WO2019238934A1 - Use of the apolipoprotein m for the treatment and diagnosis of insulin resistance - Google Patents

Abstract

The inventors examined APOM mRNA level and secretion in AT from patients in 6 independent clinical trials and in vitro in hMADS adipocytes. APOM expression and secretion were measured during dietary interventions. The inventors show that APOM is expressed in human subcutaneous and visceral AT and is released into circulation from AT. APOM expression in AT is lower in obese compared to lean individuals and reduced in subjects with metabolic syndrome and type 2 diabetes. Regardless of fat depot, there is a positive relationship between AT APOM expression and systemic insulin sensitivity. Finally, the inventors show that overexpression of APOM in the AT reduces AT inflammation and improves local insulin sensitivity during high fat diet induced obesity. Thus, the present invention relates to the present invention relates to the use of the apolipoprotein M for the treatment and diagnosis of insulin resistance.

Description

USE OF THE APOLIPOPROTEIN M FOR THE TREATMENT AND DIAGNOSIS

OF INSULIN RESISTANCE

FIELD OF THE INVENTION:

The present invention relates to the use of the apolipoprotein M for the treatment and diagnosis of insulin resistance.

BACKGROUND OF THE INVENTION:

Excess fat mass leads to being overweight or obese, which has deleterious health consequences. The development of obesity is still increasing worldwide (1). Likewise, metabolic syndrome, which is a collection of obesity-associated disorders, is associated with development of cardiovascular diseases, insulin resistance, hepatic steatosis, certain types of cancer and type 2 diabetes (2) (3). Resistance of peripheral tissues (liver, muscle, adipose tissue) to insulin action is indeed a key event in diabetes onset, so that therapeutic strategies aiming at restoring insulin sensitivity are highly relevant. However, they essentially remain unsatisfactory. For instance, thiazolidinediones, by binding peroxisome proliferator activated receptor gamma, increase insulin sensitivity, but such treatments are associated to important side effects. There is therefore still an important need to understand insulin resistance- promoting mechanisms to identify therapeutic targets.

Body fat is mainly adipose tissue which is one of the largest organs in the body. Adipose tissue is a metabolically active organ that, besides having a major role in buffering excess energy, secretes a wide panel of factors with signaling functions in homeostasis and metabolism (4). Some of these factors are polypeptides and proteins synthesized and secreted by the adipocytes, and are thus called adipokines. These molecules act locally in an autocrine- paracrine way or in an endocrine manner. Dysfunctional adipose tissue is a hallmark of systemic insulin resistance and type 2 diabetes. The study of the adipose tissue secretome has led to the identification of hundreds of adipokines with beneficial or detrimental metabolic effects, most of them having been poorly investigated so far (5, 6). Nowadays, novel adipokines are still being discovered.

Up to now, APOM expression has been described as being restricted to liver and kidneys in adults (7) and the encoded protein as a plasma apolipoprotein predominantly enriched in HDL (8). The apoM belongs to the lipocalins family (7). As such, in blood, it carries bioactive lipids such as sphingosine-l -phosphate (S1P) and retinol, both being involved several biological processes including lipid metabolism and inflammation (7, 9). ApoM-deficient mice lack HDL-associated S1P and display increased vascular permeability, which is a component of inflammatory response (10). Recently, a link between the apoM/SlP axis and energy metabolism has been demonstrated in mice (11) but the role of the apoM itself in insulin resistance has never been investigated neither in humans nor in the adipose tissue.

SUMMARY OF THE INVENTION:

The present invention relates to the use of the apolipoprotein M for the treatment and diagnosis of insulin resistance. In particular, the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

The adipose tissue (AT) is a secretory organ producing a wide variety of factors that participate in the genesis of metabolic disorders linked to excess fat mass. Weight loss improves obesity related disorders. Transcriptomic studies on human AT and a combination of analyses of transcriptome and proteome profiling of conditioned media from adipocytes and stromal cells isolated from human AT has led to the identification of apolipoprotein M (apoM) as a putative adipokine. The inventors aimed to validate apoM as novel adipokine, investigate the relationship of AT APOM expression with metabolic syndrome and insulin sensitivity, and study the regulation of its expression in AT and secretion during calorie restriction induced weight loss. Therefore, they examined APOM mRNA level and secretion in AT from patients in 6 independent clinical trials and in vitro in human multipotent adipose-derived stem (hMADS) adipocytes. APOM expression and secretion were measured during dietary interventions. The inventors show that APOM is expressed in human subcutaneous and visceral AT, mainly by adipocytes. ApoM is released into circulation from AT and plasma apoM concentrations correlate with adipose tissue APOM mRNA levels. APOM expression in AT is lower in obese compared to lean individuals and reduced in subjects with metabolic syndrome and type 2 diabetes. Regardless of fat depot, there is a positive relationship between AT APOM expression and systemic insulin sensitivity, independently of fat mass and plasma HDL. In hMADS adipocytes, APOM expression is enhanced by insulin sensitizing PPAR agonists and inhibited by TNFa, a cytokine which causes insulin resistance. In obese individuals, calorie restriction increased AT APOM expression and secretion. Finally, the inventors show that overexpression of APOM in the AT reduces AT inflammation and improves local insulin sensitivity during high fat diet induced obesity.

Methods of treatment:

Accordingly, the first object of the present invention relates to a method of treating insulin resistance in a patient in need thereof comprising administering to the patient a therapeutically effective amount of an ApoM polypeptide or a nucleic acid molecule encoding thereof

As used herein, the term“insulin resistance” has its common meaning in the art. Insulin resistance is a physiological condition where the natural hormone insulin becomes less effective at lowering blood sugars. The resulting increase in blood glucose may raise levels outside the normal range and cause adverse health effects such as metabolic syndrome, dyslipidemia and subsequently type 2 diabetes mellitus. The method of the present invention is thus particularly suitable for the treatment of type 2 diabetes. As used herein, the term "type 2 diabetes" or“non insulin dependent diabetes mellitus (NIDDM)” has its general meaning in the art. Type 2 diabetes often occurs when levels of insulin are normal or even elevated and appears to result from the inability of tissues to respond appropriately to insulin. Most of the type 2 diabetics are obese.

In some embodiments, the subject suffers from obesity. As used herein the term "obesity" refers to a condition characterized by an excess of body fat. The operational definition of obesity is based on the Body Mass Index (BMI), which is calculated as body weight per height in meter squared (kg/m²). Obesity refers to a condition whereby an otherwise healthy subject has a BMI greater than or equal to 30 kg/m², or a condition whereby a subject with at least one co-morbidity has a BMI greater than or equal to 27 kg/m². An "obese subject" is an otherwise healthy subject with a BMI greater than or equal to 30 kg/m² or a subject with at least one co-morbidity with a BMI greater than or equal 27 kg/m². A "subject at risk of obesity" is an otherwise healthy subject with a BMI of 25 kg/m² to less than 30 kg/m² or a subject with at least one co-morbidity with a BMI of 25 kg/m² to less than 27 kg/m². The increased risks associated with obesity may occur at a lower BMI in people of Asian descent. In Asian and Asian-Pacific countries, including Japan, "obesity" refers to a condition whereby a subject with at least one obesity-induced or obesity-related co-morbidity that requires weight reduction or that would be improved by weight reduction, has a BMI greater than or equal to 25 kg/m². An "obese subject" in these countries refers to a subject with at least one obesity- induced or obesity- related co-morbidity that requires weight reduction or that would be improved by weight reduction, with a BMI greater than or equal to 25 kg/m². In these countries, a "subject at risk of obesity" is a person with a BMI of greater than 23 kg/m² to less than 25 kg/m².

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]). In particular, the method of the present invention is particularly suitable for improving blood glucose control, enhancing insulin signalling in skeletal muscle and adipose tissue, reducing lipotoxicity in skeletal muscle and adipose tissue, increasing lipid oxidative capacity in skeletal muscle and adipose tissue, or maintaining long-term insulin sensitivity in the subject.

