UAB Research Foundation); U.S. Pat. No. 4,736,866 to Leder and Stewart (Harvard); and U.S. Pat. No. 4,873,316 to Meade and Lonberg (Biogen).
The non-human animals of the present invention include progeny and their descendents of first generation animals produced by the methods described herein. Such animals, including congenic animals, can be produced in accordance with known techniques, including but not limited to those described in U.S. Pat. No. 6,465,714. In general, progeny can be created by (a) providing a first (mate or female) recombinant parent animal produced as described above, and a second parent animal, wherein at least the first parent exhibits a phenotype of the invention; and then (b) crossing the first and second parent animal with one another to produce a progeny animal that exhibits that phenotype. Subsequent generations can be further produced in accordance with known techniques.
The invention also encompasses methods of identifying candidate compounds for preventing and/or treating obesity, diabetes, metabolic syndrome, hyperlipidemia, insulin resistance, hyperinsulinemia, hyperglycemia and/or glucose intolerance using the non-human animals of the invention, cells isolated therefrom, cell cultures comprising such cells or cell cultures produced by culturing such cells.
Thus, in representative embodiments, the invention provides a method of identifying a candidate compound for treating obesity, diabetes, metabolic syndrome, hyperlipidemia, insulin resistance, hyperinsulinemia, hyperglycemia and/or glucose intolerance, the method comprising: administering a compound to an obese non-human transgenic animal of the invention (e.g., an adult animal, for example, an animal that is at least about 2, 3, 4, 5, 6, 7, 8 months of age or older); and determining the body weight and/or level of obesity in the animal, wherein a reduction in body weight and/or obesity in the animal administered the compound as compared with an untreated control animal indicates that the compound is a candidate for treating obesity, diabetes, metabolic syndrome, hyperlipidemia, insulin resistance, hyperinsulinemia, hyperglycemia and/or glucose intolerance.
Generally, the compound is administered for a time sufficient for a reduction in body weight and/or level of obesity to be observed if the compound is effective (e.g., at least about 3 days, 1 week, 2 weeks, 3 weeks, four weeks, 8 weeks or longer).
The invention also provides a method of identifying a candidate compound for preventing obesity, diabetes, metabolic syndrome, hyperlipidemia, insulin resistance, hyperinsulinemia, hyperglycemia and/or glucose intolerance, the method comprising: administering a compound to a non-human transgenic animal of the invention prior to the onset of obesity (e.g., to an immature animal prior to sexual maturation, for example, an animal that is younger than about 8, 7, 6, 5, 4, 3, 2 or 1 week(s) of age) and for a time sufficient for the onset of obesity in an untreated control animal; and determining the body weight and/or degree of obesity in the non-human transgenic animal administered the compound, wherein a reduction in body weight and/or obesity in the transgenic non-human animal administered the compound relative to an untreated control animal indicates that the compound is a candidate for preventing obesity, diabetes, metabolic syndrome, hyperlipidemia, insulin resistance, hyperinsulinemia, hyperglycemia and/or glucose intolerance.
Methods of determining body weight and obesity are known in the art, for example, as described herein.
As a further aspect, the invention provides a method of identifying a candidate compound for treating obesity, diabetes, metabolic syndrome, hyperlipidemia, insulin resistance, hyperinsulinemia, hyperglycemia and/or glucose intolerance, the method comprising: administering a compound to an obese non-human transgenic animal of the invention (e.g., an adult animal, for example, an animal that is at least about 2, 3, 4, 5, 6, 7, 8 months of age or older); and determining energy expenditure in the animal, wherein an increase in energy expenditure in the animal administered the compound as compared with an untreated control animal indicates that the compound is a candidate for treating obesity, diabetes, metabolic syndrome, hyperlipidemia, insulin resistance, hyperinsulinemia, hyperglycemia and/or glucose intolerance.
Generally, the compound is administered for a time sufficient for an increase in energy expenditure to be observed if the compound is effective (e.g., at least about 3 days, 1 week, 2 weeks, 3 weeks, four weeks, 8 weeks or longer).
The invention further provides a method of identifying a candidate compound for preventing obesity, diabetes, metabolic syndrome, hyperlipidemia, insulin resistance, hyperinsulinemia, hyperglycemia and/or glucose intolerance, the method comprising: administering a compound to a transgenic non-human animal of the invention prior to the onset of impaired energy expenditure (e.g., to an immature animal prior to sexual maturation, for example, an animal that is younger than about 8, 7, 6, 5, 4, 3, 2 or 1 week(s) of age) and for a time sufficient for the onset of impairments in energy expenditure in an untreated control animal; and determining energy expenditure in the transgenic non-human animal administered the compound, wherein an increase in energy expenditure in the transgenic non-human animal administered the compound as compared with an untreated control animal indicates that the compound is a candidate for preventing obesity, diabetes, metabolic syndrome, hyperlipidemia, insulin resistance, hyperinsulinemia, hyperglycemia and/or glucose intolerance.
Methods of determining energy expenditure and impairments in energy expenditure are routine in the art and include without limitation evaluating basal metabolic rate, physical activity and/or adaptive thermogenesis.
In other representative embodiments, the invention provides a method of identifying a candidate compound for treating obesity, diabetes, metabolic syndrome, hyperlipidemia, insulin resistance, hyperinsulinemia, hyperglycemia and/or glucose intolerance, the method comprising: administering a compound to a non-human transgenic animal of the invention; and determining β-adrenergic receptor stimulated (e.g., by administering in a β-adrenergic receptor agonist such as isoproterenol to the animal) O2 consumption by the transgenic non-human animal, wherein an increase in β-adrenergic receptor stimulated O2 consumption in the transgenic non-human animal administered the compound as compared with an untreated control animal indicates that the compound is a candidate for treating obesity, diabetes, metabolic syndrome, hyperlipidemia, insulin resistance, hyperinsulinemia, hyperglycemia and/or glucose intolerance. Optionally, this embodiment of the invention can be practiced with an immature animal prior to sexual maturation, for example, an animal that is younger than about 8, 7, 6, 5, 4, 3, 2 or 1 week(s) of age. Alternatively, in some instances, this embodiment can be practiced with an adult animal, for example, an animal that is at least 2, 3, 4, 5, 6, 7, 8 months of age or older, optionally an obese adult animal.
