WO2008083157A2

WO2008083157A2 - Altering pgc-1alapha, ampk, fiaf, or the gastrointestinal microbiota as a means to modulate body fat and/or weight loss in a subject

Info

Publication number: WO2008083157A2
Application number: PCT/US2007/088822
Authority: WO
Inventors: Jeffrey Gordon; Fredrik Backhed
Original assignee: Washington University In St. Louis
Priority date: 2006-12-29
Filing date: 2007-12-26
Publication date: 2008-07-10
Also published as: WO2008083157A3

Abstract

The invention provides compositions and methods to modulate body fat and/or weightloss in a subject by altering either the amount of or the activity of a Pgc-1a polypeptide, an AMPK polypeptide, a Fiaf polypeptide, or a combination thereof, in the subject.

Description

ALTERING Pgc-1alpha, AMPK, FIAF, OR THE GASTROINTESTINAL MICROBIOTA AS A MEANS TO MODULATE BODY FAT AND/OR WEIGHT

LOSS IN A SUBJECT

GOVERNMENTAL RIGHTS IN INVENTION

[0001] This work was supported by the U.S. Department of Health and Human Services/ National Institutes of Health grant number NIH-DK79999. The U.S. Government has certain rights in this invention.

FIELD OF THE INVENTION

[0002] The current invention generally relates to the effects of the gastrointestinal microbiota on body fat and weight loss in a subject. In particular, the invention provides compositions and methods to modulate body fat and/or weightloss in a subject by altering either the amount of or the activity of a Pgc-1 α polypeptide, an AMPK polypeptide, a Fiaf polypeptide, or a combination thereof, in the subject.

BACKGROUND OF THE INVENTION

[0003] According to the Center for Disease Control (CDC), over sixty percent of the United States population is overweight, and almost twenty percent are obese. This translates into 38.8 million adults in the United States with a Body Mass Index (BMI) of 30 or above. Obesity is also a worldwide health problem with an estimated 500 million overweight adult humans [body mass index (BMI) of 25.0-29.9 kg/m²] and 250 million obese adults (Bouchard, C (2000) N Engl J Med. 343, 1888-9). This epidemic of obesity is leading to worldwide increases in the prevalence of obesity-related disorders, such as diabetes, hypertension, as well as cardiac pathology, and non-alcoholic fatty liver disease (NAFLD; Wanless, and Lentz (1990) Hepatology 12, 1106-1110. Silverman, et al, (1990) Am. J. Gastroenterol. 85, 1349-1355; Neuschwander- Tetri and, Caldwell (2003) Hepatology 37, 1202-1219). According to the National Institute of Diabetes, Digestive and Kidney Diseases (NIDDK) approximately 280,000 deaths annually are directly related to obesity. The NIDDK further estimated that the direct cost of healthcare in the U.S. associated with obesity is $51 billion. In addition, Americans spend $33 billion per year on weight loss products. In spite of this economic cost and consumer commitment, the prevalence of obesity continues to rise at alarming rates. From 1991 to 2000, obesity in the U.S. grew by 61 %.

[0004] Although the physiologic mechanisms that support development of obesity are complex, the medical consensus is that the root cause relates to an excess intake of calories compared to caloric expenditure. While the treatment seems quite intuitive, dieting is not an adequate long-term solution for most people; about 90 to 95 percent of persons who lose weight subsequently regain it. Although surgical intervention has had some measured success, the various types of surgeries have relatively high rates of morbidity and mortality.

[0005] Pharmacotherapeutic principles are limited. In addition, because of undesirable side effects, the FDA has had to recall several obesity drugs from the market. Those that are approved also have side effects. Currently, two FDA-approved anti-obesity drugs are orlistat, a lipase inhibitor, and sibutramine, a serotonin reuptake inhibitor. Orlistat acts by blocking the absorption of fat into the body. An unpleasant side effect with orlistat, however, is the passage of undigested oily fat from the body. Sibutramine is an appetite suppressant that acts by altering brain levels of serotonin. In the process, it also causes elevation of blood pressure and an increase in heart rate. Other appetite suppressants, such as amphetamine derivatives, are highly addictive and have the potential for abuse. Moreover, different subjects respond differently and unpredictably to weight-loss medications.

[0006] Because surgical and pharmacotherapy treatments are problematic, new non-cognitive strategies are needed to prevent and treat obesity and obesity-related disorders. SUMMARY OF THE INVENTION

[0007] One aspect of the present invention encompasses a method for modulating body fat and/or weight loss in a subject. The method comprises altering either the amount of or the activity of a Fiaf polypeptide in the subject and altering either the amount of or the activity of an AMPK polypeptide in the subject.

[0008] Another aspect of the present invention encompasses a method for modulating body fat and/or weight loss in a subject. The method comprises altering the microbiota population in the subject's gastrointestinal tract such that microbial-mediated regulation of an AMPK polypeptide in the subject is modified.

[0009] An additional aspect of the invention encompasses a composition. The composition comprises an agent that increases the amount of or the activity of a Pgc-1α polypeptide and an agent that increases the amount of or the activity of an AMPK polypeptide.

[0010] Yet another aspect of the invention encompasses a composition comprising an agent that increases the amount of or the activity of a Fiaf polypeptide and an agent that increases the amount of or the activity of an AMPK polypeptide.

[0011 ] Other aspects and iterations of the invention are described more thoroughly below.

BRIEF DESCRIPTION OF THE FIGURES

[0012] Figure 1 illustrates that germ-free mice are protected against diet-induced obesity. (A) Eight- to 10-week-old male C57BI/6J mice were conventionalized 3 weeks before they were switched to a high-fat Western diet. Initial weight was recorded (25.5±0.4g and 26.6±0.7g for GF and conventionalized mice, respectively). Weight gain was monitored weekly for eight weeks and compared to GF mice. Control mice remained on a low-fat diet (n=5/group). (B) Response to acute fat loading in GF and conventionalized mice maintained on a low-fat chow diet. Olive oil (400 μl) was administered by gavage to mice that had been fasted overnight. Serum triglycerides levels were measured at the indicated time points (n=5/group). (C) Locomotor activity was recorded in chow fed GF and conventionalized mice over a 3-day period (left panel), and then again after they had been fed a Western diet for 8 weeks (right panel; n=4/group). Mean values ± SE are plotted. *, P < 0.05; **, P < 0.01 and ***, P < 0.001 compared with GF.

[0013] Figure 2 illustrates that the gut microbiota suppresses

AMPK activity in the gastrocnemius muscle. (A) lmmunoblotting of protein lysates from gastrocnemius muscle harvested from 15-week-old male GF or conventionalized C57BI/6J mice fed a Western diet for 5 weeks prior to sacrifice. Representative results from two mice are shown. (B) Quantification of the results shown in panel A (n=4/group). Data are expressed relative to actin. (C) Effects of the gut microbiota on Cpt activity in freeze-clamped gastrocnemius muscle samples from the mice studied in panels A and B (n=5/group). **, P < 0.01 ; and *, P < 0.05 compared with GF.

[0014] Figure 3 illustrates that the gut microbiota suppresses

AMPK activity in liver. (A) lmmunoblotting of protein lysates from liver samples obtained from 15-week-old GF or conventionalized male mice fed a Western diet for 5 weeks before they were sacrificed. Representative results from two mice are shown. (B) Quantification of the results shown in panel A (n=4/group). Data are expressed relative to actin. Effects of the gut microbiota on glycogen levels (C) and glycogen synthase activity (D) in freeze-clamped livers (n=5/group). ***, P < 0.001 ; and **, P < 0.01 compared with GF.

[0015] Figure 4 illustrates that germ-free Fiaf-/- mice are not protected against diet-induced obesity and have lower expression of Pgc-1α and genes involved in fatty acid oxidation in their gastrocnemius muscles. (A) qRT- PCR assays of Fiaf expression in the distal small intestines and livers of GF and conventionalized wild-type male mice maintained on a low-fat diet since weaning, or given a high-fat Western diet for 8 weeks prior to being killed. Mean values ± SE are plotted. n=5 mice/group. **, P < 0.01 ; and *, P < 0.05 compared with GF mice on the chow diet. Ψ, P < 0.05 compared with GF mice on the Western diet. (B) GF Fiaf-deficient mice become obese on a Western diet. 8- to 10-week-old GF male wild-type and Fiaf-I- mice were switched to the Western diet and their body weights monitored weekly for five weeks (n=5/group). (C) Epididymal fat pad weights of the mice shown in panel B after 5 weeks on the Western diet. (D) qRT-PCR assays of gastrocnemius muscle RNAs prepared from GF Fiaf-/- mice and wild-type littermates on the Western diet (n=6/group). Mean values ± SE are plotted. **, P < 0.01 ; and *, P < 0.05 compared with wild-type animals

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The present invention provides compositions comprising

Pgc-1 α polypeptides, AMPK polypeptides, Fiaf polypeptides, or agents that modulate Pgc-1 α, AMPK, or Fiaf polypeptides, or any combination thereof. Additionally, the present invention provides methods for modulating body fat and/or weight loss, or to treat obesity and/or obesity-related disorders in a subject.

I. COMPOSITIONS FOR MODULATING BODY FAT OR WEIGHT LOSS

[0017] The invention encompasses compositions comprised of Pgc-

1 α polypeptides, AMPK polypeptides, Fiaf polypeptides, or agents that modulate Pgc-1 α, AMPK, or Fiaf polypeptides, or any combination thereof. The compositions may be used to modulate body fat and/or weight loss, or to treat obesity and/or obesity-related disorders in a subject.

(a) Fiaf polypeptides

[0018] One aspect of the present invention provides compositions comprising a Fiaf polypeptide. To decrease body fat and promote weight loss, the amount of or the activity of Fiaf may be increased in the subject. Alternatively, to increase body fat and promote weight gain, the amount of or the activity of Fiaf may be decreased in the subject.

[0019] In one embodiment, Fiaf may be increased by administering a suitable Fiaf polypeptide to the subject. Typically, a suitable Fiaf polypeptide is one that can substantially inhibit lipoprotein lipase (LPL) when administered to the subject. A number of Fiaf polypeptides known in the art are suitable for use in the present invention. Generally speaking, the Fiaf polypeptide is from a mammal. By way of non limiting example, suitable Fiaf polypeptides and nucleotides are delineated in Table Z.

TABLE Z

Species PubMed Ref.

Homo sapiens NM 139314 NM_016109

Mus musculus NM_020581

Rattus norvegicus NMJ 99115

Sus scrofa AY307772

Bos taurus AY192008

Pan troglodytes AY411895

[0020] In certain aspects, a polypeptide that is a homolog, ortholog, mimic or degenerative variant of a Fiaf polypeptide is also suitable for use in the present invention. In particular, the subject polypeptide will typically inhibit LPL when administered to the subject.