As used herein, the term“ApoM” has its general meaning in the art and refers to the apolipoprotein M encoded by the APOM gene (Gene ID: 55937). ApoM is an apolipoprotein and member of the lipocalin protein family. The term is also known as G3A or NG20. An exemplary amino acid sequence of ApoM is represented by SEQ ID NO:l and an exemplary nucleic acid sequence is represented by SEQ ID NO:2.

SEQ ID NO : 1 >sp | 095445 | APOM HUMAN Apolipoprotein M OS=Homo sapiens OX=9606 GN=APOM PE=1 SV=2

MFHQIWAALLYFYGI ILNSIYQCPEHSQLTTLGVDGKEFPEVHLGQWYFIAGAAPTKEEL

ATFDPVDNIVFNMAAGSAPMQLHLRATIRMKDGLCVPRKWIYHLTEGSTDLRTEGRPDMK

TELFSSSCPGGIMLNETGQGYQRFLLYNRSPHPPEKCVEEFKSLTSCLDSKAFLLTPRNQ

EACELSNN

SEQ ID NO: 2

1 agagtggact gagcagccag taggggagag agcagttaag gcacacagag caccagctcc 61 ctcctgcctg aagatgttcc accaaatttg ggcagctctg ctctacttct atggtattat

121 ccttaactcc atctaccagt gccctgagca cagtcaactg acaactctgg gcgtggatgg

181 gaaggagttc ccagaggtcc acttgggcca gtggtacttt atcgcagggg cagctcccac

241 caaggaggag ttggcaactt ttgaccctgt ggacaacatt gtcttcaata tggctgctgg

301 ctctgccccg atgcagctcc accttcgtgc taccatccgc atgaaagatg ggctctgtgt

361 gccccggaaa tggatctacc acctgactga agggagcaca gatctcagaa ctgaaggccg

421 ccctgacatg aagactgagc tcttttccag ctcatgccca ggtggaatca tgctgaatga

481 gacaggccag ggttaccagc gctttctcct ctacaatcgc tcaccacatc ctcccgaaaa

541 gtgtgtggag gaattcaagt ccctgacttc ctgcctggac tccaaagcct tcttattgac

601 tcctaggaat caagaggcct gtgagctgtc caataactga cctgtaactt catctaagtc

661 cccagatggg tacaatggga gctgagttgt tggagggaga agctggagac ttccagctcc

721 agctcccact caagataata aagataattt ttcaatcctc aaaaaaaaaa aaaaaaaaaa

781 aaaaaaaaaa

In some embodiments, the ApoM polypeptide of the present invention comprises an amino acid sequence having at least 70% identity with the amino acid sequence as set forth in SEQ ID NO:l. According to the invention a first amino acid sequence having at least 70% identity with a second amino acid sequence means that the first sequence has 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% identity with the second amino acid sequence. Amino acid sequence identity is typically determined using a suitable sequence alignment algorithm and default parameters, such as BLAST P (Karlin and Altschul, 1990).

According to the invention, the polypeptide of the invention is produced by conventional automated peptide synthesis methods or by recombinant expression. General principles for designing and making proteins are well known to those of skill in the art. The polypeptides of the invention may be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols as described in Stewart and Young; Tam et al, 1983; Merrifield, 1986 and Barany and Merrifield, Gross and Meienhofer, 1979. The polypeptides of the invention may also be synthesized by solid-phase technology employing an exemplary peptide synthesizer such as a Model 433A from Applied Biosystems Inc. The purity of any given protein; generated through automated peptide synthesis or through recombinant methods may be determined using reverse phase HPLC analysis. Chemical authenticity of each peptide may be established by any method well known to those of skill in the art. As an alternative to automated peptide synthesis, recombinant DNA technology may be employed wherein a nucleotide sequence which encodes a protein of choice is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression as described herein below. Recombinant methods are especially preferred for producing longer polypeptides. A variety of expression vector/host systems may be utilized to contain and express the peptide or protein coding sequence. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors (Giga-Hama et al, 1999); insect cell systems infected with virus expression vectors (e.g., baculo virus, see Ghosh et al, 2002); plant cell systems transfected with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmid; see e.g., Babe et al., 2000); or animal cell systems. Those of skill in the art are aware of various techniques for optimizing mammalian expression of proteins, see e.g., Kaufman, 2000; Colosimo et al, 2000. Mammalian cells that are useful in recombinant protein productions include but are not limited to VERO cells, HeLa cells, Chinese hamster ovary (CHO) cell lines, COS cells (such as COS-7), W138, BHK, HepG2, 3T3, RIN, MDCK, A549, PC12, K562 and 293 cells. Exemplary protocols for the recombinant expression of the peptide substrates or fusion polypeptides in bacteria, yeast and other invertebrates are known to those of skill in the art and a briefly described herein below. Mammalian host systems for the expression of recombinant proteins also are well known to those of skill in the art. Host cell strains may be chosen for a particular ability to process the expressed protein or produce certain post-translation modifications that will be useful in providing protein activity. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post- translational processing which cleaves a "prepro" form of the protein may also be important for correct insertion, folding and/or function. Different host cells such as CHO, HeLa, MDCK, 293, WI38, and the like have specific cellular machinery and characteristic mechanisms for such post-translational activities and may be chosen to ensure the correct modification and processing of the introduced, foreign protein.

In some embodiments, it is contemplated that polypeptides used in the therapeutic methods of the present invention may be modified in order to improve their therapeutic efficacy. Such modification of therapeutic compounds may be used to decrease toxicity, increase circulatory time, or modify biodistribution. For example, the toxicity of potentially important therapeutic compounds can be decreased significantly by combination with a variety of drug carrier vehicles that modify biodistribution. In example adding dipeptides can improve the penetration of a circulating agent in the eye through the blood retinal barrier by using endogenous transporters. A strategy for improving drug viability is the utilization of water- soluble polymers. Various water-soluble polymers have been shown to modify biodistribution, improve the mode of cellular uptake, change the permeability through physiological barriers; and modify the rate of clearance from the body. To achieve either a targeting or sustained- release effect, water-soluble polymers have been synthesized that contain drug moieties as terminal groups, as part of the backbone, or as pendent groups on the polymer chain. Polyethylene glycol (PEG) has been widely used as a drug carrier, given its high degree of biocompatibility and ease of modification. Attachment to various drugs, proteins, and liposomes has been shown to improve residence time and decrease toxicity. PEG can be coupled to active agents through the hydroxyl groups at the ends of the chain and via other chemical methods; however, PEG itself is limited to at most two active agents per molecule. In a different approach, copolymers of PEG and amino acids were explored as novel biomaterials which would retain the biocompatibility properties of PEG, but which would have the added advantage of numerous attachment points per molecule (providing greater drug loading), and which could be synthetically designed to suit a variety of applications. Those of skill in the art are aware of PEGylation techniques for the effective modification of drugs. In addition, to the polymer backbone being important in maintaining circulatory half-life, and biodistribution, linkers may be used to maintain the therapeutic agent in a pro-drug form until released from the backbone polymer by a specific trigger, typically enzyme activity in the targeted tissue. For example, this type of tissue activated drug delivery is particularly useful where delivery to a specific site of biodistribution is required and the therapeutic agent is released at or near the site of pathology. Linking group libraries for use in activated drug delivery are known to those of skill in the art and may be based on enzyme kinetics, prevalence of active enzyme, and cleavage specificity of the selected disease-specific enzymes. Such linkers may be used in modifying the protein or fragment of the protein described herein for therapeutic delivery.

In some embodiments, the polypeptides of the invention may be fused to a heterologous polypeptide (i.e. polypeptide derived from an unrelated protein, for example, from an immunoglobulin protein).

As used herein, the term "nucleic acid molecule" has its general meaning in the art and refers to a DNA or RNA molecule. However, the term captures sequences that include any of the known base analogues of DNA and RNA such as, but not limited to 4-acetylcytosine, 8- hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5- (carboxyhydroxylmethyl) uracil, 5-fmorouracil, 5-bromouracil, 5- carboxymethylaminomethyl-2-thiouracil, 5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1 -methyladenine, 1 -methylpseudouracil, l-methylguanine,

1- methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine,

5- methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5- methoxyamino-methyl-2-thiouracil, beta-D-mannosylqueosine, 5'- methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil- 5-oxyacetic acid methylester, uracil-5 -oxyacetic acid, oxybutoxosine, pseudouracil, queosine,

2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, -uracil-5- oxyacetic acid methylester, uracil-5 -oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.