As used herein, an “untreated control” animal can be any suitable control including but not limited to a transgenic non-human animal that has not been given any treatment or has been treated with a placebo. The untreated control can optionally be the same animal prior to treatment. In representative embodiments, the untreated control is from the same species and strain and is matched for age and, optionally, gender.
Thus, in some embodiments, the methods of the invention comprise a “base-line” measurement prior to administering the compound and one or more follow-up measurements after administration of the compound to evaluate effectiveness.
The administering step may be carried out by any suitable technique depending upon the particular compound, including parenteral administration (e.g., intraperitoneal, intramuscular, intravenous, intra-arterial administration), subcutaneous administration oral administration, inhalation administration, transdermal administration, etc.
The invention also provides cells isolated from the transgenic non-human animals of the invention. The invention further contemplates cell cultures comprising the cells isolated from the non-human transgenic animal and/or cell cultures produced by culturing the isolated cells. The cells and cell cultures derived from the non-human transgenic animals of the invention can be used in the screening methods of the invention.
Such cells and cell cultures include somatic cells and cultures including but not limited to brown adipose tissue cells and cultures, white adipose tissue cells and culture, liver cells and culture, skeletal muscle cells and culture, testis cells and culture, brain cells and culture, pancreatic cells and culture, fibroblast cells and culture, epithelial cells and culture, endodermal cells and culture, smooth muscle cells and culture, cardiac myocytes and culture, and kidney cells and culture, and germ cells and culture, etc.
In particular embodiments, the cell or cell culture is from an obese animal. In embodiments, the cell or cell culture is from an animal that exhibits impairments in energy expenditure. In embodiments of the invention, the cell or cell culture is from an adult animal. In embodiments the cell or cell culture is from an immature animal (e.g., prior to sexual maturation, for example, an animal that is younger than about 8, 7, 6, 5, 4, 3, 2 or 1 week(s) of age). In embodiments of the invention, the cell or cell culture is from an animal prior to the onset of obesity. In embodiments of the invention, the cell or cell culture is from an animal prior to the onset of impairments in energy expenditure.
The invention further provides a method of identifying a candidate compound for preventing or treating obesity, diabetes, metabolic syndrome, hyperlipidemia, insulin resistance, hyperinsulinemia, hyperglycemia and/or glucose intolerance, the method comprising: contacting a cell comprising a heterozygous or homozygous disruption of the Jhdm2a gene (described herein) with a compound; and determining:
(i) energy expenditure (e.g., oxygen consumption and/or β-oxidation),
(ii) the activity of the AMPK-PGC-1α axis (including without limitation determining expression of PGC-1α and/or its downstream targets (e.g., UCP2, MCAD, ATP5J and/or COX7A, etc.),
(iii) AMPK-mediated PGC-1α induction,
(iv) JHDM2A enrichment at the PGC-1α promoter, and/or
(v) AICAR induced H3K9me1 and/or H3K9me2 demethylation at the PGC-1α promoter,
wherein partial or complete normalization of one or more these parameters (e.g., increase in energy expenditure, JHDM2a enrichment at the PGC-1α promoter, the activity of the AMPK-PGC-1α axis and/or AICAR induced H3K9me1 and/or H3K9me2 demethylation at the PGC-1α promoter) in the cell contacted with the compound relative to an untreated control cell indicates that the compound is a candidate for preventing and/or treating obesity, diabetes, metabolic syndrome, hyperlipidemia, insulin resistance, hyperinsulinemia, hyperglycemia and/or glucose intolerance.
In representative embodiments of the foregoing method, the cell is derived from a transgenic non-human animal of the invention (cells are as described herein).
The invention further provides a method of identifying a candidate compound for preventing or treating obesity, diabetes, metabolic syndrome, hyperlipidemia, insulin resistance, hyperinsulinemia, hyperglycemia and/or glucose intolerance, the method comprising: contacting a cell comprising a heterozygous or homozygous disruption of the Jhdm2a gene (described herein) with a compound; and determining:
(i) PPARα signaling pathway activity, for example, by determining expression and/or activity of PPARα and/or one or more of its downstream targets, including without limitation UCP2, MCAD, LCAD, VLCAD, AQP7;
(ii) H3K9me2 levels at the PPRE of the PPARα gene;
(iii) β-adrenergic receptor signaling pathway activity, for example, by determining β-adrenergic receptor stimulated O2 consumption; glycerol release; expression and/or activity of one or more downstream targets involved in mitochondrial function (including without limitation PPARα, UCP3, CPT2 and/or LCAD); β-adrenergic receptor stimulated recruitment of transcription factors (including without limitation PPARγ, RXRα, PGC1α, CBP/p300 and/or SRC1) and/or co-activators to the Ucp1 gene; and/or β-adrenergic receptor stimulated upregulation of UCP1 expression and/or activity; and/or
(iv) β-adrenergic receptor mediated H3K9 demethylation at the Ucp1 enhancer region,
wherein partial or complete normalization of one or more of these parameters (e.g., increase in PPARα signaling pathway activity, β-adrenergic receptor signaling pathway activity and/or β-adrenergic receptor mediated H3K9 demethylation at the Ucp1 enhancer region and/or reduced H3K9me2 levels at the PPRE of the PPARα gene) in the cell contacted with the compound relative to an untreated control cell indicates that the compound is a candidate for preventing and/or treating obesity, diabetes, metabolic syndrome, hyperlipidemia, insulin resistance, hyperinsulinemia, hyperglycemia and/or glucose intolerance.