[0021] A number of methods may be employed to determine whether a particular homolog, mimic or degenerative variant possesses substantially similar biological activity relative to a Fiaf polypeptide. Specific activity or function may be determined by convenient in vitro, cell-based, or in vivo assays, such as measurement of LPL activity in white adipose tissue or in the heart. [0022] In addition to having a substantially similar biological function, a homolog, ortholog, mimic or degenerative variant suitable for use in the invention will also typically share substantial sequence similarity to a Fiaf polypeptide. In addition, suitable homologs, orthologs, mimics or degenerative variants preferably share at least 30% sequence homology with a Fiaf polypeptide, more preferably, 50%, and even more preferably, are greater than about 75% homologous in sequence to a Fiaf polypeptide. Alternatively, peptide mimics of Fiaf could be used that retain critical molecular recognition elements, although peptide bonds, side chain structures, chiral centers and other features of the parental active protein sequence may be replaced by chemical entities that are not native to Fiaf protein yet, nevertheless, confer activity.

[0023] In determining whether a polypeptide is substantially homologous to a Fiaf polypeptide, sequence similarity may be determined by conventional algorithms, which typically allow introduction of a small number of gaps in order to achieve the best fit. In particular, "percent homology" of two polypeptides or two nucleic acid sequences is determined using the algorithm of Karlin and Altschul [(Proc. Natl. Acad. Sci. USA 87, 2264 (1993)]. Such an algorithm is incorporated into the BLASTN and BLASTX programs of Altschul, et al. {J. MoI. Biol. 215, 403 (1990)). BLAST nucleotide searches may be performed with the BLASTN program to obtain nucleotide sequences homologous to a nucleic acid molecule of the invention. Equally, BLAST protein searches may be performed with the BLASTX program to obtain amino acid sequences that are homologous to a polypeptide of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Altschul, et al. {Nucleic Acids Res. 25, 3389 (1997)). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTX and BLASTN) are employed. See http://www.ncbi.nlm.nih.gov for more details.

[0024] Fiaf polypeptides suitable for use in the invention are typically isolated or pure and are generally administered as a composition in conjunction with a suitable pharmaceutical carrier, as detailed below. A pure polypeptide constitutes at least about 90%, preferably, 95% and even more preferably, at least about 99% by weight of the total polypeptide in a given sample.

(i) Processes for producing Fiaf polypeptides

[0025] A Fiaf polypeptide may be synthesized, produced by recombinant technology, or purified from cells using any of the molecular and biochemical methods known in the art that are available for biochemical synthesis, molecular expression and purification of Fiaf polypeptides [see e.g., Molecular Cloning, A Laboratory Manual (Sambrook, et al. Cold Spring Harbor Laboratory), Current Protocols in Molecular Biology (Eds. Ausubel, et al., Greene Publ. Assoc, Wiley-lnterscience, New York)].

[0026] Expression vectors that may be effective for the expression of Fiaf polypeptides include, but are not limited to, the PCDNA 3.1 , EPITAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors (Invitrogen, Carlsbad Calif.), PCMV-SCRIPT, PCMV-TAG, PEGSHIPERV (Stratagene, La JoIIa Calif.), and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (Clontech, Palo Alto Calif.). Fiaf polypeptides may be expressed using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or P β-actin genes), (ii) an inducible promoter (e.g., the tetracycline-regulated promoter (Gossen, et al., Proc. Natl. Acad. Sci. USA, 89, 5547 (1992); M. Gossen, et al., Science, 268, 1766 (1995); F.M., Rossi, et al.,Curr. Opin. Biotechnol. 9, 451 (1998), commercially available in the T-REX plasmid (Invitrogen)); the ecdysone-inducible promoter (available in the plasmids PVGRXR and PIND; Invitrogen); the FK506/rapanmycin inducible promoter; or the RU486/mifepristone inducible promoter (F. M. Rossi, et al., supra)), or (iii) a tissue-specific promoter or the native promoter of the endogenous gene encoding Fiaf from a normal individual. [0027] Commercially available liposome transformation kits (e.g., the PERFECT LIPID TRANSFECTION KIT, available from Invitrogen) allow one with ordinary skill in the art to deliver Fiaf polynucleotides to target cells in culture, and require minimal effort to optimize experimental parameters. Alternatively, transformation is performed using the calcium phosphate method (F. L. Graham, et al., Virology, 52, 456 (1973), or by electroporation (E. Neumann, et al., EMBO J., 1 , 841 (1982)).

[0028] A Fiaf peptide may be synthesized using traditional solid- phase methods.

(H) Modulation of Fiaf polypeptides

[0029] An agent may be delivered that specifically modulates Fiaf expression: this agent could represent a natural or synthetic compound that directly activates Fiaf gene transcription, or indirectly activates expression through interactions with components of host regulatory networks that control Fiaf transcription. Alternatively, such an agent could represent a natural or synthetic compound that directly suppresses Fiaf gene transcription, or indirectly suppresses expression through interactions with components of host regulatory networks that control Fiaf transcription. Such an agent could be identified by screening natural product and/or chemical libraries using the gnotobiotic zebrafish model as a bioassay.

[0030] In another embodiment, a chemical entity could be used that interacts with Fiaf targets such as LPL to reproduce the effects of Fiaf (e.g., in this case inhibition of LPL activity).

[0031] In still another embodiment, the amount and/or activity of

Fiaf may be increased by administering a Fiaf agonist to the subject. In one preferred embodiment, the Fiaf agonist is a peroxisome proliferator-activated receptor (PPARs) agonist. Suitable PPARs include PPARα, PPARβ/δ, and PPARy. Fenofibrate is another suitable example of a Fiaf agonist. Additional suitable Fiaf agonists and methods of administration are further described in Manards, et al., J. Biol Chem, 279, 34411 (2004), and U.S. Patent Publication No. 2003/0220373, which are both hereby incorporated by reference in their entirety. Alternatively, the amount and/or activity of Fiaf may be decreased by administering a Fiaf antagonist to the subject. A non-limiting example of a Fiaf antagonist may be an anti-Fiaf antibody.

[0032] In yet a further embodiment, Fiaf may be increased in a subject by altering the microbiota population in the subject's gastrointestinal tract such that the microbial-mediated suppression of Fiaf in the subject is decreased. Alternatively, Fiaf may be decreased in a subject by altering the microbiota population in the subject's gastrointestinal tract such that the microbial-mediated suppression of Fiaf in the subject is increased. Suitable methods for altering the microbial population are described in section Il below.

[0033] Yet another aspect of the invention encompasses methods to identify microbially produced compounds that modulate Fiaf amount or activity and non microbially produced compounds that modulate Fiaf amount or activity. Generally speaking, methods generally known in the art may be utilized to identify compounds that modulate Fiaf amount or activity. In one embodiment, a gnotobiotic zebrafish model may be used as a method for screening for a compound that is effective in altering expression of a polynucleotide encoding a Fiaf polypeptide.

[0034] In one embodiment, a method for screening for a compound that is effective in altering expression of a polynucleotide (gene) encoding a Fiaf polypeptide is provided. Effective compounds may alter polynucleotide expression by acting on transcriptional or translational regulators of Fiaf expression.

[0035] At least one, and up to a plurality, of test compounds may be screened for effectiveness in altering expression of a specific Fiaf polynucleotide. A test compound may be obtained by any method commonly known in the art, including but not limited to selection from an existing, commercially-available or proprietary library of naturally-occurring or non-natural chemical compounds; selection from a library of chemical compounds created combinatorially or randomly, or purification from a natural product, such as extracts of gut microbes grown in vitro or from conditioned medium harvested after culture of a gut microbe or collection of gut microbes. Alterations in the expression of a polynucleotide encoding a Fiaf polypeptide may be assayed by a number of methods commonly known in the art including but not limited to qRT-PCR. Detection of a change in the expression of a Fiaf polynucleotide, or its protein product, indicates that the test compound is effective in altering Fiaf gene expression. Another embodiment is to observe changes in expression of a transgene containing Fiaf transcriptional regulatory elements responsive to microbial signals, linked to an open reading frame encoding a fluorescent protein reporter, in gnotobiotic zebrafish.

[0036] Another embodiment is to test the activity of Fiaf peptides, peptidomimetics or related compounds in germ-free Fiaf-/- mice to determine whether they reduce their high body fat.

[0037] Another aspect of the invention encompasses the use of a

Fiaf polypeptide to screen for compounds that modulate the activity of the Fiaf polypeptide. Such compounds may include agonists or antagonists as detailed above. In one embodiment, an assay is performed under conditions permissive for Fiaf polypeptide activity, wherein the Fiaf polypeptide is combined with at least one test compound, and the activity of the subject polypeptide in the presence of a test compound is compared with the activity of the Fiaf polypeptide in the absence of the test compound. Activity could, for example, be defined as the capacity to inhibit LPL-catalyzed biochemical reactions in vitro. A change in the activity of Fiaf in the presence of the test compound is indicative of a compound that modulates the activity of Fiaf polypeptides. At least one and up to a plurality of test compounds may be screened.

[0038] In another embodiment, a transgene consisting of transcriptional regulatory elements that are constitutively active in the intestinal epithelium (e.g. nucleotides -1178 to +28 of the rat intestinal fatty acid binding protein gene) linked to Fiaf could be introduced into subjects, such as Fiaf-/- mice, so the effects of Fiaf activation can be studied and additional targets for pharmacologic manipulation of Fiaf-related pathways that lead to reduced adiposity can be performed.

[0039] A variety of protocols for measuring Fiaf polypeptides, including ELISAs and RIAs, may be used in any of the screening methods delineated above.

(b) Pgc-1α polypeptides

[0040] Another aspect of the present invention provides compositions comprising a Pgc-1 α polypeptide. To decrease body fat and promote weight loss, the amount of or the activity of a Pgc-1 α polypeptide may be increased in the subject. Alternatively, to increase body fat and promote weight gain the amount of or the activity of Pgc-1 α may be decreased in the subject.

[0041] In one embodiment, Pgc-1 α may be increased by administering a suitable Pgc-1 α polypeptide to the subject. Typically, a suitable Pgc-1 α polypeptide is one that can increase expression of a gene encoding a regulator of mitochondrial fatty acid oxidation, such as Cpt1 when administered to the subject. For more details, see Vega et al., MoI Cell Biol (2000) 20:1868-76. A number of Pgc-1 α polypeptides known in the art are suitable for use in the present invention. Generally speaking, the Pgc-1 α polypeptide is from a mammal. By way of non limiting example, suitable Pgc-1 α polypeptides and nucleotides are delineated in Table Y. TABLE Y

Species PubMed Ref.