In some embodiments, the nucleic acid molecule of the present invention comprises a nucleic acid sequence having has at least 70% identity with the nucleic acid sequence as set forth in SEQ ID NO:2. According to the invention a first nucleic acid sequence having at least 70% identity with a second nucleic acid sequence means that the first sequence has 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% identity with the second nucleic acid sequence.

In some embodiments, the nucleic acid molecule of the present invention is included in a suitable vector. Typically, the vector is a viral vector, and more particularly an adeno- associated virus (AAV), a retrovirus, bovine papilloma virus, an adenovirus vector, a lentiviral vector, a vaccinia virus, a polyoma virus, or an infective virus. In some embodiments, the vector is an AAV vector. As used herein, the term "AAV vector" means a vector derived from an adeno- associated virus serotype, including without limitation, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and mutated forms thereof. AAV vectors can have one or more of the AAV wild-type genes deleted in whole or part, preferably the rep and/or cap genes, but retain functional flanking ITR sequences. Retroviruses may be chosen as gene delivery vectors due to their ability to integrate their genes into the host genome, transferring a large amount of foreign genetic material, infecting a broad spectrum of species and cell types and for being packaged in special cell- lines. In order to construct a retroviral vector, a nucleic acid encoding a gene of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. In order to produce virions, a packaging cell line is constructed containing the gag, pol, and/or env genes but without the LTR and/or packaging components. When a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into this cell line (by calcium phosphate precipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media. The media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. Lentiviruses are complex retroviruses, which, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural function. The higher complexity enables the virus to modulate its life cycle, as in the course of latent infection. Some examples of lentivirus include the Human Immunodeficiency Viruses (HIV 1 , HIV 2) and the Simian Immunodeficiency Virus (SIV). Lentiviral vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted making the vector biologically safe. Lentiviral vectors are known in the art, see, e.g.. U.S. Pat. Nos. 6,013,516 and 5,994,136, both of which are incorporated herein by reference. In general, the vectors are plasmid-based or virus-based, and are configured to carry the essential sequences for incorporating foreign nucleic acid, for selection and for transfer of the nucleic acid into a host cell. The gag, pol and env genes of the vectors of interest also are known in the art. Thus, the relevant genes are cloned into the selected vector and then used to transform the target cell of interest. Recombinant lentivirus capable of infecting a non-dividing cell wherein a suitable host cell is transfected with two or more vectors carrying the packaging functions, namely gag, pol and env, as well as rev and tat is described in U.S. Pat. No. 5,994,136, incorporated herein by reference. This describes a first vector that can provide a nucleic acid encoding a viral gag and a pol gene and another vector that can provide a nucleic acid encoding a viral env to produce a packaging cell. Introducing a vector providing a heterologous gene into that packaging cell yields a producer cell which releases infectious viral particles carrying the foreign gene of interest. The env preferably is an amphotropic envelope protein which allows transduction of cells of human and other species. Typically, the nucleic acid molecule or the vector of the present invention include "control sequences'", which refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these control sequences need always be present so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appropriate host cell. Another nucleic acid sequence, is a "promoter" sequence, which is used herein in its ordinary sense to refer to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene which is capable of binding R A polymerase and initiating transcription of a downstream (3’-direction) coding sequence. Transcription promoters can include "inducible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), "repressible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and "constitutive promoters”.

By a "therapeutically effective amount" is meant a sufficient amount of the polypeptide or the nucleic acid molecule encoding thereof to prevent for use in a method for the treatment of the disease (e.g. haemophilia) at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Preferably, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

According to the invention, the polypeptide or the nucleic acid molecule (inserted or not into a vector) of the present invention is administered to the subject in the form of a pharmaceutical composition. Typically, the polypeptide or the nucleic acid molecule (inserted or not into a vector) of the present invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The polypeptide or the nucleic acid molecule (inserted or not into a vector) of the present invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the typical methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The preparation of more, or highly concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small tumor area. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

Methods of diagnosis: A further object of the present invention relates to a method of determining whether a subject has or is at risk of having insulin resistance comprising i) determining the expression level of ApoM in a biological sample obtained from the subject and ii) comparing the expression level determined at step i) with a predetermined reference value wherein detecting differential between the expression level determined at step i) with the predetermined reference value indicated whether the subject has or is at risk of having insulin resistance.

The method of the present invention is thus particularly suitable for determining whether the subject has or is at risk of having a cardiometabolic disease. As used herein, the term "cardiometabolic disease" has its general meaning in the art and relates to cardiovascular diseases associated with metabolic syndrome, such as obesity, diabetes/insulin resistance, hypertension and dyslipidemia. The term "cardiometabolic diseases" refers to cardiac consequences of metabolic syndrome such as atherosclerosis, coronary heart disease, obesity- associated heart disease, insulin resistance- associated heart disease, hypertensive heart disease, cardiac remodeling, heart failure and cardiometabolic diseases disclosed in Hertle et al, 2014; Hua and Nair, 2014; U.S. Pat. Application No. 2012/0214771 and International Patent Application No. 2008/094939.

As used herein, the term "risk" in the context of the present invention, relates to the probability that an event will occur over a specific time period and can mean a subject's "absolute" risk or "relative" risk. Absolute risk can be measured with reference to either actual observation post-measurement for the relevant time cohort, or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period. Relative risk refers to the ratio of absolute risks of a subject compared either to the absolute risks of low risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed. Odds ratios, the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p/(l-p) where p is the probability of event and (1- p) is the probability of no event) to no- conversion. "Risk evaluation," or "evaluation of risk" in the context of the present invention encompasses making a prediction of the probability, odds, or likelihood that an event or disease state may occur, the rate of occurrence of the event or conversion from one disease state to another. Risk evaluation can also comprise prediction of future clinical parameters, traditional laboratory risk factor values, or other indices of relapse, either in absolute or relative terms in reference to a previously measured population. The methods of the present invention may be used to make continuous or categorical measurements of the risk of conversion, thus diagnosing and defining the risk spectrum of a category of subjects defined as being at risk of conversion. In the categorical scenario, the invention can be used to discriminate between normal and other subject cohorts at higher risk. In some embodiments, the present invention may be used so as to discriminate those at risk from normal.

In some embodiments, the biological sample is a blood sample. As used herein the term “blood sample” means any blood sample derived from the subject. Collections of blood samples can be performed by methods well known to those skilled in the art. In some embodiments, the blood sample is a serum sample or a plasma sample.

The measurement of the level of ApoM in the blood sample is typically carried out using standard protocols known in the art. For example, the method may comprise contacting the blood sample with a binding partner capable of selectively interacting with ApoM in the sample. In some embodiments, the binding partners are antibodies, such as, for example, monoclonal antibodies or even aptamers. For example the binding may be detected through use of a competitive immunoassay, a non-competitive assay system using techniques such as western blots, a radioimmunoassay, an ELISA (enzyme linked immunosorbent assay), a“sandwich” immunoassay, an immunoprecipitation assay, a precipitin reaction, a gel diffusion precipitin reaction, an immunodiffusion assay, an agglutination assay, a complement fixation assay, an immunoradiometric assay, a fluorescent immunoassay, a protein A immunoassay, an immunoprecipitation assay, an immunohistochemical assay, a competition or sandwich ELISA, a radioimmunoassay, a Western blot assay, an immunohistological assay, an immunocytochemical assay, a dot blot assay, a fluorescence polarization assay, a scintillation proximity assay, a homogeneous time resolved fluorescence assay, a IAsys analysis, and a BIAcore analysis. The aforementioned assays generally involve the binding of the partner (ie. antibody or aptamer) to a solid support. Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e.g., in membrane or microtiter well form); polyvinylchloride (e.g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like. An exemplary biochemical test for identifying specific proteins employs a standardized test format, such as ELISA test, although the information provided herein may apply to the development of other biochemical or diagnostic tests and is not limited to the development of an ELISA test (see, e.g., Molecular Immunology: A Textbook, edited by Atassi et al. Marcel Dekker Inc., New York and Basel 1984, for a description of ELISA tests). Therefore ELISA method can be used, wherein the wells of a microtiter plate are coated with a set of antibodies which recognize ApoM. A sample containing or suspected of containing ApoM is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate(s) can be washed to remove unbound moieties and a detectably labelled secondary binding molecule added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate washed and the presence of the secondary binding molecule detected using methods well known in the art. Measuring the level of ApoM (with or without immunoassay-based methods) may also include separation of the compounds: centrifugation based on the compound’s molecular weight; electrophoresis based on mass and charge; HPLC based on hydrophobicity; size exclusion chromatography based on size; and solid-phase affinity based on the compound's affinity for the particular solid-phase that is used. Once separated, said one or two biomarkers proteins may be identified based on the known "separation profile" e.g., retention time, for that compound and measured using standard techniques. Alternatively, the separated compounds may be detected and measured by, for example, a mass spectrometer. Typically, levels of immunoreactive ApoM in a sample may be measured by an immunometric assay on the basis of a double-antibody "sandwich" technique, with a monoclonal antibody specific for ApoM. According to said embodiment, said means for measuring ApoM level are for example i) a ApoM buffer, ii) a monoclonal antibody that interacts specifically with ApoM, iii) an enzyme- conjugated antibody specific for ApoM and a predetermined reference value of ApoM.