In representative embodiments, the cell is a cell derived from a transgenic non-human animal of the invention (cells are as described herein).
In particular embodiments, a nucleic acid comprising the appropriate regulatory region(s) (e.g., promoter and/or enhancer) of the PPARα gene operably associated with the coding sequence for a reporter protein (e.g., Green Fluorescent Protein, luciferase, β-galactosidase, alkaline phosphatase) is transiently and/or stably introduced into the cell or cell culture. The reporter protein can provide a simple read-out of PPARα expression and/or PPARα pathway activity.
Likewise, in other embodiments, a nucleic acid comprising the appropriate regulatory region(s) (e.g., promoter and/or enhancer) of the Ucp1 gene operably associated with the coding sequence for a reporter protein (e.g., Green Fluorescent Protein, luciferase, β-galactosidase, alkaline phosphatase) is transiently and/or stably introduced into the cell. The reporter protein can provide a simple read-out of UCP1 expression and/or β-adrenergic pathway activity.
In other representative embodiments, the invention further provides a method of identifying a candidate compound for preventing or treating obesity, diabetes, metabolic syndrome, hyperlipidemia, insulin resistance, hyperinsulinemia, hyperglycemia and/or glucose intolerance, the method comprising: contacting a cell comprising a heterozygous or homozygous disruption of the Jhdm2a gene (described herein) with a compound; and determining mitochondrial metabolism, wherein increased mitochondrial metabolism in the cell contacted with the compound relative to an untreated control cell indicates that the compound is a candidate for preventing and/or treating obesity, diabetes, metabolic syndrome, hyperlipidemia, insulin resistance, hyperinsulinemia, hyperglycemia and/or glucose intolerance.
In representative embodiments, the cell is derived from a transgenic non-human animal of the invention (cells are as described herein).
The screening methods of the invention can also be practiced with cells and cell cultures in which Jhdm2a expression is reduced, for example, using “knockdown” methods such as RNAi knockdown (e.g., using shRNA, siRNA, etc.), miRNA or antisense RNA approaches. Alternatively, the Jhdm2a gene can be disrupted. Methods of gene disruption are known in the art (for example, as described herein). Such cells and cell cultures include somatic cells and cultures including but not limited to brown adipose tissue cells and cultures, white adipose tissue cells and culture, liver cells and culture, skeletal muscle cells and culture, testis cells and culture, brain cells and culture, pancreatic cells and culture, fibroblast cells and culture, epithelial cells and culture, endodermal cells and culture, smooth muscle cells and culture, cardiac myocytes and culture, and kidney cells and culture, and germ cells and culture, etc. The cells and cell culture can further be embryonic cells and culture. The cell or cell culture can be contacted with the RNAi, miRNA or antisense RNA transiently by exogenously adding the RNAi to the cell or cell culture. Alternatively, the genome of the cell or cultured cells can be altered to express the RNAi, miRNA or antisense RNA molecule.
According to this embodiment, the cells can be derived from animals of any age including embryonic, neonatal, immature, senescent and adult animals and can further be from obese animals. The cells can also be derived from immortalized cell lines.
The invention also provides a method of identifying a candidate compound for preventing or treating obesity, diabetes, metabolic syndrome, hyperlipidemia, insulin resistance, hyperinsulinemia, hyperglycemia and/or glucose intolerance, the method comprising: contacting a Jhdm2a “knockdown” cell or cell culture with a compound; and determining:
(i) energy expenditure (e.g., oxygen consumption and/or β-oxidation),
(ii) the activity of the AMPK-PGC-1α axis (including without limitation determining expression of PGC-1α and/or its downstream targets (e.g., UCP2, MCAD, ATP5J and/or COX7A, etc.),
(iii) AMPK-mediated PGC-1α induction,
(iv) JHDM2A enrichment at the PGC-1α promoter, and/or
(v) AICAR H3K9me1 and/or H3K9me2 demethylation at the PGC-1α promoter,
wherein partial or complete normalization of one or more these parameters (e.g., increase in energy expenditure, JHDM2a enrichment at the PGC-1α promoter, the activity of the AMPK-PGC-1α axis and/or AICAR induced H3K9me1 and/or H3K9me2 demethylation at the PGC-1α promoter) in the cell or cell culture contacted with the compound relative to an untreated control cell or cell culture indicates that the compound is a candidate for preventing and/or treating obesity, diabetes, metabolic syndrome, hyperlipidemia, insulin resistance, hyperinsulinemia, hyperglycemia and/or glucose intolerance.
The invention also provides a method of identifying a candidate compound for preventing or treating obesity, diabetes, metabolic syndrome, hyperlipidemia, insulin resistance, hyperinsulinemia, hyperglycemia and/or glucose intolerance, the method comprising: contacting a Jhdm2a “knockdown” cell or cell culture with a compound; and determining:
(i) PPARα signaling pathway activity, for example, by determining expression and/or activity of PPARα and/or one or more of its downstream targets, including without limitation UCP2, MCAD, LCAD, VLCAD, AQP7;
(ii) H3K9me2 levels at the PPRE of the PPARα gene;
(iii) β-adrenergic receptor signaling pathway activity, for example, by determining β-adrenergic receptor stimulated O2 consumption; glycerol release; expression and/or activity of one or more downstream targets involved in mitochondrial function (including without limitation PPARα, UCP3, CPT2 and/or LCAD); β-adrenergic receptor stimulated recruitment of transcription factors (including without limitation PPARγ, RXRα, PGC1α, CBP/p300 and/or SRC1) and/or co-activators to the Ucp1 gene; and/or β-adrenergic receptor stimulated upregulation of UCP1 expression and/or activity; and/or
(iv) β-adrenergic receptor mediated H3K9 demethylation at the Ucp1 enhancer region,
wherein partial or complete normalization of one or more of these parameters (e.g., increase in PPARα signaling pathway activity, β-adrenergic receptor signaling pathway activity and/or β-adrenergic receptor mediated H3K9 demethylation at the Ucp1 enhancer region and/or reduced H3K9me2 levels at the PPRE of the PPARα gene) in the cell or cell culture contacted with the compound relative to an untreated control cell or cell culture indicates that the compound is a candidate for preventing and/or treating obesity, diabetes, metabolic syndrome, hyperlipidemia, insulin resistance, hyperinsulinemia, hyperglycemia and/or glucose intolerance.