Homo sapiens NP 037393 NP_055877

Mus musculus NP_032930

Rattus norvegicus NPJ 12637

Sus scrofa BAC66011

Bos taurus AAQ82595

[0042] In certain aspects, a polypeptide that is a homolog, ortholog, mimic or degenerative variant of a Pgc-1 α polypeptide is also suitable for use in the present invention. In particular, the subject polypeptide will typically increase expression of a gene encoding a regulator of mitochondrial fatty acid oxidation when administered to the subject.

[0043] A number of methods may be employed to determine whether a particular homolog, mimic or degenerative variant possesses substantially similar biological activity relative to a Pgc-1 α polypeptide. Specific activity or function may be determined by convenient in vitro, cell-based, or in vivo assays. In order to determine whether a particular Pgc-1 α polypeptide increases expression of a gene encoding a regulator of mitochondrial fatty acid oxidation, the procedures detailed in Vega et al., MoI Cell Biol (2000) 20:1868- 76, hereby incorporated by reference, may be followed.

[0044] In addition to having a substantially similar biological function, a homolog, ortholog, mimic or degenerative variant suitable for use in the invention will also typically share substantial sequence similarity to a Pgc-1 α polypeptide. In addition, suitable homologs, orthologs, mimics or degenerative variants preferably share at least 30% sequence homology with a Pgc-1 α polypeptide, more preferably, 50%, and even more preferably, are greater than about 75% homologous in sequence to a Pgc-1 α polypeptide. Alternatively, peptide mimics of Pgc-1 α could be used that retain critical molecular recognition elements, although peptide bonds, side chain structures, chiral centers and other features of the parental active protein sequence may be replaced by chemical entities that are not native to Pgc-1 α protein yet, nevertheless, confer activity.

[0045] In determining whether a polypeptide is substantially homologous to a Pgc-1α polypeptide, sequence similarity may be determined by conventional algorithms, which typically allow introduction of a small number of gaps in order to achieve the best fit. In particular, "percent homology" of two polypeptides or two nucleic acid sequences is determined using the algorithm of Karlin and Altschul [(Proc. Natl. Acad. Sci. USA 87, 2264 (1993)] as described above.

[0046] Pgc-1 α polypeptides suitable for use in the invention are typically isolated or pure and are generally administered as a composition in conjunction with a suitable pharmaceutical carrier, as detailed below. A pure polypeptide constitutes at least about 90%, preferably, 95% and even more preferably, at least about 99% by weight of the total polypeptide in a given sample.

(i) Processes for producing Pgc-1a polypeptides

[0047] The Pgc-1 α polypeptide may be synthesized, produced by recombinant technology, or purified from cells using any of the molecular and biochemical methods known in the art that are available for biochemical synthesis, molecular expression and purification of the Pgc-1 α polypeptides [see e.g., Molecular Cloning, A Laboratory Manual (Sambrook, et al. Cold Spring Harbor Laboratory), Current Protocols in Molecular Biology (Eds. Ausubel, et al., Greene Publ. Assoc, Wiley-lnterscience, New York)].

[0048] Expression vectors that may be effective for the expression of Pgc-1α are commonly known in the art. Such vectors may include, but are not limited to, the vectors listed in section l(a)(i) above. Commercially available liposome transformation kits (e.g., the PERFECT LIPID TRANSFECTION KIT, available from Invitrogen) allow one with ordinary skill in the art to deliver Pgc-1 α polynucleotides to target cells in culture, and require minimal effort to optimize experimental parameters. Alternatively, transformation may be performed using the calcium phosphate method (F. L. Graham, et al., Virology, 52, 456 (1973), or by electroporation (E. Neumann, et al., EMBO J., 1 , 841 (1982)).

[0049] A Pgc-1 α peptide may be synthesized using traditional solid-phase methods.

(H) Modulating Pgc-1α polypeptides

[0050] An agent may be delivered that specifically modulates Pgc-

1 α expression: this agent could represent a natural or synthetic compound that directly activates Pgc-1 α gene transcription, or indirectly activates expression through interactions with components of host regulatory networks that control Pgc-1 α transcription. For instance, a Fiaf polypeptide may be used to increase expression of Pgc-1 α. Alternatively, such an agent could represent a natural or synthetic compound that directly suppresses Pgc-1 α gene transcription, or indirectly suppresses expression through interactions with components of host regulatory networks that control Pgc-1 α transcription. Such an agent may be identified by screening natural product and/or chemical libraries.

[0051] In another embodiment, a chemical entity could be used that interacts with Pgc-1 α targets such as Cpt1 to reproduce the effects of Pgc-1α (e.g., in this case increase of Cpt1 expression).

[0052] In still another embodiment, the amount and/or activity of

Pgc-1 α may be increased by administering a Pgc-1 α agonist to the subject. In one embodiment, the Pgc-1 α agonist may be a Fiaf agonist. The Fiaf agonist may be a peroxisome proliferator-activated receptor (PPARs) agonist, as described in section l(a)(ii) above. Alternatively, the amount and/or activity of Pgc-1 α may be decreased by administering a Pgc-1 α antagonist to the subject. In one embodiment, the Pgc-1 α antagonist may be a Fiaf antagonist. In another embodiment, the Pgc-1 α antagonist may be an anti-Pgc-1 α antibody. [0053] In yet a further embodiment, Pgc-1 α may be increased in a subject by altering the microbiota population in the subject's gastrointestinal tract such that the microbial-mediated suppression of Pgc-1 α in the subject is decreased. Alternatively, Pgc-1 α is increased in a subject by altering the microbiota population in the subject's gastrointestinal tract such that the microbial-mediated suppression of Fiaf in the subject is decreased. In another alternative, Pgc-1 α may be decreased in a subject by altering the microbiota population in the subject's gastrointestinal tract such that the microbial-mediated suppression of Pgc-1 α in the subject is increased. Suitable methods for altering the microbial population are described in section Il below.

[0054] Yet another aspect of the invention encompasses methods to identify microbially produced compounds that modulate Pgc-1 α amount or activity and non microbially produced compounds that modulate Pgc-1 α amount or activity. Generally speaking, methods generally known in the art may be utilized to identify compounds that modulate Pgc-1α amount or activity.

[0055] In one embodiment, a method for screening for a compound that is effective in altering expression of a polynucleotide (gene) encoding a Pgc- 1 α polypeptide is provided. Effective compounds may alter polynucleotide expression by acting on transcriptional or translational regulators of Pgc-1 α expression.

[0056] At least one, and up to a plurality, of test compounds may be screened for effectiveness in altering expression of a specific Pgc-1α polynucleotide. A test compound may be obtained by any method commonly known in the art, including but not limited to selection from an existing, commercially-available or proprietary library of naturally-occurring or non-natural chemical compounds; selection from a library of chemical compounds created combinatohally or randomly, or purification from a natural product, such as extracts of gut microbes grown in vitro or from conditioned medium harvested after culture of a gut microbe or collection of gut microbes. Alterations in the expression of a polynucleotide encoding a Pgc-1 α polypeptide may be assayed by a number of methods commonly known in the art including but not limited to qRT-PCR. Detection of a change in the expression of a Pgc-1 α polynucleotide, or its protein product, indicates that the test compound is effective in altering Pgc-1 α gene expression. Another embodiment is to observe changes in expression of a transgene containing Pgc-1 α transcriptional regulatory elements responsive to microbial signals, linked to an open reading frame encoding a fluorescent protein reporter, in a gnotobiotic subject.

[0057] Another embodiment is to test the activity of Pgc-1 α peptides, peptidomimetics or related compounds in germ-free Fiaf-/- mice to determine whether they reduce their high body fat.

[0058] Another aspect of the invention encompasses the use of a

Pgc-1 α polypeptide to screen for compounds that modulate the activity of the Pgc-1 α polypeptide. Such compounds may include agonists or antagonists as detailed above. In one embodiment, an assay is performed under conditions permissive for Pgc-1 α polypeptide activity, wherein the Pgc-1 α polypeptide is combined with at least one test compound, and the activity of the subject polypeptide in the presence of a test compound is compared with the activity of the Pgc-1 α polypeptide in the absence of the test compound. Activity could, for example, be defined as the capacity to increase expression of a gene encoding a regulator of mitochondrial fatty acid oxidation. A change in the activity of Pgc-1 α in the presence of the test compound is indicative of a compound that modulates the activity of Pgc-1 α polypeptides. At least one and up to a plurality of test compounds may be screened.

[0059] In another embodiment, a transgene consisting of transcriptional regulatory elements that are constitutively active in the intestinal epithelium (e.g. nucleotides -1178 to +28 of the rat intestinal fatty acid binding protein gene) linked to Pgc-1 α could be introduced into a subject so the effects of Pgc-1 α activation may be studied and additional targets for pharmacologic manipulation of Pgc-1 α -related pathways that lead to reduced adiposity may be performed. [0060] A variety of protocols for measuring Pgc-1 α polypeptides, including ELISAs and RIAs, may be used in any of the screening methods delineated above.

(c) AMPK polypeptides

[0061 ] One aspect of the present invention provides compositions comprising an AMPK polypeptide. To decrease body fat and promote weight loss, the amount of or the activity of AMPK may be increased in the subject. Alternatively, to increase body fat and promote weight gain, the amount of or the activity of AMPK may be decreased in a subject.

[0062] In one embodiment, AMPK may be increased by administering a suitable AMPK polypeptide to the subject. Generally speaking, an AMPK polypeptide may be comprised of an α, β, or Y AMPK chain, or a combination thereof. Additionally, the invention contemplates AMPK polypeptides comprised of different isoforms of AMPK α, β, or Y chains, including α1 and α2. For more information, see Kahn et al. Cell Metabolism (2005) 1 :15-25, or US Patent Nos. 6,124,125 and 6,258,547. Typically, a suitable AMPK polypeptide is one that can activate acetylCoA carboxylase (Ace). A number of AMPK polypeptides known in the art are suitable for use in the present invention. Generally speaking, the AMPK polypeptide is from a mammal. By way of non limiting example, suitable AMPK polypeptides and nucleotides may be found in US Patent Nos. 6,124,125 and 6,258,547.

[0063] In certain aspects, a polypeptide that is a homolog, ortholog, mimic or degenerative variant of an AMPK polypeptide is also suitable for use in the present invention. In particular, the subject polypeptide will typically activate Ace when administered to the subject.

[0064] A number of methods may be employed to determine whether a particular homolog, mimic or degenerative variant possesses substantially similar biological activity relative to an AMPK polypeptide. Specific activity or function may be determined by convenient in vitro, cell-based, or in vivo assays, such as measurement of Ace activation.