In some embodiments, the predetermined reference value is a threshold value or a cut off value. Typically, a "threshold value" or "cut-off value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. For example, retrospective measurement of expression level of the gene in properly banked historical patient samples may be used in establishing the predetermined reference value. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. For example, after determining the level of the marker in a group of reference, one can use algorithmic analysis for the statistic treatment of the measured levels of the marker in samples to be tested, and thus obtain a classification standard having significance for sample classification. The full name of ROC curve is receiver operator characteristic curve, which is also known as receiver operation characteristic curve. It is mainly used for clinical biochemical diagnostic tests. ROC curve is a comprehensive indicator that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1 -specificity). It reveals the relationship between sensitivity and specificity with the image composition method. A series of different cut-off values (thresholds or critical values, boundary values between normal and abnormal results of diagnostic test) are set as continuous variables to calculate a series of sensitivity and specificity values. Then sensitivity is used as the vertical coordinate and specificity is used as the horizontal coordinate to draw a curve. The higher the area under the curve (AUC), the higher the accuracy of diagnosis. On the ROC curve, the point closest to the far upper left of the coordinate diagram is a critical point having both high sensitivity and high specificity values. The AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic result gets better and better as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is moderate. When AUC is higher than 0.9, the accuracy is quite high. This algorithmic method is preferably done with a computer. Existing software or systems in the art may be used for the drawing of the ROC curve, such as: MedCalc 9.2.0.1 medical statistical software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER.SAS, CREATE-ROC.SAS, GB STAT VIO.O (Dynamic Microsystems, Inc. Silver Spring, Md., USA), etc.

In some embodiments, the predetermined reference value is the expression level of ApoM determined in a population of healthy individuals. Typically, it is concluded that the subject has or is at risk of having insulin resistance when the expression level of is at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100 fold lower than the expression level determined in a population of healthy individuals.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1. The apolipoprotein M in human adipose tissue.

A. ApoM concentration was measured in paired samples of arterial and venous plasma samples from obese women. Data are plotted as the ratio between venous and arterial blood (AV) for each of the women (cohort A, n=8). *, P<0.05; from Wilcoxon test comparing venous to arterial blood.

B. APOM mRNA level was measured in paired samples (n=l2) of freshly isolated adipocytes and stroma-vascular fraction (SVF) from human subcutaneous abdominal adipose tissue. Data are presented as mean ± SEM. ***, P <0.001; *, P <0.05 from Wilcoxon test. C. ApoM concentration was measured in media from isolated adipocytes (n=l8) and stroma- vascular cells (SVF) (n=l2) from human subcutaneous abdominal adipose tissue fractions cultured for 24h. Data are presented as mean ± SEM. *, P <0.05; from Mann- Whitney test.

D. Differentiated hMADS adipocytes were treated for 4 days with 10 ng/ml TNFa, or the vehicle (n=6).

P <0.01 from Mann and Whitney test compared to control cells.

E. Differentiated hMADS adipocytes were treated for 4 days with 100 nM Rosiglitazone (n=9), 300 nM GW7647, or the vehicle (n=9). **, P <0.01; *, P <0.05 from Mann- Whitney test compared to control cells.

Figure 2. APOM expression in adipose tissues from individuals with a wide range of BMI.

A. APOM mRNA level in subcutaneous adipose tissue was compared between overweight (25< BMI <30, n=66) and obese (30< BMI <35, n=l50; 35 < BMI <40, n=74; BMI>40, n=46) individuals according to obesity grade (cohort E, 111 men and 225 women). Data are presented as mean ± SEM. *, P <0.05 compared to BMI<30 from ANOVA with Dunnett’s post-hoc test compared to overweight group.

B. APOM mRNA level in paired subcutaneous (SAT) and visceral (VAT) adipose tissue was compared between lean (mean BMI= 20.5±3.8, n=l 1) and obese (mean BMI= 39.0 ± 5.9, n=l2) women (cohort B). Data are presented as mean ± SEM. ***, p<0.00l; from 2 way ANOVA test with Dunnett’s post-hoc test compared to lean group.

C. APOM mRNA level was measured in subcutaneous adipose tissue of lean (n=l l), overweight (n=7) and obese (n=27) women (cohort B). Linear regression analysis of APOM expression and body fat mass with correlation coefficient and adjusted P-values according to Benj amini-Hochberg.

Figure 3. Human APOM mRNA level in subcutaneous adipose tissue according to the presence of metabolic syndrome.

A. Adipose tissue APOM expression in relation to adipose morphology in 29 obese (14 with adipose tissue hyperplasia and 15 with adipose tissue hypertrophy) and 26 non obese (13 with adipose tissue hyperplasia and 13 with adipose tissue hypertrophy) women (cohort F).

B-D. APOM mRNA level was measured in subcutaneous adipose tissue of lean (n=l 1), overweight (n=7) and obese (n=27) women (cohort B). Linear regression analysis of subcutaneous adipose tissue APOM expression and waist circumference (B), plasma HDL- cholesterol (C) and plasma triglycerides (D) with correlation coefficient and adjusted p-values according to Benj amini-Hochberg is displayed in each graph. E. APOM mRNA level in subcutaneous adipose tissue was compared between BMI- matched obese (30<BMI<35) women with the metabolic syndrome (MetS, n=28) and obese women without metabolic syndrome (no-MetS, n=28) (cohort E). Data are presented as mean ± SEM. *, P <0.05 from Mann- Whitney test.

Figure 4. Adipose tissue APOM mRNA level and circulating apoM relationship to insulin sensitivity.

A. APOM mRNA level in subcutaneous adipose tissue was compared between normal glucose tolerant (NGT, h=210), impaired fasting glucose and/or impaired glucose tolerance (IFG+IGT, h=101) and type 2 diabetes (T2D, n=l4) individuals (cohort E, 111 men and 225 women). Data are presented as mean ± SEM. *, P <0.05 compared to NGT from ANOVA with Dunnett’s post-hoc test compared to NGT group.

B. Relationship of adipose tissue APOM expression to QUICKI insulin sensitivity index. APOM mRNA level was measured in paired samples of subcutaneous (SAT) and visceral (VAT) adipose tissue of lean (n=l l-l4), overweight (n=7-l l) and obese (n=27-3 l) women (cohort C). Linear regression analysis of APOM gene expression and the quantitative insulin-sensitivity check index (QUICKI). Linear correlation coefficient and adjusted P-values according to Benjamini-Hochberg is displayed in each graph.

Figure 5. Effect of calorie-restriction induced weight loss on adipose tissue apoM.

A. APOM mRNA level was measured in subcutaneous adipose tissue of 314 obese individuals (cohort E) before (baseline) and after an 8-week very low-calorie diet (VLCD). Data are presented as mean ± SEM. ***, P <0.001; from Wilcoxon test.