In particular embodiments, a nucleic acid comprising the appropriate regulatory region(s) (e.g., promoter and/or enhancer) of the PPARα gene operably associated with the coding sequence for a reporter protein (e.g., Green Fluorescent Protein, luciferase, β-galactosidase, alkaline phosphatase) is transiently and/or stably introduced into the cell or cell culture. The reporter protein can provide a simple read-out of PPARα expression and/or PPARα pathway activity.
Likewise, in other embodiments, a nucleic acid comprising the appropriate regulatory region(s) (e.g., promoter and/or enhancer) of the Ucp1 gene operably associated with the coding sequence for a reporter protein (e.g., Green Fluorescent Protein, luciferase, β-galactosidase, alkaline phosphatase) is transiently and/or stably introduced into the cell or cell culture. The reporter protein can provide a simple read-out of UCP1 expression and/or β-adrenergic pathway activity.
In other representative embodiments, the invention provides a method of identifying a candidate compound for preventing or treating obesity, diabetes, metabolic syndrome, hyperlipidemia, insulin resistance, hyperinsulinemia, hyperglycemia and/or glucose intolerance, the method comprising: contacting a Jhdm2a “knockdown” cell or cell culture with a compound; and determining mitochondrial metabolism, wherein increased mitochondrial metabolism in the cell or cell culture contacted with the compound relative to an untreated control cell or cell culture indicates that the compound is a candidate for preventing and/or treating obesity, diabetes, metabolic syndrome, hyperlipidemia, insulin resistance, hyperinsulinemia, hyperglycemia and/or glucose intolerance. Methods of measuring mitochondrial metabolism are known in the art (e.g., as described herein).
The foregoing embodiments have been described with respect to cells having a knockdown in Jhdm2a expression, but can also be practiced with any cell having a genetic modification that results in a reduction in Jhdm2a gene expression.
Those skilled in the art will appreciate that the inventive screening methods can be practiced with the animals and cells of the invention by detecting the activity of any suitable parameter (e.g., any of the molecular, biochemical, metabolic, physiological and/or phenotypic changes described herein) and evaluating the effect of a candidate compound on that parameter, for example, to determine if the compound can partially or completely normalize the affected parameter as compared with an appropriate control animal or cell.
In reference to the screening methods of the invention, the terms “reduce,” “reduced,” “reducing” and the like refer to a decrease of at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98% or more in the relevant parameter.
In reference to the screening methods of the invention, the terms “increase,” “increases,” “increasing” and like terms refer to an elevation of at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150% or more in the relevant parameter.
In particular embodiments, by the terms “normalize,” “normalized,” “normalizing” and the like, it is meant that a parameter that is reduced in the animals and cells of the invention is increased to at least about 60%, 70%, 75%, 80%, 85%, 90%, 95% or more of the value in a suitable control animal (e.g., a wild-type animal of the same species, strain, general age and/or gender, for example, a wild-type littermate). In particular embodiments, the terms “normalize,” “normalized,” “normalizing” and the like indicate that a parameter that is increased in the animals and cells of the invention is reduced to less than about 200%, 150%, 140%, 130%, 125%, 120%, 110% or less of the value in a suitable control animal. In representative embodiments, by the terms “normalize,” “normalized,” “normalizing” and the like, it is meant that the value is essentially the same as in a suitable control animal.
The transgenic non-human animals of the invention are phenotypically similar to humans with diabetes (e.g., type II diabetes), obesity and/or metabolic syndrome. Thus, genetic markers in the Jhdm2a gene can be used to identify subjects at increased risk for developing obesity, diabetes (e.g., type II diabetes), metabolic syndrome, hyperinsulinemia, insulin resistance, glucose intolerance, hyperglycemia and/or hyperlipidemia.
Accordingly, the invention provides a method of identifying a mammalian subject (e.g., a human) at increased risk for developing obesity, diabetes (e.g., type II diabetes), metabolic syndrome, hyperinsulinemia, insulin resistance, glucose intolerance, hyperglycemia and/or hyperlipidemia, the method comprising detecting in the subject the presence of a genetic marker in the Jhmda2a gene, wherein the genetic marker is correlated with an increased risk of developing obesity, diabetes (e.g., type II diabetes), metabolic syndrome, hyperinsulinemia, insulin resistance, glucose intolerance, hyperglycemia and/or hyperlipidemia, thereby identifying a mammalian subject having increased risk of developing obesity, diabetes (e.g., type II diabetes), metabolic syndrome, hyperinsulinemia, insulin resistance, glucose intolerance, hyperglycemia and/or hyperlipidemia.
The invention also provides a method of identifying a mammalian subject at increased risk for developing obesity, diabetes (e.g., type II diabetes), metabolic syndrome, hyperinsulinemia, insulin resistance, glucose intolerance, hyperglycemia and/or hyperlipidemia, the method comprising: (a) correlating the presence of a genetic marker in the Jhdm2a gene with an increased risk of developing obesity, diabetes (e.g., type II diabetes), metabolic syndrome, hyperinsulinemia, insulin resistance, glucose intolerance, hyperglycemia and/or hyperlipidemia; and (b) detecting the presence of the genetic marker of (a) in a mammalian subject, thereby identifying a mammalian subject at increased risk for developing obesity, diabetes (e.g., type II diabetes), metabolic syndrome, hyperinsulinemia, insulin resistance, glucose intolerance, hyperglycemia and/or hyperlipidemia.