[0065] In addition to having a substantially similar biological function, a homolog, ortholog, mimic or degenerative variant suitable for use in the invention will also typically share substantial sequence similarity to an AMPK polypeptide. In addition, suitable homologs, orthologs, mimics or degenerative variants preferably share at least 30% sequence homology with an AMPK polypeptide, more preferably, 50%, and even more preferably, are greater than about 75% homologous in sequence to an AMPK polypeptide. Alternatively, peptide mimics of AMPK could be used that retain critical molecular recognition elements, although peptide bonds, side chain structures, chiral centers and other features of the parental active protein sequence may be replaced by chemical entities that are not native to AMPK protein yet, nevertheless, confer activity.

[0066] In determining whether a polypeptide is substantially homologous to an AMPK polypeptide, sequence similarity may be determined by conventional algorithms, which typically allow introduction of a small number of gaps in order to achieve the best fit. In particular, "percent homology" of two polypeptides or two nucleic acid sequences is determined using the algorithm of Karlin and Altschul [(Proc. Natl. Acad. Sci. USA 87, 2264 (1993)] as described above.

[0067] AMPK polypeptides suitable for use in the invention are typically isolated or pure and are generally administered as a composition in conjunction with a suitable pharmaceutical carrier, as detailed below. A pure polypeptide constitutes at least about 90%, preferably, 95% and even more preferably, at least about 99% by weight of the total polypeptide in a given sample.

(i) Process for producing AMPK polypeptides

[0068] The AMPK polypeptide may be synthesized, produced by recombinant technology, or purified from cells using any of the molecular and biochemical methods known in the art that are available for biochemical synthesis, molecular expression and purification of AMPK polypeptides [see e.g., Molecular Cloning, A Laboratory Manual (Sambrook, et al. Cold Spring Harbor Laboratory), Current Protocols in Molecular Biology (Eds. Ausubel, et al., Greene Publ. Assoc, Wiley-lnterscience, New York)].

[0069] Expression vectors that may be effective for the expression of AMPK are commonly known in the art. Such vectors may include, but are not limited to, the vectors listed in section l(a)(i) above. Commercially available liposome transformation kits (e.g., the PERFECT LIPID TRANSFECTION KIT, available from Invitrogen) allow one with ordinary skill in the art to deliver AMPK polynucleotides to target cells in culture, and require minimal effort to optimize experimental parameters. Alternatively, transformation is performed using the calcium phosphate method (F. L. Graham, et al., Virology, 52, 456 (1973), or by electroporation (E. Neumann, et al., EMBO J., 1 , 841 (1982)).

[0070] An AMPK peptide may be synthesized using traditional solid- phase methods.

(H) Modulation of AMPK polypeptides

[0071] An agent may be delivered that specifically modulates AMPK expression: this agent could represent a natural or synthetic compound that directly activates AMPK gene transcription, or indirectly activates expression through interactions with components of host regulatory networks that control AMPK transcription. Alternatively, such an agent could represent a natural or synthetic compound that directly suppresses AMPK gene transcription, or indirectly suppresses expression through interactions with components of host regulatory networks that control AMPK transcription. Such an agent may be identified by screening natural product and/or chemical libraries.

[0072] In another embodiment, an agent may be delivered that specifically increases AMPK activity: this agent could represent a natural or synthetic compound that directly increases AMPK activity, or indirectly increases activity through interactions with components of host regulatory networks that control AMPK activity. Alternatively, such an agent could represent a natural or synthetic compound that directly decreases AMPK activity, or indirectly decreases activity through interactions with components of host regulatory networks that control AMPK activity. For instance, the agent may be AMPKK. Alternatively, the agent may be AMP. In another alternative, the agent may be a compound that phosphorylates AMPK.

[0073] In another embodiment, a chemical entity could be used that interacts with AMPK targets such as Ace to reproduce the effects of AMPK (e.g., in this case activation of Ace).

[0074] In another alternative of this embodiment, AMPK expression and/or activity may be increased by administering an AMPK agonist or antagonist to the subject. In one embodiment, an AMPK agonist may be an AMPKK agonist. In another embodiment, an AMPK antagonist may be an anti-AMPK antibody. For instance, see US Patent No. 6,258,547.

[0075] In yet a further alternative of this embodiment, AMPK is increased in a subject by altering the microbiota population in the subject's gastrointestinal tract such that the microbial-mediated suppression of AMPK in the subject is decreased. Alternatively, the microbiota population in the subject's gastrointestinal tract may be altered such that the microbial-mediated suppression of AMPK in the subject is increased. Suitable methods for altering the microbial population are described in section Il below.

[0076] Yet another aspect of the invention encompasses methods to identify microbially produced compounds that modulate AMPK amount or activity and non microbially produced compounds that modulate AMPK amount or activity. Generally speaking, methods generally known in the art may be utilized to identify compounds that modulate AMPK transcription or activity.

[0077] In one embodiment, a method for screening for a compound that is effective in altering expression of a polynucleotide (gene) encoding an AMPK polypeptide is provided. Effective compounds may alter polynucleotide expression by acting on transcriptional or translational regulators of AMPK expression.

[0078] At least one, and up to a plurality, of test compounds may be screened for effectiveness in altering expression of a specific AMPK polynucleotide. A test compound may be obtained by any method commonly known in the art, including but not limited to selection from an existing, commercially-available or proprietary library of naturally-occurring or non-natural chemical compounds; selection from a library of chemical compounds created combinatohally or randomly, or purification from a natural product, such as extracts of gut microbes grown in vitro or from conditioned medium harvested after culture of a gut microbe or collection of gut microbes. Alterations in the expression of a polynucleotide encoding an AMPK polypeptide may be assayed by a number of methods commonly known in the art including but not limited to qRT-PCR. Detection of a change in the expression of an AMPK polynucleotide, or its protein product, indicates that the test compound is effective in altering AMPK gene expression. Another embodiment is to observe changes in expression of a transgene containing AMPK transcriptional regulatory elements responsive to microbial signals, linked to an open reading frame encoding a fluorescent protein reporter, in a gnotobiotic subject.

[0079] Another embodiment is to test the activity of AMPK peptides, peptidomimetics or related compounds in germ-free Fiaf -/- mice to determine whether they reduce their high body fat.

[0080] Another aspect of the invention encompasses the use of an

AMPK polypeptide to screen for compounds that modulate the activity of the AMPK polypeptide. Such compounds may include agonists or antagonists as mentioned above. In one embodiment, an assay is performed under conditions permissive for AMPK polypeptide activity, wherein the AMPK polypeptide is combined with at least one test compound, and the activity of the subject polypeptide in the presence of a test compound is compared with the activity of the AMPK polypeptide in the absence of the test compound. Activity could, for example, be defined as the capacity to activate Ace. A change in the activity of AMPK in the presence of the test compound is indicative of a compound that modulates the activity of AMPK polypeptides. At least one and up to a plurality of test compounds may be screened.

[0081] In another embodiment, a transgene consisting of transcriptional regulatory elements that are constitutively active in the intestinal epithelium (e.g. nucleotides -1178 to +28 of the rat intestinal fatty acid binding protein gene) linked to AMPK could be introduced into a subject so the effects of AMPK activation may be studied and additional targets for pharmacologic manipulation of AMPK-related pathways that lead to reduced adiposity may be performed.

[0082] A variety of protocols for measuring AMPK polypeptides, including ELISAs and RIAs, may be used in any of the screening methods delineated above.

[0083] In each of the above embodiments, the AMPK polypeptide may be phosphorylated.

(d) Peptide compositions

[0084] The present invention encompasses compositions that may be used to modulate body fat and/or weight loss. For instance, some compositions may be used to decrease body fat and/or promote weight loss. Other compositions may be used to increase body fat and/or promote weight gain. An increase in body fat and/or weight gain may be advantageous in subjects that are underweight, for instance, due to illness such as HIV infection, AIDS, or cancer.

[0085] Some compositions comprise an isolated Pgc-1α polypeptide and an agent that modulates the amount of or the activity of a Fiaf polypeptide. The amount of or the activity of a Fiaf polypeptide may be modulated by an agent as described above. In some embodiments, the amount of or the activity of a Fiaf polypeptide is increased (i.e. for decreased body fat and/or weight loss). In other embodiments, the amount of or the activity of a Fiaf polypeptide is decreased (i.e. for increased body fat and/or weight gain). For instance, the agent may be a compound that alters the gastrointestinal microbiota such that the amount of or the activity of a Fiaf polypeptide is modulated, as described in section Il below. Alternatively, the agent may be a Fiaf agonist. In certain embodiments, the Fiaf agonist may be a PPAR agonist. The agent may also be a Fiaf polypeptide in some embodiments.

[0086] Other compositions comprise an isolated Pgc-1 α polypeptide and an agent that modulates the amount of or the activity of an AMPK polypeptide. The amount of or the activity of an AMPK polypeptide may be modulated by an agent as described above. In some embodiments, the amount of or the activity of an AMPK polypeptide is increased (i.e. for decreased body fat and/or weight loss). In other embodiments, the amount of or the activity of an AMPK polypeptide is decreased (i.e. for increased body fat and/or weight gain). For instance, the agent may by a compound that alters the gastrointestinal microbiota such that the amount of or the activity of an AMPK polypeptide is modulated as described in section Il below. Alternatively, the agent may be an AMPKK polypeptide. The agent may also be an AMPK polypeptide in some embodiments. In each of the above embodiments, the AMPK polypeptide may be phosphorylated.

[0087] Some compositions comprise an isolated Fiaf polypeptide and an agent that modulates the amount of or the activity of an AMPK polypeptide, as described above. Certain compositions comprise an isolated AMPK polypeptide and an agent that increases the amount of or the activity of a Fiaf polypeptide, as described above. The AMPK polypeptide may be phosphorylated.

[0088] Additionally, the invention encompasses compositions comprised of any combination of a Fiaf polypeptide, a Pgc-1α polypeptide, an AMPK polypeptide, an agent that modulates the amount of or the activity of a Fiaf, Pgc-1 α, or AMPK polypeptide, or any combination thereof. [0089] In other embodiments, any of the proteins, polypeptides, or agonists of the invention, as detailed in section I, may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. Generally speaking, agents will include those that modulate body fat and/or weight loss by a mechanism other the mechanisms detailed herein. In one embodiment, acarbose may be administered with any compound described herein. Acarbose is an inhibitor of α-glucosidases and is required to break down carbohydrates into simple sugars within the gastrointestinal tract of the subject. In another embodiment, an appetite suppressant such as an amphetamine or a selective serotonin reuptake inhibitor such as sibutramine may be administered with any compound described herein. In still another embodiment, a lipase inhibitor such as orlistat or an inhibitor of lipid absorption such as Xenical may be administered with any compound described herein. The combination of therapeutic agents may act synergistically to modulate body fat and/or weight loss. In yet still another embodiment, a compound that increases body fat and/or weight gain may be administered with any compound described herein. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.