B. ApoM concentration was measured in media from subcutaneous adipose tissue explants from 10 women (cohort D) at baseline and after a 28-day very low-calorie diet (VLCD). Data are presented as mean ± SEM. *, P <0.05; from Wilcoxon test.

Figure 6. Effect of human APOM overexpression in the perigonadic adipose tissue in high fat fed mice. A. Effect of adenovirus transduction on target genes expression. Perigonadic adipose tissues (PG-AT) were injected with adenoviruses (2xl0⁹ particles/fat pad) containing human APOM (black bars) or green fluorescent protein (white bars, GFP) coding sequences. Gene expression for GFP, human APOM and mouse apom was measured in PG-AT and liver. h=10 per group. B. Effect of adenovirus transduction on body weight. Body weight was measured at baseline (white bars) then after 30 days of high fat diet (black bars) in mice with perigonadic adipose tissues transduced with adenoviruses (2xl0⁹ particles/fat pad) containing human APOM (ad-hAPOM) or green fluorescent protein (ad-GFP), or saline (sham). Data are means ± SEM; h=10 per group; ***, p<0.00l . C. Effect of adenovirus transduction on glucose tolerance. Area under the curve of plasma glucose concentration versus time after an intraperitoneal glucose (lmg g/g body weight) bolus at baseline (DO) and after 21 days of high fat diet (D21) in mice with perigonadic adipose tissues transduced with adenoviruses (10⁹ particles/fat pad) containing human APOM (ad-hAPOM) or green fluorescent protein (ad- GFP), or saline (sham). DO (baseline), n=28; D21, sham n=9, ad-GFP h=10, ad-h APOM n=9. D. Perigonadic adipose tissue phosphorylation of Akt signaling. Perigonadic adipose tissue was incubated ex vivo with 100 nM insulin (black bars), or vehicle (white bars) in Krebs Ringer Hepes supplemented with 0.2% bovine serum albumin for 20 min at 37°C. Proteins were extracted from tissue lysates and quantified according to Bradford. Fifteen pg proteins were separated by SDS-PAGE and after transfer to nitrocellulose membranes were immunoblotted using antibodies raised against Ser(P)-Akt 473 or total- Akt. Data are means ± SEM; Sham, n=4; ad-GFP n=5, ad-h APOM n=6; **, p<0.0l . E. Effect of AAVs transduction of hAPOM in perigonadic adipose tissue on inflammatory signature. Perigonadic adipose tissue was injected with AAVs (5 x 10^L10 particles/fat pad) containing human APOM (3), or mCherry (2), or with NaCl 0.9% (1). Gene expression for interleukinl (II- 1 b), macrophage chemoattractant protein (M cpl) and tumor necrosis factor (Jnf-a) was measured in perigonadic adipose tissue. Data are means ± SEM; n=9 per group; *, p<0.05, **, p<0.0l in a 2 -way ANOVA with Tukey's multiple comparisons test.

Figure 7. . Effect of AAVs transduction of h APOM in perigonadic adipose tissue on phosphorylation of Akt signaling. Perigonadic adipose tissue was incubated ex vivo with 100 nM insulin (black bars), or vehicle (white bars) in Krebs Ringer Hepes supplemented with 0.2% bovine serum albumin for 20 min at 37°C. Proteins were extracted from tissue lysates and quantified according to Bradford. Fifteen pg proteins were separated by SDS-PAGE and after transfer to nitrocellulose membranes were immunoblotted using antibodies raised against Ser(P)-Akt 473 or total- Akt. Data are means ± SEM; Control NaCl 0.9% (sham), n=l5; AAV- mCherry n=l l, AAV-h APOM n=l4. *, p-value<0.05 in a 2-way ANOVA with Tukey's multiple comparisons test.

EXAMPLE 1: apolipoprotein M: a novel adipokine decreasing with obesity and upregulated by calorie restriction.

Methods

Clinical cohorts

The premenopausal women included in cohort A, B, C and D were recruited at the Third Faculty of Medicine, Charles University Hospital in Prague (Czech Republic). The studies were approved by the Ethic Committee of the Third Faculty of Medicine, Charles University, Prague. Individuals in cohort E were included in the multicenter, randomized, controlled dietary intervention study, DiOGenes, registered in ClinicalTrials.gov (NCT00390637). Individuals in cohort F were recruited at the Karolinska Institutet, Stockholm (Sweden), and the study was approved by the Regional Ethics committee in Stockholm. All projects were conducted in accordance with the guidelines in The Declaration of Helsinki and individual written informed consent was obtained from all participants involved in the studies. Clinical investigations were performed at rest after an overnight fast. Details for trials and clinical assessments are described in the corresponding references.

Cohort A included 8 obese women (BMI 33.9 ± 0.1 kg/m²) with stable weight during the 3 months preceding the study. As previously described in (12), exclusion criteria were hypertension, diabetes, hyperlipidemia, or any drug treatment.

Cohort B comprised 56 women scheduled to have abdominal surgery (laparoscopic or laparotomic cholecystectomy, hysterectomy and gastric banding) at the Departments of Surgery and Gynecology at Kralovske Vinohrady Faculty Hospital in Prague. BMI ranged from 17.3 to

48.5 kg/m². Clinical investigation was carried out 7-14 days prior to the surgery, as described in (13). Insulin sensitivity was assessed using a euglycemic hyperinsulinemic clamp. During surgery, paired samples of subcutaneous and visceral adipose tissue were obtained and stored at -80°C until processing.

Cohort C included 14 obese (BMI 34.1 ± 4.7 kg/m2) women with or without polycystic ovary syndrome (PCOS) recruited during 2004-2005. Anthropometric measurements, blood sampling, euglycemic-hyperinsulinemic clamp and subcutaneous abdominal adipose tissue needle biopsies were performed as described in (14).

Cohort D included 10 metabolically healthy overweight and obese women (BMI 34.1 ±

4.5 kg/m²) that followed an 800 kcal/d very low-calorie diet (VLCD) lasting 28 days. Inclusion and exclusion criteria for study participation were previously outlined (15). Clinical measures and subcutaneous abdominal adipose tissue biopsies were taken at baseline and at the end of the VLCD. The participants were instructed not to change their habitual activity during the study. Anthropometric measurements, blood sampling and subcutaneous abdominal adipose tissue needle biopsies were performed at rest after an overnight fast. Adipose tissue explants were maintained in culture media as described below.

Cohort E included a subgroup of 336 (111 men and 225 women) overweight and obese individuals (BMI 34.1 ± 4.7 kg/m2) from the DiOGenes Study in 8 European countries (16). Individuals followed a VLCD for 8 weeks, and those that lost at least 8% of their baseline weight were randomized to one of five ad libitum follow-up diets for 6 months. Clinical measures and subcutaneous abdominal adipose tissue biopsies were taken at each time-points. The participants were instructed not to change their habitual activity during the study. Only baseline and post-VLCD fat samples and clinical data were investigated in the present study.

Cohort F included 55 women without diabetes who had participated in a cross-sectional study conducted at Karolinska Institutet, Stockholm (Sweden) and were investigated as reported in (17). Subcutaneous adipose tissue gene expression was assessed by Gene 1.0 ST Affymetrix arrays and adipocytes isolated from the same fat biopsy were used to measure the mean volume of the adipocytes. By comparing the mean size of the adipocytes with the total amount of body fat, the morphology of adipose tissue could be quantitatively determined (D values), as described in detail in (18). Negative D values indicate hyperplasia (many small adipocytes), and positive D values indicate hypertrophy (few but large adipocytes).

The risk score for metabolic syndrome was calculated according to (19). For assessment of insulin sensitivity, where euglycemic hyperinsulinemic clamp data were not available, the quantitative insulin-sensitivity check index (QUICKI), calculated as l/[log(I) + log(G)], where I is fasting insulin (pun its/m L) and G is fasting glucose (mg/dL), was used since it has been reported to be more reproducible and has a better correlation to the reference glucose clamp method than other surrogates including HOMA-IR, especially for longitudinal studies (20).