The invention also provides a method of identifying a genetic marker in the Jhdma2 gene correlated with increased risk of a mammalian subject developing obesity, diabetes (e.g., type II diabetes), metabolic syndrome, hyperinsulinemia, insulin resistance, glucose intolerance, hyperglycemia and/or hyperlipidemia, the method comprising: (a) identifying a mammalian subject that has developed obesity, diabetes (e.g., type II diabetes), metabolic syndrome, hyperinsulinemia, insulin resistance, glucose intolerance, hyperglycemia and/or hyperlipidemia; (b) detecting in the mammalian subject the presence of a genetic marker in the Jhdm2a gene; and (c) correlating the presence of the genetic marker of step (b) with the development of obesity, diabetes (e.g., type II diabetes), metabolic syndrome, hyperinsulinemia, insulin resistance, glucose intolerance, hyperglycemia and/or hyperlipidemia, thereby identifying a genetic marker in the Jhdm2a gene correlated with increased risk of a mammalian subject developing obesity, diabetes (e.g., type II diabetes), metabolic syndrome, hyperinsulinemia, insulin resistance, glucose intolerance, hyperglycemia and/or hyperlipidemia.
As a further aspect the invention provides a method of identifying a genetic marker in the Jhdma2 gene correlated with increased risk of a mammalian subject developing obesity, diabetes (e.g., type II diabetes), metabolic syndrome, hyperinsulinemia, insulin resistance, glucose intolerance, hyperglycemia and/or hyperlipidemia, the method comprising: (a) identifying a mammalian subject that has developed obesity, diabetes (e.g., type II diabetes), metabolic syndrome, hyperinsulinemia, insulin resistance, glucose intolerance, hyperglycemia and/or hyperlipidemia; (b) determining the nucleotide sequence of the Jhdm2a gene of the mammalian subject of (a); (c) comparing the nucleotide sequence of (b) with the wild-type nucleotide sequence of the Jhdm2a gene (e.g., a “reference sequence”); (d) detecting a genetic marker in the nucleotide sequence of (b); and (e) correlating the presence of the genetic marker of step (b) with the development of obesity, diabetes (e.g., type II diabetes), metabolic syndrome, hyperinsulinemia, insulin resistance, glucose intolerance, hyperglycemia and/or hyperlipidemia in the mammalian subject of (a).
In particular embodiments, the methods of the invention further comprise performing a population based study to detect the polymorphisms in a group of subjects with obesity, diabetes (e.g., type II diabetes), metabolic syndrome, hyperinsulinemia, insulin resistance, glucose intolerance, hyperglycemia and/or hyperlipidemia and racially and/or ethnically matched controls.
The term “genetic marker” as used herein refers to a region of a nucleotide sequence (e.g., in a chromosome) that is subject to variability (i.e., the region can be polymorphic for a variety of alleles). For example, a single nucleotide polymorphism (SNP) in a nucleotide sequence is a genetic marker that is polymorphic for two alleles. Other examples of genetic markers of this invention can include but are not limited to microsatellites, restriction fragment length polymorphisms (RFLPs), repeats (i.e., duplications), insertions, deletions, and the like.
The mammalian subject can be any animal that is susceptible to obesity, diabetes (e.g., type II diabetes), metabolic syndrome, hyperinsulinemia, insulin resistance, glucose intolerance, hyperglycemia and/or hyperlipidemia. Examples of subjects of this invention can include, but are not limited to, humans, non-human primates, dogs, cats, horses, cattle, goats, pigs, sheep, guinea pigs, mice, rats and rabbits, as well as any other domestic, commercially or clinically valuable animal, including animal models.
Human subjects can be male and/or female and can be of any age including neonates, infants, juveniles, adults and/or senescent individuals.
In the methods described herein, the detection of a genetic marker in a subject can be carried out according to methods well known in the art. For example, nucleic acid (e.g., DNA or RNA) is obtained from any suitable sample from the subject that will contain nucleic acid and the nucleic acid is then prepared and analyzed according to well-established protocols for the presence of genetic markers according to the methods of this invention. In some embodiments, analysis of the nucleic acid can be carried out by amplification of the region of interest according to amplification protocols well known in the art (e.g., polymerase chain reaction, ligase chain reaction, strand displacement amplification, transcription-based amplification, self-sustained sequence replication (3SR), Qβ replicase protocols, nucleic acid sequence-based amplification (NASBA), repair chain reaction (RCR) and boomerang DNA amplification (BDA)). The amplification product can then be visualized directly in a gel by staining or the product can be detected by hybridization with a detectable probe. When amplification conditions allow for amplification of all allelic types of a genetic marker, the types can be distinguished by a variety of well-known methods, such as hybridization with an allele-specific probe, secondary amplification with allele-specific primers, by restriction endonuclease digestion, or by electrophoresis. Thus, the present invention can further provide oligonucleotides for use as primers and/or probes for detecting and/or identifying genetic markers according to the methods of this invention.
The genetic markers of this invention can be correlated with obesity, diabetes (e.g., type II diabetes), metabolic syndrome, hyperinsulinemia, insulin resistance, glucose intolerance, hyperglycemia and/or hyperlipidemia according to methods well known in the art. In general, identifying such correlation involves conducting analyses that establish a statistically significant association and/or a statistically significant correlation between the presence of a genetic marker or a combination of markers and the phenotypic trait in the subject. An analysis that identifies a statistical association (e.g., a significant association) between the marker or combination of markers and the phenotype establishes a correlation between the presence of the marker or combination of markers in a subject and the particular phenotype being analyzed.