[0090] An additional embodiment of the invention relates to the administration of a composition that generally comprises an active ingredient formulated with a pharmaceutically acceptable excipient. Excipients may include, for example, sugars, starches, celluloses, gums, and proteins. Various formulations are commonly known and are thoroughly discussed in the latest edition of Reminton's Pharmaceutical Sciences (Maack Publishing, Easton Pa.). Such compositions may comprise a Fiaf polypeptide or Fiaf peptidomimetic, a Pgc-1 α polypeptide or Pgc-1α peptidomimetic, or an AMPK polypeptide or AMPK peptidomimetic. [0091] The compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.

[0092] The actual effective amounts of compound described herein can and will vary according to the specific composition being utilized, the mode of administration and the age, weight and condition of the subject. Dosages for a particular individual subject can be determined by one of ordinary skill in the art using conventional considerations. Those skilled in the art will appreciate that dosages may also be determined with guidance from Goodman & Gilman's The Pharmacological Basis of Therapeutics, Ninth Edition (1996), Appendix II, pp. 1707-1711 and from Goodman & Gilman's The Pharmacological Basis of Therapeutics, Tenth Edition (2001 ), Appendix II, pp. 475-493.

II. METHODS OF THE INVENTION

[0093] The present invention provides methods of modulating body fat and/or weight loss in a subject. Also, the present invention provides methods of treating obesity or an obesity-related disorder in a subject.

(a) Modulating body fat and/or weight loss

[0094] One embodiment encompasses a method for modulating body fat and/or weight loss in a subject. The method may comprise altering either the amount of or the activity of a Pgc-1 α polypeptide of the invention, as described above. The amount of or the activity of a Pgc-1 α polypeptide may be increased to decrease body fat and promote weight loss. Alternatively, the amount of or the activity of a Pgc-1 α polypeptide may be decreased to increase body fat and promote weight gain. By way of non-limiting example, the amount of a Pgc-1 α polypeptide may be increased in the subject by administering an effective amount of a Pgc-1 α polypeptide to the subject. Alternatively, the amount of or the activity of a Pgc-1 α polypeptide may be increased in a subject by increasing the amount of or the activity of a Fiaf polypeptide in the subject. One way the amount of or the activity of a Fiaf polypeptide may be increased is by administering an effective amount of a Fiaf polypeptide to the subject. Another way is to administer a Fiaf agonist to the subject. For instance, a PPAR agonist may be administered to a subject to increase the amount of or the activity of a Fiaf polypeptide. In one embodiment, the method comprises increasing the amount of or the activity of an AMPK polypeptide in a subject, in addition to a Pgc-1 α polypeptide in the subject. In another embodiment, the method comprises increasing the amount of or the activity of a Fiaf polypeptide in a subject, in addition to a Pgc-1 α polypeptide in the subject. In yet another embodiment, the method comprises increasing the amount of or the activity of a Fiaf polypeptide, a Pgc-1 α polypeptide, and an AMPK polypeptide in a subject.

[0095] In another embodiment, the method of modulating body fat and/or weight loss may comprise (a) altering either the amount of or the activity of a Fiaf polypeptide in the subject, and (b) altering either the amount of or the activity of an AMPK polypeptide in the subject. The amount of or the activity of a Fiaf polypeptide and an AMPK polypeptide may be increased to decrease body fat and promote weight loss. Alternatively, the amount of or the activity of a Fiaf polypeptide and an AMPK polypeptide may be decreased to increase body fat and promote weight gain. The amount of or the activity of a Fiaf polypeptide may be altered as described above. Similarly, the amount of or the activity of an AMPK polypeptide may be altered as described above. By way of non-limiting example, the amount of an AMPK polypeptide may be altered by administering an effective amount of an AMPK polypeptide to the subject. Alternatively, the amount of or the activity of the AMPK polypeptide may be increased by administering an effective amount of an AMPKK polypeptide to the subject. In each of the above embodiments, the AMPK polypeptide may be phosphorylated.

[0096] Another aspect of the present invention provides a method to modulate body fat and weight loss in a subject by altering the microbial population in the subject's gastrointestinal tract. In one embodiment, the microbiota population in the subject's gastrointestinal tract is altered such that microbial-mediated suppression of a Pgc-1 α polypeptide in the subject is decreased. In another embodiment, the microbiota population in the subject's gastrointestinal tract is altered such that microbial-mediated suppression of an AMPK polypeptide in the subject is decreased. Methods of altering the microbiota population in the subject's gastrointestinal tract are known in the art, and may include administering an antibiotic or probiotic to the subject. Alternatively, the microbiota population may be altered by increasing or decreasing archeabacteria within the subject's gastrointestinal tract.

[0097] The microbiota population may also be altered such that microbial-mediated transcriptional suppression of a LPL inhibitor, such as Fiaf, is decreased in the subject and results in a decrease of triglyceride storage in the adipocytes of the subject. In a certain embodiment, Fiaf may be selectively increased only in the gastrointestinal tract of the subject. In yet another embodiment, the microbiota population may be altered such that a signaling pathway that regulates hepatic lipogenesis is substantially inhibited, thereby resulting in a decrease of triglyceride storage in the adipocytes of the subject. In one embodiment, hepatic lipogenesis is substantially inhibited as a result of a decrease in microbial processing of dietary polysaccharides.

[0098] The presence of microbes that suppress Fiaf transcription may be decreased. For instance, the presence of saccharolytic microbes, such as Bacteroides, may be decreased. (Saccharolytic microbes typically degrade complex, otherwise indigestible dietary polysaccharides that the subject cannot.) In an alternative embodiment, the presence of microbes that ferment sugars to short chain fatty acids may be decreased. In still another embodiment, the presence of microbes that increase the uptake of microbial and diet-derived monosaccharides (e.g., glucose, fructose and galactose) by the host may be decreased. [0099] To alter the gastrointestinal microbiota, methods generally known in the art may be utilized. In one embodiment, a suitable probiotic is administered to the subject. Generally speaking, suitable probiotics include those that alter the representation or biological properties of microbiota populations that are involved in a subject's uptake of energy. By way of non- limiting example, suitable probiotics include Lactobacillus, Acidophilus and Bifidobacteria, each of which is commercially available from several sources. In another embodiment, microbes that induce Fiaf, Pgc-1α, or AMPK expression in the subject's gastrointestinal tract may be administered to the subject. Alternatively, microbes that suppress Fiaf, Pgc-1 α, or AMPK expression in the subject's gastrointestinal tract may be administered to the subject. In yet another embodiment, selective reduction in the representation of components of the microbiota, such as saccharolytic bacteria, is achieved by administering an antibiotic to the subject. In yet another embodiment, selective reduction in the representation of components of the microbiota, such as saccharolytic bacteria, is achieved with antibiotics.

[0100] In yet another embodiment, a subject may be administered a diet that alters the microbiota population so as to modulate body fat and/or weight loss in the subject.

(b) Treating obesity or obesity-related disorders

[0101 ] A further aspect of the invention encompasses the use of the methods to modulate body fat and/or weight loss in a subject as a means to treat obesity or an obesity-related disorder. The method comprises, in part, diagnosing a subject in need of treatment for obesity or an obesity-related disorder.

[0102] Typically, a subject in need of treatment for obesity will have at least one of three criteria: (i) BMI over 30; (ii) 100 pounds overweight; or (iii) 100% above an "ideal" body weight. In addition, non-limiting examples of obesity-related disorders that may be treated by the methods of the invention include metabolic syndrome, type Il diabetes, hypertension, cardiovascular disease, and nonalcoholic fatty liver disease. A subject in need of treatment for obesity is diagnosed and is then administered any of the treatments detailed herein, such as in section Il (a) above.

[0103] In one embodiment, obesity or an obesity-related disorder may be treated by modulating the amount of or the activity of a Pgc-1 α polypeptide. The amount of or the activity of a Pgc-1 α polypeptide may be modulated as described above. In another embodiment, obesity or an obesity- related disorder may be treated by altering either the amount of or the activity of a Fiaf polypeptide in the subject and altering either the amount of or the activity of an AMPK polypeptide in the subject. The amount of or the activity of a Fiaf or an AMPK polypeptide may be modulated as described above.

[0104] In another embodiment, obesity or an obesity-related disorder may be treated by altering a subject's gastrointestinal microbial population, as detailed above. For instance, a subject's gastrointestinal microbial population may be altered such that microbial-suppression of a Pgc-1 α polypeptide in the subject is decreased. Or alternatively, a subject's gastrointestinal microbial population may be altered such that microbial- suppression of an AMPK polypeptide in the subject is decreased.

[0105] In still another embodiment, obesity or an obesity-related disorder may be treated by administering a combination of the therapies listed above.

DEFINITIONS

[0106] Ace stands for acetyl-CoA carboxylase.

[0107] The term "agent" and "compound" are used interchangeably herein.

[0108] The term "antagonist" refers to a molecule that inhibits or attenuates the biological activity of a Fiaf polypeptide and in particular, the ability of Fiaf to inhibit LPL. Antagonists may include proteins such as antibodies, nucleic acids, carbohydrates, small molecules, or other compounds or compositions that modulate the activity of a Fiaf polypeptide either by directly interacting with the polypeptide or by acting on components of the biological pathway in which Fiaf participates.

[0109] The term "agonist" refers to a molecule that enhances or increases the biological activity of a Fiaf polypeptide and in particular, the ability of Fiaf to inhibit LPL. Agonists may include proteins, peptides, nucleic acids, carbohydrates, small molecules (e.g., such as metabolites), or other compounds or compositions that modulate the activity of a Fiaf polypeptide either by directly interacting with the polypeptide or by acting on components of the biological pathway in which Fiaf participates.

[0110] The term "altering" as used in the phrase "altering the microbiota population" is to be construed in its broadest interpretation to mean either a change in the representation of microbes in the gastrointestinal tract of a subject or a change in the capacity of the microbiota to harvest energy. The change may be a decrease or an increase in the presence of a particular microbial species.

[0111] "BMI" as used herein is defined as a human subject's weight

(in kilograms) divided by height (in meters) squared.

[0112] "Conservative amino acid substitutions" are those substitutions that are predicted to least interfere with the properties of the original protein, i.e., the structure and especially the function of the protein is conserved and not significantly changed by such substitutions.

[0113] An "effective amount" is a therapeutically-effective amount that is intended to qualify the amount of agent that will achieve the goal of a decrease in body fat, or in promoting weight loss.