Adipose tissue fractionation and ex vivo cell culture

The individuals included in the ex vivo studies are described in Table 1. Human subcutaneous AT samples were collected from patients who underwent abdominal dermo- lipectomy during restorative surgery according to Declaration of Helsinki and to the guidelines of the Ethical Committee of Rangueil Hospital of Toulouse and the Committee for the Protection to Persons (n°: DC-2014-2039). Adipose tissue fractionation and ex vivo culture of cells for 24h are described in (21). Immunoselection/depletion of the distinct adipose tissue cell fractions and the 24h cell culture is described in (22). Media and cell fractions were stored at - 80°C until analysis.

Secretome and transcriptome analyses

For proteome analyses, NanoLC/ESI LTQ-Orbitrap MS/MS was used for analysis of 24h culture media of the different human adipose tissue cell types. Details are reported in (23).

For transcriptome studies, microarray assays were performed using the Agilent Whole Human Genome Microarray 4x44K v2 according to manufacturer’s recommendations (Agilent Technologies) and analysed as described in (21).

Subcutaneous adipose tissue catheterization To measure arteriovenous differences across subcutaneous abdominal adipose tissue, venous and arterial blood were sampled in 8 obese women (BMI 33.9 ± 0.1 kg/m²) as described in (12) (cohort A).

hMADS cell culture

The Human multipotent adipose-derived stem cell (hMADS) cells were cultured and differentiated as previously described (24). Briefly, after six days of proliferation, the cells were incubated in the differentiation medium (serum- free DMEM low glucose/ Ham’s F-12 medium containing l0pg/ml of transferrin, 5pg/ml of insulin, 0.2 nM triiodothyronine, 100mM 3- isobutyl-l-methylxanthine, 1 mM dexamethasone, and 100 nM rosiglitazone). Three days after (day3), dexamethasone and 3 -isobutyl- l-methylxanthine were omitted from the medium. Rosiglitazone was omitted at day 9. By day 13, cells were fully differentiated into adipocytes. At day 14, cells were treated with 100 nM of the PPARy agonist rosiglitazone (Alexis Biochemicals), or 300 nM of the PPARa agonist GW7647 (Sigma), or 10 ng/ml TNFa (Sigma), or the vehicle, for 4 days.

Adipose tissue explants

Adipose tissue samples of about 400 mg obtained from 10 obese women were cut into small pieces and incubated at 37°C for 4h in 4ml of Krebs/Ringer phosphate buffer supplemented with lg/F glucose and 20g/F bovine serum albumin as described in (15) (cohort D).

RNA extraction, cDNA synthesis and quantitative PCR

Total RNA was extracted from tissues and cells using the RNeasy mini kit (Qiagen, GmbH, Hilden, Germany). Total RNA (500 ng) was reverse transcribed for 120 min at 37°C using Superscript II reverse transcriptase (Invitrogen, St Aubin, France) in the presence of random hexamers. A minus RT reaction was performed in parallel to ensure the absence of genomic DNA contamination.

Real-time PCR was performed in duplicate using a set of TaqMan (Thermo Fisher Scientific) probes and primers assays (Hs002l9533_ml). 20 ng cDNA was analyzed on a StepOne Plus Real-Time PCR system (Applied Biosystems, Carlsbad, CA) or, for longitudinal dietary interventions, using the Bio mark™ HD system (BioMark) as described in (21). Adipose tissue and cell data were normalized to GUSB (Hs00939627_ml) or LRP10 (Hs0l047362_ml), respectively.

ELISA assay

ApoM protein level was measured in duplicate using a human apoM EFISA kit (E-EF- H0473, Elabscience, Clinisciences, Nanterre, France), following manufacturer’s instructions. ApoM concentration was calculated using sigmoidal standard curve fitted by nonlinear regression analysis for each test.

Statistical analyses

Gaussian distribution was tested using the D'Agostino & Pearson test. For comparing data from two groups, where a normal distribution was unattainable, a Wilcoxon or Mann- Whitney test was employed for paired or unpaired comparisons respectively. For a comparison using data from more than two groups, ANOVA was performed with Dunnett’s post-hoc test or Kruskal- Wallis with Dunn’s post-hoc test for non-normal data distribution. Linear regression was performed with adipose tissue APOM expression as the dependent variable and the Benjamini-Hochberg procedure was applied to account for multiple testing. Multiple regression was performed with all variables significantly correlated with adipose tissue APOM expression. Partial correlations were computed to assess direct correlations independently of other confounding variables.

Results

APOM is released by human adipose tissue and secreted by adipocytes.

In addition to mature adipocytes, the adipose tissue contains different cell types including preadipocytes and immune, endothelial and progenitor cells corresponding to the stroma vascular fraction. A comparative proteomic analysis of the proteins secreted by these 5 distinct human adipose tissue cell populations led to the identification of 626 proteins secreted by adipocytes, 123 of which have never been described in the literature as produced by the adipose tissue and were found specifically in the media of mature adipocytes. We compared this list of potentially new adipokines with the data of the transcriptome analyses on the same different adipose tissue cellular fractions. Forty of the proteins above, including apoM, were identified as adipocyte markers according to their gene expression profile. We combined this list with previous transcriptome analyses of adipose tissue from individuals with varying amounts of fat mass (13) and identified apoM as a potential adipokine with an expression regulated according to BMI.

To validate apoM as an adipokine, we measured apoM in venous and arterial blood obtained by catheterization of the adipose tissue (cohort A) and investigated APOM expression in adipose tissue fractions and secretion in media of isolated adipocytes and stroma vascular fraction (Table 1).

Table 1. Ex vivo studies Gender Age, years BMI range

Description (M/F) (mean±SD) (kg/m²)

Adipose tissue cells selection for omics 0/6 54.7±9.l 25.0-30.4

Expression adipocytes vs. stromal cells 0/12 45.l±5.3 20.4-33.1

Secretion adipocytes vs. stromal cells 0/22 44.9±l2.3 20.2-39.1

When apoM was measured in plasma from venous and arterial blood obtained by catheterization of the subcutaneous abdominal adipose tissue, the ratio of apoM concentration was greater than 1 for each women (Figure 1A). On average, the concentration of apoM in venous plasma was significantly 12% higher than in arterial plasma, indicating that adipose tissue produces apoM and releases it into the circulation.

APOM expression and secretion were compared between isolated adipocytes and stroma vascular fractions. Compared to the stroma vascular fraction of adipose tissue, APOM was expressed (Figure IB) and secreted (Figure 1C) mainly by adipocytes.

In cohort B, there was a positive relationship between plasma apoM and subcutaneous adipose tissue APOM expression (cohort B, data not shown).

During adipogenic differentiation of hMADS cells APOM expression increased in a time-dependent manner (data not shown). This cell line was used to investigate the effect of prominent regulators of adipocyte secretory activities (25). In differentiated hMADS adipocytes, APOM mRNA level was reduced by TNFa treatment with (Figure ID) and enhanced by a treatment with PPAR agonists (PPARy, rosiglitazone; PPARa, GW 7647)

(Figure IE).

Higher APOM expression in adipose tissue from non-obese compared to obese individuals.

Comparison of APOM expression in subcutaneous adipose tissue between overweight and obese individuals according to obesity grade showed a decreased APOM expression with increasing obesity grade (cohort E, Figure 2A). No difference in subcutaneous adipose tissue

APOM expression was found between men and women (data not shown). Adipose tissue

APOM expression was analysed in paired samples of subcutaneous and visceral adipose tissue from lean and obese women (cohort B). Irrespective of the fat depot, a higher APOM expression was found in lean compared to obese individuals (Figure 2B). As previously reported (26), plasma apoM was lower in overweight and obese compared to lean women (data not shown). Moreover, a strong negative correlation between body fat and APOM expression in both subcutaneous and visceral adipose tissue was found (cohort B, Figure 2C).

Low adipose tissue APOM expression is associated with a dysmetabolic phenotype.

Obesity is an important risk factor for metabolic syndrome as the enlargement of fat mass impacts adipocyte biology. Adipocyte size is linked to metabolic syndrome (27).

Figure 3A shows that subcutaneous adipose tissue characterized by large fat cells (hypertrophy) displayed lower APOM expression than fat from subjects matched for BMI but with many small adipocytes (hyperplasia), irrespective of body weight status (cohort F). A negative correlation between adipose tissue APOM expression and adipocyte volume was found accordingly (data not shown).