The correlation can involve one or more than one genetic marker (e.g., two, three, four, five, or more) in any combination. In some embodiments of this invention, one or more or all of the genetic markers are located in the JHDM2a gene.
The genetic markers can be used individually or in combination. Thus, in some embodiments, the methods of the invention can include correlations between various genetic markers located in the Jhdm2s gene and obesity, diabetes (e.g., type II diabetes), metabolic syndrome, hyperinsulinemia, insulin resistance, glucose intolerance, hyperglycemia and/or hyperlipidemia.
The genetic marker can be a single nucleotide polymorphism (SNP). Exemplary single nucleotide polymorphisms include but are not limited to T for G, T for A, C for A, C for T, A for G, A for C, A for T, G for A and G for T substitutions. SNPs within the human Jhdm2a gene are known (see
The present invention also provides a method wherein the genetic marker is a combination of the single nucleotide polymorphisms, or haplotypes, within the Jhdm2a gene, that is correlated with an aspect of obesity, diabetes (e.g., type II diabetes), metabolic syndrome, hyperinsulinemia, insulin resistance, glucose intolerance, hyperglycemia and/or hyperlipidemia.
The present invention is explained in greater detail in the following non-limiting Examples.
An embryonic stem (ES) cell clone with Jhdm2a gene trap allele (YHA186) was purchased from BayGenomics. Disruption of the Jhdm2a gene in ES cells was accomplished by the insertion of a β-Geo/Neo cassette in intron 10 (or intron 7 of splicing variant 2), see
Homozygous mice, verified by Southern blot analysis, were viable and born at Mendelian ratios (data not shown). Expression of the Jhdm2a (exon1-10)-β-GEO fusion transcript in homozygous (Jhdm2aG/G) and heterozygous (Jhdm2a+/G) mice was confirmed by RT-PCR (data not shown). However, a low level (−10%) of wild-type mRNA was also detected in Jhdm2aG/G mice, see
Using a bacterial artificial chromosome (BAC) clone, a targeting vector was constructed. In this vector, a loxP site and a β-Geo cassette flanked by two loxP sites were introduced into the mouse Jhdm2a locus, see
All results are presented as the mean±standard error (S.E.). Statistical comparisons were by Student's t tests. Statistical significance was set at p<0.05, where NS indicates not significant.
All animal experiments were performed according to procedures approved by the Institutional Animal Care and Use Committee (IACUC). Mice were maintained on a diet of standard rodent chow or a high-fat diet containing 60% fat-derived calories (58Y1, TestDiet, Richmond, Ind.) with 12 hour light and dark cycles. Body weight was measured weekly for a period of 8 months. For diet-induced obesity, 4 week-old mice were fed a high-fat diet for 2 months and body fat was measured weekly. The food intake of mice was measured using singly housed mice. Before measurement of food intake, the mice were acclimated to the housing environment for at least a week, and the intake data was collected during a 2-week period.
Primer sequences used for genotyping, qRT-PCR and ChIP analysis are listed in Table 1.
The results demonstrated that both male and female Jmdm1a−/− mice become obese in adulthood when compared with their wild-type littermates, see
Jhdm2a 1lox/1lox (knockout) mice were confirmed by PCR (
Body fat deposition in 4-month-old KO mice was determined using Magnetic Resonance Imaging. The analysis revealed marked body fat deposition in KO mice. However, non-adipose tissues (lean) were comparable between wild-type and Jhdm2a knockout mice, see
To determine whether Jhdm2a KO mice were prone to diet-induced obesity, 4-week-old mice were fed a high-fat diet (HFD) for 2 months after weaning and their body weight was measured weekly. The results shown in
In humans, excessive body fat deposition is closely linked to systemic insulin resistance, and diet-induced obesity is a primary risk factor for Type 2 diabetes (Qatanani, Genes Dev. 21, 1443 (2007); Kahn, J. Clin. Invest. 106, 473 (2000)). To determine whether Jhdm2a-deficiency also causes systemic insulin resistance, glucose and insulin tolerance tests (GTT and ITT, respectively) were performed. Glucose (1.5 mg/g) was intra-peritoneously infused for GTT. To measure glucose levels, blood was collected from the tail before and 15, 30, 60, 90, and 120 minutes after infusion. Glucose levels were determined using an Assure3 glucose meter (HYPOGUARD). For ITT, mice were fasted for 4 hours prior to intra-peritoneal injection of insulin (1 IU/kg). Body composition was evaluated by EchoMRI-100 (Echo Medical Systems). GTT showed that Jhdm2a KO mice were glucose-intolerant, see
The body weight of an animal is maintained through a balance between food intake and energy expenditure (Spiegelman, Cell 104, 531 (2001); Evans, Nat Med 10, 355 (2004)). To understand how Jhdm2a-deficiency results in obesity, food intake was analyzed before the onset of obesity using 6-week-old mice. It was found that caloric intake was not elevated in Jhdm2a KO mice, see
The effect of Jhdm2a deficiency on energy expenditure was analyzed. Systemic energy expenditure includes basal metabolic rate, physical activity, and adaptive thermogenesis (Spiegelman, Cell 104, 531 (2001)). Metabolic studies were performed using TSE LabMaster calorimetry Module (TSE-System) in the Clinical Nutrition Research Unit at the University of North Carolina. For respiration measurements, cultured cells were resuspended in PBS. Oxygen consumption was measured using a Clarke-type electrode (Diamond General) at baseline, and after treatment with 10 μM isoproterenol (ISO) (Wu, Cell 98, 115 (1999)). For normalization of the respiration rates, an aliquot of the cells was lysed and protein concentration was measured. It was found that loss of JHDM2A function results in decreased metabolic rate as evidenced by decreased oxygen (O2) consumption and heat generation, see
Given that the human counterpart of mouse Jhdm2a has been previously demonstrated to function as a transcriptional co-activator (Yamane, Cell 125, 483 (2006)), gene expression profiles of wild-type and Jhdm2a knockout skeletal muscles were compared using AFFYMETRIX microarray technology. The analysis was performed for skeletal muscle because of the high expression of Jhdm2a and also its role in energy expenditure.