[0114] Fiaf stands for fasting-induced adipocyte factor.

[0115] A "gene" is a hereditary unit that has one or more specific effects upon the phenotype of the organism, and that can mutate to various allelic forms.

[0116] GF stands for germ free. [0117] LPL stands for lipoprotein lipase.

[0118] A "nucleic acid" is a nucleotide polymer of DNA or RNA or a synthetic mimic. It may consist of a purine or pyrimidine base, e.g. with associated pentose sugars, and phosphate groups.

[0119] PPAR stands for peroxisome proliferator-activator receptor.

[0120] "Peptide" is defined as a compound formed of two or more amino acids, with an amino acid defined according to standard definitions.

[0121] The term "pharmaceutically acceptable" is used adjectivally herein to mean that the modified noun is appropriate for use in a pharmaceutical product; that is the "pharmaceutically acceptable" material is relatively safe and/or non-toxic, though not necessarily providing a separable therapeutic benefit by itself. Pharmaceutically acceptable cations include metallic ions and organic ions. More preferred metallic ions include, but are not limited to appropriate alkali metal salts, alkaline earth metal salts and other physiologically acceptable metal ions. Exemplary ions include aluminum, calcium, lithium, magnesium, potassium, sodium and zinc in their usual valences. Preferred organic ions include protonated tertiary amines and quaternary ammonium cations, including in part, trimethylamine, diethylamine, N₁N'- dibenzyl ethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. Exemplary pharmaceutically acceptable acids include without limitation hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, methanesulfonic acid, acetic acid, formic acid, tartaric acid, maleic acid, malic acid, citric acid, isocitric acid, succinic acid, lactic acid, gluconic acid, glucuronic acid, pyruvic acid, oxalacetic acid, fumaric acid, propionic acid, aspartic acid, glutamic acid, benzoic acid, and the like.

[0122] A "polypeptide" is a polymer made up of less than 350 amino acids.

[0123] "Protein" is defined as a molecule composed of one or more polypeptide chains, each composed of a linear chain of amino acids covalently linked by peptide bonds. Most proteins have a mass between 10 and 100 kilodaltons. A protein is often symbolized by its mass in kDa.

[0124] "Subject" as used herein typically is a mammalian species.

The subject may be a rodent, a human, a livestock animal, a companion animal, or a zoological animal. In one embodiment, the subject is a rodent, i.e. a mouse, a rat, a guinea pig, etc. In another embodiment, the subject is a human. In a yet another embodiment the subject is a livestock animal. Non-limiting examples of livestock animals include pigs, cows, horses, goats, sheep, llamas and alpacas. In still another embodiment, the subject is a companion animal. Non-limiting examples of companion animals include pets, such as dogs, cats, rabbits, and birds. In still yet another embodiment, the subject is a zoological animal. As used herein, a "zoological animal" refers to an animal that may be found in a zoo. Such animals may include non-human primates, large cats, wolves, and bears. In a further embodiments, subjects that may be treated by the methods of the invention include a human, a dog, a cat, a cow, a horse, a rabbit, a pig, a sheep, a goat, as well as non-mammalian species including an avian species and a fish species.

[0125] A "vector" is a self-replication DNA molecule that transfers a

DNA segment to a host cell.

[0126] As various changes could be made in the above compounds, products and methods without departing from the scope of the invention, it is intended that all matter contained in the above description and in the examples given below, shall be interpreted as illustrative and not in a limiting sense.

EXAMPLES

Example 1. Germ-free Mice Are Protected Against Diet-induced Obesity.

[0127] The trillions of microbes that colonize adult human intestine function collectively as a metabolic organ that communicates with, and complements, the human metabolic apparatus. Given the worldwide epidemic in obesity, there is interest in how interactions between human and microbial metabolomes may affect our energy balance. By comparing germ-free (GF) and conventionally-raised mice, it has been shown that the gut microbiota functions as an environmental factor that regulates fat storage. Colonization of adult mice with a microbiota harvested from the distal intestine (cecum) of conventionally- raised (CONV-R) animals (a process known as conventionalization), produces a 60% increase in body fat content, and relative insulin-resistance within 14 days, despite reduced food intake (Backhed et al. (2004) Proc. Natl. Acad. Sci. USA 101 :15718-15723). In addition, the microbiota suppresses expression of fasting- induced adipose factor (Fiaf, also known as angiopoietin-like protein-4), a secreted lipoprotein lipase (LPL) inhibitor: this suppression is confined to the intestinal epithelium and does not occur at other sites where Fiaf is produced (liver and fat) (Backhed et al. 2004). LPL functions in a number of cell lineages as the rate limiting step for uptake of triglycehde-derived fatty acids. By suppressing Fiaf, colonization increases LPL activity in adipocytes, and enhances storage of liver-derived triglycerides (Backhed et al. 2004). To further examine the relationship between microbiota and obesity, the effects of a Western-style high fat, high-sugar diet were compared in GF mice and conventionalized mice.

[0128] Animals. GF wild-type C57BI/6J animals were maintained in gnotobiotic isolators under a strict 12-h light cycle (lights on at 0600 hours), and fed an autoclaved low-fat, polysacchahde-hch chow diet (B & K Universal, East Yorkshire, U.K.) ad libitum. GF mice were colonized at 8-10 weeks of age with cecal contents harvested from an adult conventionally raised (CONV-R) mouse, and kept in their gnotobiotic isolators. Mice were either switched to an irradiated Western diet (TD96132; Harlan Teklad, Madison, Wl) 2-3 weeks after conventionalization, or maintained on their autoclaved low-fat chow diet. Body weight and food consumption were monitored weekly. Only male animals were used in this study, which was performed using protocols approved by the Washington University Animal Studies Committee. [0129] Assays of sera. Standard biochemical methods were used to assay sera for glucose, cholesterol, triglycerides, and nonesterified fatty acids (Backhed et al. 2004). Insulin and leptin levels were determined by ELISA (Crystal Chemical, Sofia, Bulgaria). Glucose and insulin tolerance tests were performed as described in Backhed et al. (2004).

[0130] Statistical analysis. Data were analyzed using Student's t- test or ANOVA with Tukey's post-hoc analysis.

[0131] Locomotor activity measurements. Mice were anesthetized before a transmitter (minimitter PDT-4000; Mini Mitter, Bend OR) was implanted intra-abdominally. Mice were allowed to recover for 7 days after implantation, and locomotor activity data were collected continuously during the following 3 days. To do so, the signal emitted by the transmitter was detected by receivers positioned underneath the plastic gnotobiotic isolators. Data were then converted into activity counts by VitalView software (Mini Mitter). The experimental group of mice was subsequently switched to a Western diet, while the control group remained on the standard polysaccharide rich chow diet: at the end of 8 weeks, mice in both groups were again monitored, continuously, over a 3d interval.

[0132] Results To determine whether GF mice are protected against diet-induced obesity, 10 week old C57BI/6J males, maintained since weaning on an autoclaved low-fat chow diet (5% lipids; caloric density 4.1 kcal/d), were conventionalized with an unfractionated cecal microbiota from a CONV-R donor. Three weeks later, half of the animals were switched to a 'Western-diet' where 41 % of the calories were in the form of fat, 41 % as sucrose, maltodexthn and corn starch, and 18% as protein (caloric density 4.8 kcal/g). Chow consumption and weight gain were recorded weekly. After 8 weeks, conventionalized animals on the Western diet had gained significantly more weight than their GF counterparts (5.3±0.8g versus 2.1 ±0.5g; n=5 mice/group; P < 0.05 according to Student's t-test) (Fig. 1A). Weight gain in the GF group was not significantly different from the weight gain observed in GF and conventionalized mice that had been maintained on the standard low-fat polysaccharide-rich diet (Fig. 1A). Epididymal fat pad weights were also significantly heavier in conventionalized mice fed the Western diet compared to their GF counterparts (37±5 versus 22±1 mg/g body weight; P < 0.05).

[0133] GF and conventionalized mice consumed similar amounts of the Western diet (2.61 ±0.05 vs. 2.48±0.07 g/mouse/day, n=5/group; P = 0.24), and there were no significant differences in the energy content of their feces, as defined by bomb calorimetry (3.76±0.01 versus 3.81 ±0.09 kcal/g; n=5/group; P = 0.53). Fatty acid absorption was also similar in the two groups: when GF and conventionalized mice were given a single gavage of olive oil after a overnight fast, serum triglycerides rose rapidly over a two hour period reaching equivalent levels in the two groups (Fig. 1 B). However, while triglycerides were subsequently cleared from the circulation in conventionalized mice, they remained elevated in GF mice, a phenomenon that can be attributed to their reduced LPL activity (Backhed et al. 2004). This decrease in LPL activity was also manifested by higher fasting serum triglyceride levels in GF compared to conventionalized mice on the Western diet (Table 1 ).

Table 1. Biochemical and ELISA studies of sera obtained after a four hour fast from GF and conventionalized wild-type C57BI/6J mice fed low- versus high-fat diets.

Low-fat diet Western diet

GF Conventionalized P GF Conventionalized P

Serum levels (n = 5) (n = 5) value (n = 8) (n = 8) value

Glucose, mM 5.46 ± 7.28 ± 0.12 4E- 10.4 ± 13.9 ± 1.2 0.04

0.16 05 1.6

Insulin, ng/ml 0.39 ± 0.85 ± 0.08 0.006 0.43 ± 0.75 ± 0.07 0.007

0.03 0.07

Leptin, ng/ml 1.65 ± 2.70 ± 0.02 0.023 2.01 ± 5.89 ± 0.60 0.004

0.14 0.32

Triglycerides, 49 ± 4 39 ± 5 0.19 68.5 ± 50.4 ± 4.1 0.006 mg/dl 3.8

Cholesterol, 110 ± 103 ± 18 0.17 173.5 185.1 ± 7.9 0.51 mg/dl 3 ± 5.2

Free fatty 1.09 ± 1.01 ± 0.10 0.60 0.87 ± 1.07 ± 0.07 0.03 acids, mM 0.12 0.08 Mean values ± SE are shown

[0134] Using an implantable detector of locomotion, it was found that the absence of a microbiota is associated with significantly increased movement in GF mice, whether these age- and gender-matched animals were on a standard chow or a Western diet (n=4/group) (Fig. 1C).

Example 2. Germ-free mice have increased levels of phosphorylated AMPK in muscle and liver.