There was a negative correlation between subcutaneous adipose tissue APOM expression and waist circumference and fasting plasma triglycerides, and a positive relationship with plasma HDL (cohort B Figure 3B-D). The same features were found for APOM expression in visceral adipose tissue (cohort B, data not shown).

Accordingly, when APOM expression in subcutaneous adipose tissue was compared in obese women matched for BMI and differing in presence of metabolic syndrome (19), a lower APOM expression was found in women with metabolic syndrome (cohort E, Figure 3E).

High adipose tissue APOM expression is associated with insulin sensitivity

Subcutaneous adipose tissue APOM expression was lower in subjects with type 2 diabetes and prediabetes (impaired fasting glucose and/or impaired glucose tolerance, assessed as in (28)) compared to normal glucose-tolerant individuals (cohort E, Figure 4A).

Since euglycemic hyperinsulinemic clamp data and several bioclinical parameters used for calculation of various insulin sensitivity indexes (29) were available for cohort B, we used this cohort to investigate the relationship between adipose tissue APOM expression and these different indexes. Significant correlations of APOM mRNA levels with QUICKI and HOMA- IR, fasting plasma glucose, revised QUICKI (30), and glucose disposal rate were found, irrespective of fat depot (Figure 4B and data not shown). The correlation to QUICKI, revised QUICKI and glucose disposal rate remained significant when corrected for plasma HDL or fat mass (data not shown). Accordingly, there was a positive correlation between APOM and GLUT4 expression in both subcutaneous and visceral fat depots (data not shown). As previously reported, higher plasma apoM concentration was associated with higher insulin- sensitivity (data not shown).

The link between adipose tissue APOM expression and insulin sensitivity was confirmed in a supplementary independent cohort including obese women with or without PCOS (cohort C). The presence of PCOS is linked to insulin resistance and functional derangements in adipose tissue (31). Here, women with PCOS had a lower insulin sensitivity and adipose tissue APOM expression compared to obese women without PCOS matched for fat mass and HDL (data not shown).

Effect of longitudinal dietary interventions on adipose tissue expression and secretion

As adipose tissue apoM expression and plasma levels are associated with obesity and comorbidities developed in obesity, we aimed to study if a weight-reducing diet which improves metabolic profile could influence the levels of apoM expression and production.

We measured APOM expression in subcutaneous adipose tissue of obese individuals before and after an 8-week VLCD (cohort E). The VLCD, which induced a decrease in fat mass, plasma triglycerides, cholesterol and HDL, and an increase in insulin sensitivity, as previously reported (32), resulted in a significant increase in adipose tissue APOM expression (Figure 5A). However, no significant association was found between the magnitude of changes in APOM expression and changes in fat mass, plasma HDL or triglycerides, or QUICKI, though, there was a weak but significant negative association between changes in APOM expression and changes in the C-reactive protein (r= -0.14, P= 0.0088, «=287).

The secretion of apoM by subcutaneous adipose tissue explants was assessed in obese women during a 1 -month VLCD (cohort D). The VLCD induced weight and body fat loss and an improvement in insulin sensitivity. ApoM concentration in media from adipose tissue explants significantly increased (+22%) at the end of the VLCD (Figure 5B).

Discussion:

A combination of microarray studies and proteomic profiling of conditioned media from freshly isolated adipocytes and cell populations in the stroma vascular fraction from human adipose tissue has led to the identification of apoM as a novel adipokine. The present study shows that the human adipose tissue is a site of expression of the APOM gene and that adipose tissue and predominantly adipocytes do secrete apoM.

With reference to other apolipoproteins, the apoM, which was identified in 1999 (33), is comparatively recent. The apoM has been mainly studied in liver where it is produced in hepatocytes; also, kidney proximal tubule cells express apoM (7). Here, we show that apoM is released from adipose tissue in vivo and ex vivo, particularly from adipocytes. The apoM concentration in media from adipose tissue explants in culture is comparable to other adipokines, such as leptin or IL6 (15). In addition, analyses of blood obtained by catheterization of the subcutaneous abdominal adipose tissue confirmed the production of apoM by adipose tissue in vivo (i.e. higher apoM concentration in the venous drainage of adipose tissue compared to paired arterial blood from same individuals). Our study indicates that the adipose tissue might be another producer of apoM in humans; still, the question remains about the extent of contribution of the adipose tissue to circulating apoM. We also found that APOM expression in adipose tissue positively correlates to plasma apoM. This suggests that the large adipose tissue mass is likely to contribute to circulating apoM.

Weight gain in adults is accompanied by an increase in adipocyte size. Adipocytes of obese subjects are dysfunctional, especially regarding metabolism and secretory functions (34). Large adipocytes were reported to be associated with metabolic disease, insulin resistance and type 2 diabetes in multiple studies (35). Here, we found a negative association between adipose tissue APOM mRNA level and fat cell volume. Moreover, in both obese and non-obese women, hypertrophic adipose tissue, containing few but large adipocytes, displayed lower APOM expression compared to hyperplastic tissue that includes more adipocytes but with smaller size. The APOM gene expression thus seems to be a hallmark of healthy adipose tissue.

Indirect data could account for lower APOM expression in obesity. Obesity is now regarded as a chronic low-grade inflammatory state with increased synthesis of acute phase factors by liver and pro -inflammatory cytokines released by immune cells within adipose tissue (36). Here, we found that TNFa down-regulated APOM expression in hMADS adipocytes. Also, in cohort E, we observed a nominal negative association (^-0.143, P=0.0108) between adipose tissue APOM expression and plasma C-reactive protein (data not shown). Since TNFa is locally increased with obesity (36), it is likely that adipose tissue APOM expression could be locally regulated by this pro -inflammatory cytokine.

Unhealthy adipose tissue, deficient in beneficial adipokines such as adiponectin and producing excess of harmful factors, is one of the features of metabolic syndrome and diseases such as insulin resistance and type 2 diabetes. The present study has shown that, in contrast to most adipokines (5, 21) and similarly to adiponectin (37), APOM gene expression in adipose tissue was lower with increasing adiposity. This trend is in accordance with the plasma concentration of apoM lower in obese compared to lean individuals, as previously reported (26).

Furthermore, we show that APOM mRNA level is lower in adipose tissue obtained from subjects with metabolic syndrome, independently of fat mass, and that adipose tissue APOM mRNA level is associated with several metabolic syndrome features (waist circumference, plasma HDF and triglycerides). In humans, plasma apoM has been described as a component of the pre-b HDL (8) and reduced with metabolic syndrome (38), in accordance with the present report.

It was shown previously that type 2 diabetes was also associated with reduced plasma apoM levels compared to glucose tolerance (39, 40). ApoM serum concentration was suggested as a marker for MODY diabetes additionally (41). Here, a positive relationship between adipose tissue APOM mRNA level and systemic insulin sensitivity, irrespective of fat depot, was found in 3 different cohorts. Interestingly, the association with risk factors for diabetes were independent of body fat or plasma HDL. Overall, adipose tissue APOM mRNA level is reduced with prediabetes and diabetes. More evidence is provided by another independent longitudinal study where lean men were investigated before and after a 4-d treatment with the glucocorticoid analogue, dexamethasone. The treatment induced insulin resistance without change in fat mass (42). The adipose tissue microarray data included APOM and showed that glucocorticoid exposure downregulated (mean fold change 0.6) APOM gene expression in subcutaneous fat

(42). Interestingly, here, when all parameters identified in association with adipose tissue APOM expression in univariate analysis were analyzed in a multiple linear regression model with adipose tissue APOM as dependent variable, only QUICKI was an independent predictor (b=0.500, P= 0.04) of adipose tissue APOM mRNA level (data not shown).

Defects in insulin action within adipose tissue are a common feature of insulin resistance

(43). Adipose tissue and the adipocyte itself have emerged as major regulators of whole body insulin action and fuel homeostasis (44). Targeted defects in insulin action in adipocytes lead to systemic insulin resistance (45). In vitro, we show that APOM gene expression in adipocytes is regulated by compounds such as TNFa, and PPAR agonists which influence adipose tissue inflammation and also regulate insulin sensitivity and adipokine production (25). The antidiabetic drug Rosiglitazone is a PPARy agonist that improves insulin sensitivity (46). Treatment of hMADS adipocytes with Rosiglitazone increased APOM mRNA level. The positive effect of this insulin sensitizing agent raises the possibility that apoM may play a causative role in insulin sensitivity.