Seven μg of total RNA was used to synthesize cDNA. A custom cDNA kit from Life Technologies was used with a T7-(dT)24 primer for this reaction. Biotinylated cRNA was then generated from the cDNA reaction using the BIOARRAY High Yield RNA Transcript Kit. The cDNA was then fragmented in fragmentation buffer (5× fragmentation buffer: 200 mM Tris-acetate, pH 8.1, 500 mM KOAc, 150 mM MgOAc) at 94° C. for 35 minutes before the chip hybridization. Fifteen μg of fragmented cDNA was then added to a hybridization cocktail (0.05 μg/μl fragmented cDNA, 50 pM control oligonucleotide B2, BioB, BioC, BioD, and cre hybridization controls, 0.1 mg/ml herring sperm DNA, 0.5 mg/ml acetylated BSA, 100 mM MES, 1 M [Na+], 20 mM EDTA, 0.01% TWEEN 20). Ten μg of cDNA was used for hybridization. Arrays were hybridized for 16 hours at 45° C. in the GENECHIP Hybridization Oven 640. The arrays were washed and stained with R-phycoerythrin streptavidin in the GENECHIP Fluidics Station 400. After this, the arrays were scanned with the HEWLETT PACKARD GENEARRAY Scanner. AFFYMETRIX GENECHIP Microarray Suite 5.0 software was used for washing, scanning, and basic analysis. Sample quality was assessed by examination of 3′ to 5′ intensity ratios of certain genes. Data analysis was carried out in GENESPRING GX 10.0 software (Agilent Technologies) using the RMA algorithm. Probes exhibiting at least a 2-fold reduction in mRNA level in Jhdm2a KO samples versus WT samples were subjected to pathway analysis and gene ontology enrichment analysis with significance threshold set to p<0.05. Data mapping onto the PPAR pathway was carried out using GENMAPP 2.0 (Gladstone Institutes).
Of the 43,000 probes analyzed, 602 probes (1.4%) were down-regulated at least two-fold and 220 probes (0.05%) were up-regulated at least two-fold in jhdm2a knockout soleus muscle. Gene ontology enrichment analysis indicated that the largest proportion of down-regulated genes were involved in metabolic processes (P=0.03), see
Consistent with down-regulation of Aqp7 (
In skeletal muscles, leptin promotes energy expenditure such as fat oxidation through activation of AMPK signaling, a major energy sensor at the cellular level (Minokoshi, Nature 415, 339 (2002); Kahn, Cell Metab. 1, 15 (2005); Hardie, Bioessays 23, 1112 (2001)). Activation of AMPK signaling was determined by treating C2C12 myotubes with 800 μM AICAR (Sigma Aldrich) for 16 hours. To prepare the C2C12 myotubes, primary myoblasts were isolated and established from neonatal mice as reported (Rando, J Cell Biol 125, 1275 (1994)). The primary myoblasts were then cultured in DMEM with 2% horse serum for the differentiation to primary myotubes. C2C12 cells were cultured in DMEM containing 10% FCS. Consistent with increased serum leptin levels in the Jhdm2a-deficient mice, see
To gain further support for the involvement of JHDM2A in AMPK-mediated mitochondrial function, the effect of Jhdm2a-deficiency on AMPK activation-mediated respiration in primary myotubes was measured. The results shown in
To understand how the loss of Jhdm2a function results in the down-regulation of PPARα and its target genes, the presence of Jhdm2a around defined PPRE sequences was analyzed using chromatin immunoprecipitation (ChIP) (Gulick, et al., Proc. Natl. Acad. Sci. USA 91:11012 (1994); Lopez, et al., Mol. Cell. Endocrinol. 205:169 (2003); Pineda Torra, et al., Mol. Endocrinol. 16:1013 (2002); Tugwood, et al., EMBO J. 11:433 (1992)). Results indicate that Jhdm2a binds to the PPRE of PPARα, but not to the PPREs of MCAD, Acox1 or Scp2, see
To demonstrate that the reduced PPARα expression in Jhdm2a knock-out muscle cells was a cell-intrinsic effect, qRT-PCR and ChIP analysis were performed using in vitro-cultured primary myocytes derived from newborn wild-type and knock-out mice. Results shown in
Because the brown adipose tissue (BAT) is the primary cellular component responsible for adaptive thermogenesis, the effect of Jhdm2a KO on BAT was analyzed. Brown adipose tissue was dissected 5 hours after cold exposure and subjected to quantitative RT-PCR. Total RNA was purified using an RNEASY or RNEASY lipid tissue kit (Qiagen). After DNase treatment, first-strand cDNA was synthesized using the IMPROM II Reverse Transcription System (Promega). Quantitative RT-PCR was performed using SYBR GREENER (Invitrogen). In addition to skeletal muscle, Jhdm2a is also highly expressed in BAT (
The phenotypic similarity between Jhdm2a knock-out mice and mice lacking β-adrenergic receptors (Bachman, et al., Science 297, 843 (2002)) raised the possibility that Jhdm2a might be a critical factor in β-adrenergic signaling. Consistent with this, β-adrenergic-stimulated oxygen consumption and glycerol release were greatly reduced in the Jhdm2a knock-out BAT, see
As one of the most important molecules involved in cold-induced thermogenesis in brown fat, the transcriptional regulation of Ucp1 has been extensively characterized (Lowell, et al. Nature 404, 652 (2000)). In addition to PPARα and PPARγ, other transcription factors and co-activators known to be involved in Ucp1 activation include Rxrα, Atf2, p300, Src1 and Pgc1α. qRT-PCR demonstrated that the expression of these genes was not significantly altered in the Jhdm2a knock-out BAT; however cold-induced upregulation of PPARα was defective in knock-out BAT, see