[0135] Although LPL is the rate-limiting enzyme for import and subsequent storage of thglyceride-dehved fatty acids in adipocytes, genetically- engineered mice that express LPL only in their myocytes gain weight normally and have a normal body mass composition. Instead of importing triglycerides from the circulation, they increase de novo fatty acid synthesis in adipose tissue. This finding raises the question of whether the lean phenotype of GF mice involves mechanisms beyond a Fiaf-mediated reduction in LPL activity. AMP- activated protein kinase (AMPK) is a heterothmehc enzyme that is conserved from yeast to humans, and functions as a 'fuel gauge' that monitors cellular energy status: it is activated in response to metabolic stresses that result in an increased intracellular ratio of AMP to ATP (e.g., exercise, hypoxia, glucose deprivation). Adipocyte-dehved leptin, adiponectin, and an elevated NAD:NADH ratio also increase AMPK activity. Activation of AMPK occurs by phosphorylation of Thr172 in its catalytic α subunit, leading to suppression of ATP-consuming anabolic pathways and induction of ATP-generating catabolic pathways.

[0136] To investigate whether AMPK is involved in mediating the resistance of GF mice to diet-induced obesity, the levels of phosphorylated (active) AMPK were compared in gastrocnemius muscles harvested from GF and conventionalized animals on the Western diet (described in Example 1 ).

[0137] Isolation and initial processing of tissues. After animals were sacrificed, their small intestine was removed and divided into 16 equal-sized segments. Segments 13-14, liver, gastrocnemius muscle, and epididymal fat pads, from each animal were snap-frozen, and total RNA was isolated (Qiagen RNeasy kit; Valencia, CA) for real-time quantitative (q) RT-PCR assays. Additional gastrocnemius and liver tissue samples (-50 mg each) were directly placed in 1 ml of lysis buffer [20 mM Tris, (pH 7.5), 150 mM NaCI, 1 mM EDTA, 1 % Triton X-100] containing Complete® protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN) and phosphatase inhibitor cocktail 1 (Sigma- Aldrich, St. Louis, MO). Following homogenization at 4°C, extracts were centhfuged for 10 min at 4°C at 15,000 x g to remove insoluble debris and the protein concentration in the resulting supernatant fraction was determined (DC protein assay; Bio-Rad, Hercules, CA). Tissue samples used for assaying enzyme activities and metabolites were harvested after freeze clamping (Backhed et al. 2004) and processed as described below.

[0138] Immunoblotting. Soluble proteins from liver and gastrocnemius muscle were separated on 10% Bis-Tris gels (Invitrogen, Carlsbad, CA) and transferred to PVDF membranes. Membranes were placed in 5% BSA/0.1 % Tween-20/PBS for 60 min at room temperature and then incubated overnight at 4°C in 1 %BSA/0.1 % Tween/PBS together with one of the following antibodies: rabbit anti-phospho-acetylCoA carboxylase, rabbit anti- phospho-AMPK, and rabbit anti-α-subunit of AMPK (all from Cell Signaling Technology, Beverly, MA; final dilution, 1 :1000), or rabbit anti-actin (Sigma- Aldrich; 1 :3000).

[0139] QRT-PCR. RNA prepared from each tissue sample was reverse-transcribed by using Superscript Il (Invitrogen) and a dT15 primer (Roche Diagnostics), as described in Backhed et al. (2004). qRT-PCR assays were performed in 25 μl reactions containing gene-specific primers [900 nM; Mead forward, δ'-GCTGGAGACATTGCCAATCA-S' (SEQ ID NO:1 ), Mead reverse, δ'-TCTTGGCGTCCCTCATCAG-S' (SEQ ID NO:2), L32 forward, 5'- CCTCTGGTGAAGCCCAAGATC-3' (SEQ IS NO:3), L32 reverse, 5'- TCTGGGTTTCCGCCAGTTT-3' (SEQ ID NO:4)], and SYBR green (Abgene, Epsom U.K.). Data were normalized to L32 mRNA (ΔΔCT analysis). [0140] Biochemical assays of enzyme activities and metabolite levels. Pyridine nucleotide cycling methods were used to measure NADH, NAD, AMP, ADP, and ATP in freeze-clamped gastrocnemius muscle and liver (Backhed et al. 2004). Glycogen was determined according to Lin et al. (2001 ) J. Biol. Chem 276:36000-36007. Carnitinepalmitoyl-transferase (Cpt) activity was measured as follows. Muscle samples were homogenized on ice in 0.5 ml_ of extraction buffer (20 mM sodium phosphate, pH 7.4, 0.02% BSA, 0.5 mM EDTA, 5 mM β-mercaptoethanol, 25% glycerol, 0.6 M KCI and 0.5% Triton X-100). A 1 μl_ aliquot of the resulting homogenate was added to 0.5 ml_ of assay reagent A [100 mM Tris HCI pH 8.1 , 1 mM MgCI₂, 1 mM carnitine (Sigma) and 0.25 mM palmitoylCoA (Sigma)] and the samples were incubated for 60 min at 20⁰C before adding 0.5 ml_ assay reagent B [100 mM Tris succinate pH 7.7, 1 mM MgCI₂, 0.4 mM guanosine 5' triphosphate, 0.4 mM phosphoenolpyruvate, 0.3 M KCI, 30 μM NADH, 5 μg/ml beef heart lactate dehydrogenase (Sigma; specific activity, 500 units/mg protein), 40 μg/ml rabbit muscle pyruvate kinase (Sigma; 400 units/mg protein) and 40 μg/ml pig heart succinyl CoA synthetase (Sigma; 130 units/mg protein)]. Samples were incubated for 5 min at 25°C before reduction of NADH was measured using a Farrand filter fluorometer (excitation 365 nm; emission 355 nm). CoA standards (2-5 nmol) were used to quantify enzyme activity, which was normalized to protein content (Bradford assay; BioRad, Hercules, CA).

[0141] To measure glycogen synthase activity, liver samples were homogenized on ice in 0.5 ml_ of lysis buffer consisting of 20 mM sodium phosphate pH 7.4, 5 mM EDTA, 5 mM β-mercaptoethanol, 25% glycerol, 0.5% Thton-X100 and 50 mM potassium fluoride. Enzyme activity was determined in the resulting homogenate according to Lin et al. 2001.

[0142] Results, lmmunoblots disclosed that phospho-AMPK concentrations were 40% higher in GF animals (n=4/group, P < 0.05; Fig. 2A,B). There were no significant differences in the total level of immunoreactive AMPK α subunit (Fig. 2A,B). Consistent with the elevations in phospho-AMPK, biochemical assays disclosed 50% higher levels of AMP in the gastrocnemius muscles of GF compared to conventionalized mice, and no differences in ADP or ATP concentrations (Table 2).

Table 2. Biochemical assays of various metabolites in gastrocnemius muscle and liver harvested from GF and conventionalized wild-type C57BI/6J mice fed a Western diet.

AMP 6.14 ± 4.09 ± 0.42 0.01 20.08 ± 20.87 ± 1.60 0.68

0.39 0.85

ADP 6.36 ± 7.19 ± 0.18 0.35 4.25 ± 5.19 ± 0.52 0.22

0.89 0.46

ATP 26.78 ± 28.56 ± 3.29 0.40 3.41 ± 4.13 ± 1.04 0.62

1.79 0.73

NAD+ 1.99 ± 1.88 ± 0.12 0.86 1.98 ± 1.15 ± 0.21 0.02

0.06 0.18

NADH 0.06 ± 0.09 ± 0.03 0.27 0.29 ± 0.34 ± 0.06 0.46

0.02 0.03

Mean values ± SE are shown (n=5).

[0143] Phosphorylated AMPK stimulates fatty acid oxidation in peripheral tissues by directly phosphorylating acetylCoA carboxylase (Ace; converts acetyl CoA to malonylCoA). Phosphorylation of Ace inhibits its activity, leading to decreased malonylCoA levels. Because malonylCoA inhibits carnitine:palmitoyl transferase-1 (Cpt1 ), which catalyzes the rate-limiting step for entry of long-chain fatty acylCoA into mitochondria, diminished malonylCoA concentrations result in increased Cpt1 activity and increased fatty acid oxidation.

[0144] A 43% increase in the levels of phospho-Acc was documented in the gastrocnemius muscle of GF animals by using an immunoblot assay (P < 0.01 ; Fig. 2A, B), and a modest, but statistically significant 17% increase in Cpt1 activity, as defined by a biochemical assay (Fig. 2C). In addition, a 31 ±4.5% increase in medium chain acylCoA dehydrogenous (Mead) expression was detected in GF gastrocnemius by qRT-PCR (n=4/group, P < 0.05). Mead is a mitochondrial enzyme that catalyzes the initial step in β- oxidation of C8-C12 fatty acids. Together, these findings suggest that the presence of a gut microbiota suppresses skeletal muscle fatty acid oxidation through a metabolic pathway that may involve phosphorylation of AMPK.

[0145] A similar increase in phosphorylated AMPK was found in the livers of these GF animals (Fig. 3A, B). Previous research revealed that short- term, adenoviral-mediated over-expression of a constitutively active form of AMPK in the livers of CONV-R mice produces mild hypoglycemia, and reduced hepatic glycogen stores (Foretz et al. (2005) Diabetes 54:1331-1339). GF mice fed a 'Western' diet for five weeks also had significantly reduced hepatic glycogen levels and decreased glycogen synthase activity (Fig. 3C,D). In addition, significantly reduced serum glucose and insulin levels were observed in GF compared to conventionalized mice on the Western diet (Table 1 ). Glucose and insulin tolerance tests confirmed their increased insulin sensitivity relative to their conventionalized obese counterparts.

[0146] Although phospho-AMPK was increased in the livers of GF mice, there were no significant differences in hepatic AMP:ATP ratios between GF and conventionalized animals. However, biochemical assays disclosed that GF mice had 72% higher levels of NAD+ (Table 2), which also activates AMPK. Similar regulation of AMPK and its targets in both muscle and liver is consistent with recent reports that metabolic crosstalk exists between these distinct tissues. Collectively, these findings suggest that insulin-sensitive GF mice are protected against diet-induced obesity at least in part because of increased AMPK activity and increased fatty acid oxidation in their peripheral tissues. Example 3. GF Fiaf-deficient mice have lost their resistance to diet-induced obesity.

[0147] To examine the role of Fiaf in the obesity-resistant phenotype of GF mice, GF wild-type and Fiaf -I- mice were fed a Western diet using the protocol described above in Example 1.

[0148] Methods. Mice were maintained as described in Example 1.

Fiaf -I- mice on a mixed C57BI/J:129/Sv background were backcrossed one generation to C57BI/6J animals and re-derived as GF, as described in Backhed et al. (2004). Wild-type and Fiaf-deficient littermates were used in these studies. Quantitative RT-PCR was performed as described above in Example 2, except Fiaf primers were used [forward, δ'-CAATGCCAAATTGCTCCAATT-S' (SEQ ID NO:5), reverse δ'-TGGCCGTGGGCTCAGT-S' (SEQ ID NO:6)]. Locomotor activity was measured as described in Example 1.