Reduction in calorie intake results in fat mass loss and alleviates metabolic disorders in obese individuals (47). Diet-induced weight loss also modifies the secretory profile of adipose tissue (14, 48). Contrary to most adipokines (21, 49), including adiponectin (50), we find here that fat mass loss induced by calorie restriction increases expression and secretion of apoM in adipose tissue. No correlation between changes in subcutaneous adipose tissue APOM mRNA level and changes in bioclinical parameters found associated to adipose tissue APOM expression in univariate analysis was found to be significant. However, the increase in adipose tissue APOM expression was associated with the decrease in plasma C-reactive protein, indicating a link between adipose tissue apoM and obesity-related inflammation. Being a lipocalin, apoM was reported as a carrier of several anti-inflammatory lipids (7).

In mice, the apoM/SlP protects against atherosclerosis (9) and controls the metabolic activity of brown fat (11). The ability of apoM to bind anti-inflammatory factors could convey a local effect on the inflammatory status of the adipose tissue. Though, this highlights apoM as a good candidate for monitoring healthy adipose tissue status.

To summarize, the present study reports apoM production by adipocytes for the first time. It shows that APOM mRNA level is reduced in adipose tissue from obese subjects and independently of body fat mass, is also lower in individuals with insulin resistance, type 2 diabetes or dyslipidemia. Patterns of expression and secretion in adipose tissue indicate that apoM is also regulated in response to calorie-restriction induced weight loss.

Altogether this study indicates that apoM is a new adipokine, which may reflect changes in adipose tissue mass and whole body metabolic dysfunction, and which presence in the white adipose tissue is positively associated with insulin sensitivity.

EXAMPLE 2: Effect of human APOM overexpression in the perigonadic adipose tissue in high fat fed mice.

Methods:

Thirty 5-week old C57BL6/J male mice fed ad libitum were randomized in 3 groups of 10 mice according to body weight and glucose tolerance tests (lmg glucose/g body weight, intraperitoneal). Mice were housed at room temperature and manipulated according to Inserm guidelines and European Directive 2010/63/UE in the local animal care facility (agreements A 31 555 04 and C 31 555 07). Mice were fed with a 60% high fat diet for 4 weeks. After 2 weeks, mice were anaesthetized and perigonadic adipose tissues (PG-AT) were injected with recombinant adenoviruses (2xl0⁹ particles/fat pad) containing coding sequences of human APOM (ad-hAPOM) or green fluorescent protein (ad-GFP), or with saline (sham) or red fluorescent protein (mCherry). Two weeks later, glucose tolerance tests were performed in order to ensure that the high fat diet induced insulin resistance, at least in control mice. Then, after 30 days of high fat diet mice were weighed, and euthanatized before dissection of perigonadic and subcutaneous adipose tissues, and liver. Part of the tissues were snap frozen and stored at -80°C until total RNA extraction. To assess insulin sensitivity, the remaining adipose tissue samples were incubated ex vivo with insulin (100 nM), or vehicle, in Krebs Ringer Hepes supplemented with 0.2% bovine serum albumin. After 20 min at 37°C, tissue samples were rinced, snap frozen and stored at -80°C until protein extraction. Moreover, the perigonadic adipose tissue response to insulin was investigated ex vivo (20 min. insulin at 100 nM vs control) with the subsequent study of the Akt signaling pathway which full activation requires phosphorylation of S473 (Akt-P473).

Results:

Direct injection of adenoviruses into perigonadic fat pad induced overexpression of target genes, human apoM and the control GFP, in the perigonadic fat with no leak in liver and no change in mouse apoM expression (Figure 6A). Murine apoM was nearly undetectable in adipose tissue. As expected, the high fat diet induced weight gain and worsened glucose tolerance (Figures 6B and 6C). There was no difference between groups. In perigonadic fat of mice with human apoM overexpression, insulin-induced Akt phosphorylation was higher compared to mice with GFP overexpression and sham mice (Figure 6D). The expression of genes encoding inflammatory factors (interleukin 1 (II- 1 b), macrophage chemoattractant protein (Mcpl) and tumor necrosis factor (Tnf-a)) in perigonadic adipose tissue is displayed on Figure 6E. Despite no significant change in 116 gene expression was found across the 3 treatments, the perigonadic adipose tissue transduced using AAVmCherry exhibited an inflammatory signature evidenced by an increase in Tnf-a, Ill -b and Mcpl mRNA levels. When the perigonadic adipose tissue was transduced using AAVhAPOM, no inflammatory signature was found. This indicates that local overexpression of human APOM may prevent from an inflammatory signature.

Transduction of perigonadic adipose tissue with AAVs induced no change in control (baseline) Akt-P473. However, a higher Akt-P473 level under insulin treatment in AAVhAPOM, compared to AAVmCherry suggests a positive effect of local overexpression of human APOM on insulin signaling (Figure 7).

Discussion:

Obesity is associated with insulin resistance, a pathological condition and a factor in type 2 diabetes. Many of the mechanisms that bridge the pathogenesis of insulin resistance and obesity have been associated with ectopic lipid accumulation and/or obesity-induced inflammation. Pioneer research (Hotamisligil etal, Science 1993;259:87-91) demonstrated that tumor necrosis factor (TNF-a), an inflammatory mediator produced extensively by activated macrophages, is an insulin resistance pathogenicity factor. In lean individuals, adipose tissue secretes high levels of anti-inflammatory mediators such as interleukin- 10 (IL-10) or adiponectin. On the contrary, in individuals with obesity, adipose tissue secretes high levels of proinflammatory adipokines such as I L- 1 b, monocyte chemoattractant protein 1 (MCP-l), and TNF-a. This preponderance of proinflammatory versus anti-inflammatory adipokines is a hallmark of obesity-associated low-grade inflammation which leads to macrophage infiltration. Similar results were obtained using AAVs instead of adenoviruses, when local overexpression of human apoM prevented high fat diet induced impairement of insulin signaling and expression of pro-inflammatory cytokines.

The present study shows that overexpression of APOM in the AT reduces AT inflammation and improves local insulin sensitivity during high fat diet induced obesity.

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Claims

CLAIMS:

1. A method of treating insulin resistance in a patient in need thereof comprising administering to the patient a therapeutically effective amount of an ApoM polypeptide or a nucleic acid molecule encoding thereof.

2. The method of claim 1 wherein the patient suffers from type 2 diabetes.

3. The method of claim 1 wherein the patient suffers from obesity.

4. The method of claim 1 wherein the ApoM polypeptide of the present invention comprises an amino acid sequence having at least 70% identity with the amino acid sequence as set forth in SEQ ID NO: 1.

5. The method of claim 1 wherein the nucleic acid molecule of the present invention comprises a nucleic acid sequence having has at least 70% identity with the nucleic acid sequence as set forth in SEQ ID NO:2.

6. The method of claim 1 wherein the nucleic acid molecule of the present invention is included in a suitable vector.

7. The method of claim 6 wherein the vector is an AAV vector.

8. A method of determining whether a subject has or is at risk of having insulin resistance comprising i) determining the expression level of ApoM in a biological sample obtained from the subject and ii) comparing the expression level determined at step i) with a predetermined reference value wherein detecting differential between the expression level determined at step i) with the predetermined reference value indicated whether the subject has or is at risk of having insulin resistance.

9. The method of claim 1 wherein the biological sample is a blood sample.

10. The method of claim 1 wherein the predetermined reference value is the expression level of ApoM determined in a population of healthy individuals.

11. The method of claim 10 wherein it is concluded that the subject has or is at risk of having insulin resistance when the expression level of is at least 0.5, 1, 1.5, 2, 2.5, 3,

3.5, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100 fold lower than the expression level determined in a population of healthy individuals.

12. Use of the method of claim 8 for determining whether the subject has or is at risk of having a cardio metabolic disease.