Given that cold-induced Ucp1 upregulation is intact in the PPARα-deficient mice (Kersten, et al., J. Clin. Invest. 103:1489 (1999)), the possibility was explored that Jhdm2a directly regulates Ucp1 expression in response to cold exposure. To ascertain that the effect of Jhdm2a on Ucp1 expression was cell-intrinsic, short-hairpin RNA (shRNA)-mediated was performed in the brown adipose cell line HIB1B using lentivirus-based shRNA. To generate a Jhdm2a knock-down HIB1B cell line, undifferentiated cells were infected with a lentiviral virus expressing a shRNA for Jhdm2a or control. The shRNA sequence for Jhdm2a was 5′-GCA GGT GTC ACT AGC CTT AAT-3′ (SEQ ID NO:76). The differentiation of HIB1B cells was performed as previously described (Ross, et al., Proc. Natl. Acad. Sci. USA 89:7561 (1992)). Although Jhdm2a knock-down did not affect HIB1B differentiation (data not shown), the differentiated knock-down cells had impaired Ucp1 activation by isoproterenol (ISO), a general β-adrenergic receptor agonist, see
The possibility that Jhdm2a directly regulates Ucp1 expression was subsequently examined. ChIP analysis demonstrated that Jhdm2a can bind to the Ucp1 enhancer region (Cassard-Doulcier, et al., Mol. Endocrinol. 7:497 (1993)) in a β-adrenergic receptor ligand-dependent manner. The binding was site-specific as Jhdm2a was not detected within the Ucp1 coding region, see
Previous studies have indicated that PPARγ- and Rxrα-mediated Ucp1 activation requires the recruitment of co-activators (Wang, et al., Cell Metab. 3, 111 (2006)). In addition to removing the repressive H3K9me2 mark at the Ucp1 enhancer, Jhdm2a could contribute to Ucp1 activation by affecting the recruitment of transcription factors and co-activators. To examine this possibility, the effect of the loss of Jhdm2a on the recruitment of transcription factors (PPARγ, Rxrα and Atf2) and co-activators (Pgc1α, CBP/p300 and Src1) was analyzed. It was found that the binding of PPARγ, Rxrα and Atf2 to the Ucp1 enhancer was increased in response to ISO treatment, see
In the present study, PPARα and Ucp1 were identified as direct targets of Jhdm2a. Notably, the expression of these two genes, as well as Jhdm2a, is induced after β-adrenergic stimulation (Lowell, et al., Nature 404, 652 (2000); Lomax, et al., Endocrinology 148, 461 (2007)), see
Jhdm2a knock-down was also carried out in C2C12 cells. To generate a Jhdm2a knock-down (KD) C2C12 cell line, myoblasts were infected with a lentiviral vector containing an RNAi construct for Jhdm2a or control, and were selected with puromycin. The RNAi construct for Jhdm2a was generated using the sequence: 5′-GCA GGT GTC ACT AGC CTT AAT-3′ (SEC) ID NO:76). Following puromycin selection, virally infected cell lines were grown to confluence and cultured in DMEM containing 2% horse serum to induce myotube differentiation. Consistent with a cell intrinsic effect, knock-down of Jhdm2a significantly reduced the expression levels of PGC-1α and its down stream targets, including UCP2, MCAD, ATP5J, and COX7A, see
Rescue experiments were performed to determine whether the demethylase activity of JHDM2A was required for AMPK-mediated PGC-1α induction by attempting to rescue PGC-1α induction using FLAG-tagged constructs encoding either a wild-type or an enzymatic defective mutant JHDM2A. For the rescue experiments, pcDNA3-human Jhdm2a or mutant jhdm2a (H1120Y) plasmid was transfected into Jhdm2a knock-down C2C12 myoblasts using LIPOFECTAMINE2000 (Invitrogen). Because the AMPK signaling is constitutively active in the primary Jhdm2a KO muscle cells (data not shown), Jhdm2a KD cells were chosen for the rescue experiment to avoid experimental bias due to pre-activation of AMPK. Importantly, over-expression of the wild-type human JHDM2A protein in the Jhdm2a knock-down C2C12 cells restored the expression of PGC-1α, see
The results herein indicate that JHDM2A directly contributes to the expression of PGC-1α in myoblasts. To determine whether PGC-1α is a direct target of JHDM2A, ChIP assays were performed. Ctrl or Jhdm2a KD C2C12 myotubes with or without AICAR treatment were used for ChIP analysis with histone modification-specific antibodies and anti-JHDM2A as described previously (Okada, Nature 450, 119 (2007)). The results demonstrated that JHDM2A is indeed localized to the promoter region (amplicon A), but not in the coding region (amplicon B), see
To further confirm that binding of JHDM2A to the PGC-1α promoter maintains a lower level of H3K9 methylation, the H3K9me1/2 levels in control and Jhdm2a KD C2C12 cells with or without the treatment of AICAR were analyzed. The results shown in
Single nucleotide polymorphisms in the human JHDM2A (JMJD1A) gene are listed in Table 3.
rs7598058
The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.
This application claims the benefit of U.S. Provisional Application No. 61/075,576; Filed Jun. 25, 2008, and U.S. Provisional Application No. 61/149,174 filed Feb. 2, 2009, the disclosure of which is incorporated by reference herein in its entirety.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US09/03733 | 6/23/2009 | WO | 00 | 2/28/2011 |
| Number | Date | Country | |
|---|---|---|---|
| 61075576 | Jun 2008 | US | |
| 61149174 | Feb 2009 | US |