[0149] Results. The obesity-resistant phenotype of GF mice can also be attributed to their increased intestinal expression of Fiaf (Fig. 4A): no differences in hepatic Fiaf expression were noted between the groups. Moreover, when GF wild-type and Fiaf -/- mice were fed a Western diet, Fiaf- deficient animals gained significantly more weight than their wild-type littermates (6.2±0.9 g versus 2.7±1.0 g over a 5 week period; n=5/group, P < 0.05) (Fig. 4B), and had significantly greater epididymal fat pad weights (Fig. 4C).

[0150] Consistent with their increased adiposity, GF Fiaf -I- mice on the Western diet had higher serum levels of leptin and insulin than their GF wild- type littermates (Table 3). Serum triglycerides but not free fatty acids were significantly reduced in GF Fiaf-/- mice on the Western diet (Table 3), consistent with the fact they lack this circulating inhibitor of LPL.

[0151 ] GF Fiaf +/+ and Fiaf-/- littermates on the standard chow diet had no statistically significant differences in their locomotor activity, despite significant differences in their adiposity. Table 3. Biochemical and ELISA studies of sera, obtained after a four hour fast, from 15 week-old GF and conventionalized Fiaf-/- mice and their wild-type littermates maintained on a low-fat diet.

Fiaf +/+ Fiaf-/-

Serum levels (n - 8) (n - 6) P value

Glucose, mM 6,4 ± 1.6 6.8 ± 0.8 0.49

Insulin, ng/ml 0 .39 ± 0.05 0.54 ± 0.04 0.02

Leptin, ng/ml 2 .90 ± 0.51 4.93 ± 0.77 0.04

Triglycerides, mg/dl & 4.5 ± 16.3 54.7 ± 3.6 0.0005

Cholesterol, mg/dl 1 84.4 ± 8.4 137.8 ± 9.2 0.01

Free fatty acids 0 .95 ± 0.04 0.99 ± 0.07 0.65

Mean values ± SE are shown.

Example 4. Fiaf regulates Pgc-1α expression in gastrocnemius muscle.

[0152] The expression of genes encoding key enzymes involved in fatty acid oxidation in muscle was examined in Fiaf +/+ and Fiaf-/- littermates. Quantitative RT-PCR was performed as described in Example 2, using Mead, Fiaf, Cpt1 [forward, δ'-AGCACACCAGGCAGTAGCTT-S' (SEQ ID NO:7), reverse, δ'-AGGATGCCATTCTTGATTCG-S' (SEQ ID NO:8)] and Pgc-1α [forward, 5'-AACCACACCCACAGGATCAGA-3; (SEQ ID NO:9), reverse, 5'- TCTTCGCTTTATTGCTCCATGA-3' (SEQ ID NO:10)] primers.

[0153] Fiaf-deficiency in GF animals fed a Western diet was associated with statistically significant 24-46% decreases in the expression of genes encoding key enzymes involved in fatty acid oxidation in muscle (Cpt1 and Mead; see Fig. 4D). This effect does not appear to involve AMPK: there were no statistically significant differences in phospho-AMPK, total AMPK, phospho-Acc, AMP, ADP, ATP, NAD+, or NADH levels in the gastrocnemius muscles and livers of GF Fiaf knockout compared to their wild-type littermates (n=4 mice/group; Table 4). However, a significant reduction in expression of the PPAR (peroxisomal proliferator activated receptor) co-activator 1 α (Pgc-1 α) was found in GF Fiaf -/- gastrocnemius muscle (24±7 % compared to GF Fiaf +/+ littermates; n=6 mice/group; P < 0.05; Fig. 4D). Pgc-1α is capable of co- activating nearly all known nuclear receptors, as well as many other transcription factors: it is known to increase expression of genes encoding regulators of mitochondrial fatty acid oxidation, including Cpt1 and Mead.

Table 4. Biochemical assays of various metabolites in gastrocnemius muscle and liver harvested from GF wild-type and F/af-deficient littermates.

Metabolite, Muscle Liver μmol/g P I P

FiRi F ₊/+ Flat +/+ Fiaf -A protein Fiaf -/- value va lue

AMP 2 ,70 ± 0. 18 2, 00 ± 0,33 0,11 5 .72 ± 0. .41 5. 24 ± 0. 28 0 .30

ADP 4 ,33 ± 0. 45 4, 36 ± 0,62 0,97 3 .48 ± 0. .32 3. 44 ± 0. 27 0 .92

ATP 24 .02 ± 1. 42 27 .13 i ± 0.87 0.08 9 .29 ± 0. .43 13 .22 i ± 1 .25 0 .07

NAD÷ 1 .48 ± 0. 05 1. 50 ± 0.06 0.78 0 .68 ± 0, .17 0, 61 ± 0, 26 0 .81

NADH 0 .26 ± 0. 02 0. 24 ± 0.02 0.50 0 .36 ± 0, .02 0, 37 ± 0, 03 0 .71

Mean values ± SE are shown (n=6 per group).

[0154] Thus, GF animals are protected from diet-induced obesity by two complementary but independent mechanisms that result in increased fatty acid metabolism: (i) elevated levels of Fiaf, which induces Pgc-1α, and (ii) increased AMPK activity. Together, these findings underscore the importance of considering the human metabolome in a supraorganismal context in order to achieve a full view of the factors that regulate energy balance.

Claims

CLAIMSWhat is claimed is:

1. A method for modulating body fat and/or weight loss in a subject, the method comprising (a) altering either the amount of or the activity of a Fiaf polypeptide in the subject; and (b) altering either the amount of or the activity of an AMPK polypeptide in the subject.

2. The method of claim 1 , wherein the amount of Fiaf polypeptide is altered in the subject by administering an effective amount of a Fiaf polypeptide to the subject.

3. The method of claim 1 , wherein the amount of or the activity of the Fiaf polypeptide is altered by administering a PPAR agonist to the subject.

4. The method of claim 1 , wherein altering either the amount of or the activity of a Fiaf polypeptide alters the activity of a Pgc-1 α polypeptide.

5. The method of claim 1 , wherein the amount of AMPK polypeptide is altered in the subject by administering an effective amount of an AMPK polypeptide to the subject.

6. The method of claim 1 , wherein the AMPK polypeptide is phosphorylated.

7. The method of claim 1 , wherein the amount of or the activity of the AMPK polypeptide is altered by administering an effective amount of an AMPKK polypeptide to the subject.

8. The method of claim 1 , wherein the amount of or the activity of the Fiaf polypeptide is altered by altering the microbiota population in the subject's gastrointestinal tract, such that the microbial-mediated regulation of the Fiaf polypeptide in the subject is modified.

9. The method of claim 8, wherein microbial-mediated transcriptional suppression of the Fiaf polypeptide occurs only in the gastrointestinal tract of the subject.

10. The method of claim 8, wherein the microbiota population is altered by decreasing the presence of at least one genera of saccharolytic microbe.

11. The method of clam 10, wherein the microbiota population is altered by decreasing the presence of B. thetaiotaomicron.

12. The method of claim 10, wherein the presence of a microbe is decreased by administering a probiotic selected from the group consisting of Lactobacillus, Acidophilus, Bifidobacteria and other components of the gut microbiota.

13. The method of claim 1 , wherein the amount of or the activity of the AMPK polypeptide is altered by altering the microbiota population in the subject's gastrointestinal tract, such that the microbial-mediated regulation of the AMPK polypeptide in the subject is modified.

14. The method of claim 13, wherein the microbiota population is altered by decreasing the presence of at least one genera of saccharolytic microbe.

15. The method of clam 14, wherein the microbiota population is altered by decreasing the presence of B. thetaiotaomicron.

16. The method of claim 14, wherein the presence of a microbe is decreased by administering a probiotic selected from the group consisting of Lactobacillus, Acidophilus, Bifidobacteria and other components of the gut microbiota.

17. The method of claim 13, wherein the AMPK polypeptide is phosphorylated.

18. A method for modulating body fat and/or weight loss in a subject, the method comprising altering the microbiota population in the subject's gastrointestinal tract such that microbial-mediated regulation of an AMPK polypeptide in the subject is modified.

19. The method of claim 18, further comprising altering the microbiota population in the subject's gastrointestinal tract such that microbial- mediated transcriptional suppression of a lipoprotein lipase inhibitor in the subject is decreased.

20. The method of claim 19, wherein the lipoprotein lipase inhibitor is a Fiaf polypeptide.

21. The method of claim 20, wherein microbial-mediated transcriptional suppression of the Fiaf polypeptide occurs only in the gastrointestinal tract of the subject.

22. The method of claim 18, wherein the microbiota population is altered by decreasing the presence of at least one genera of saccharolytic microbe.

23. The method of clam 22, wherein the microbiota population is altered by decreasing the presence of B. thetaiotaomicron.

24. The method of claim 22, wherein the presence of a microbe is decreased by administering a probiotic selected from the group consisting of Lactobacillus, Acidophilus, Bifidobacteria and other components of the gut microbiota.

25. The method of claim 18, further comprising administering to the subject an effective amount an agent that alters the amount of or the activity of a Fiaf polypeptide.

26. The method of claim 18, further comprising administering to the subject an effective amount of an agent that alters the amount of or the activity of an AMPK polypeptide to the subject.

27. The method of claim 26, wherein the AMPK polypeptide is phosphorylated.

28. A composition comprising an agent that increases the amount of or the activity of a Pgc-1 α polypeptide and an agent that increases the amount of or the activity of an AMPK polypeptide.

29. The composition of claim 28, wherein the agent that increases the amount of or the activity of a Pgc-1 α polypeptide is a Pgc-1 α polypeptide.

30. The composition of claim 28, wherein the AMPK polypeptide is phosphorylated.

31. The composition of claim 28, wherein the agent that increases the amount of or the activity of an AMPK polypeptide is an AMPK polypeptide.

32. A composition comprising an agent that increases the amount of or the activity of a Fiaf polypeptide and an agent that increases the amount of or the activity of an AMPK polypeptide.

33. The composition of claim 31 , wherein the agent that increases the amount of or the activity of a Fiaf polypeptide is a PPAR agonist.

34. The composition of claim 31 , wherein the agent that increases the amount of or the activity of a Fiaf polypeptide is a Fiaf polypeptide.

35. The composition of claim 31 , wherein the AMPK polypeptide is phosphorylated.

6. The composition of claim 31 , wherein the agent that increases the amount of or the activity of an AMPK polypeptide is an AMPK polypeptide.