WO2020136193A1 - Agonistes peptidiques du récepteur de l'adiponectine 1 et 2 - Google Patents

Agonistes peptidiques du récepteur de l'adiponectine 1 et 2 Download PDF

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WO2020136193A1
WO2020136193A1 PCT/EP2019/087006 EP2019087006W WO2020136193A1 WO 2020136193 A1 WO2020136193 A1 WO 2020136193A1 EP 2019087006 W EP2019087006 W EP 2019087006W WO 2020136193 A1 WO2020136193 A1 WO 2020136193A1
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peptide
adiporl
cells
mice
glucose
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Burtea CARMEN
Déborah CROMBEZ
Sophie Laurent
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Universite de Mons
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Universite de Mons
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention pertains to the technical field of agonists of adiponectin receptors 1 and 2 (Adipo Rl/2) and their clinical use in subjects.
  • diabetes mellitus The global prevalence of diabetes mellitus is approximately 360 million affected individuals, and is expected to reach 550 million by 2030, or one adult in ten. The prevalence has doubled over the past three decades as a result of obesity epidemic, and poses major health and socio-economic burden. Obesity is nearly invariably associated with insulin resistance in muscle, liver and fat.
  • Type 2 diabetes T2D
  • Type 1 diabetes T1D, formerly known as insulin-dependent, juvenile or childhood-onset
  • T2D non-insulin-dependent or adult-onset
  • Metabolic syndrome assembles several risk factors that concur in the development of various life-threatening disorders, such as the coronary artery disease, stroke, and T2D. Although the molecular basis of MS remains to be elucidated, all the risk factors are related to obesity. Among the risk factors, the central obesity and insulin resistance are the most important ones.
  • the adipose tissue acts as an endocrine organ that secretes several types of adipokines, such as free fatty acids, adiponectin, adipsin, leptin, plasminogen activator inhibitor-1, resistin, and TNF-a. They may link obesity to MS and the consequent morbid disorders due to their implication in energy and vascular homeostasis, but also in the immune pathways.
  • adipokines such as free fatty acids, adiponectin, adipsin, leptin, plasminogen activator inhibitor-1, resistin, and TNF-a.
  • the pro-inflammatory adipokines are overproduced in obesity, whereas anti inflammatory or insulin-sensitizing adipokines, such as adiponectin, are decreased.
  • the modulation of the altered production of adipokines may thus have therapeutic potential in the management of MS.
  • Adiponectin has been reported to improve insulin sensitivity and exert antidiabetic, anti-inflammatory and antiatherogenic effects. Adiponectin binding to its main receptors, AdipoRl and AdipoR2, triggers the oxidation of FFA and the glucose uptake by skeletal muscle, while liver gluconeogenesis is prevented. AdipoRl is principally expressed in skeletal muscle, where the signaling pathway of 5' adenosine monophosphate-activated protein kinase (AMPK) is activated. AdipoR2 is mostly expressed in the liver, where it activates the pathway of peroxisome proliferator-activated receptor alpha (PPARa). The two receptors are also co expressed in many other cell and tissue types. For instance, pancreatic beta cells express both AdipoRl/R2 and regulate the antiapoptotic effects of adiponectin by activating the MEK-ERK1/2 and PI3K-Akt pathways.
  • AMPK 5' adenosine monophosphate
  • Modulation of adiponectin signaling could have an effect on diseases such as obesity, type 2 diabetes and metabolic syndrome as well as provide a dietary solution.
  • Sunghwan et al., 2018; WO 2012/142142; Miki Okada-Iwabu et al., 2013 and 2015; and Laszlo Otvos Jr et al., 2011 all describe small molecules able to act as agonists of adiponectin.
  • Sunghwan et al., 2018 and Miki Okada-Iwabu et al., 2013 and 2015 discuss the use of these agonists for use in diabetes treatment.
  • only Sunghwan et al., 2018 is directed to a peptide. While Sunghwan et al., 2018 suggests that the peptide is able to bind to both AdipoRl and AdipoR2, it is apparent from the specification that binding to AdipoR2 is far less effective.
  • the current invention aims to provide a therapy or solution for at least one of the problems mentioned above, and which has a broad spectrum. Summary of the invention
  • the present invention provides for peptides according to claim 1 or a molecule according to claim 7. These peptides or molecules are capable of binding to AdipoRl and AdipoR2 and are agonists of AdipoRl and AdipoR2.
  • the current invention also relates to within AdipoRl and AdipoR2, wherein said region has an amino acid sequence according to SEQ ID n°13.
  • the current invention provides a composition according to claim 9.
  • the current invention provides for a use of the peptides or compositions as described above in a subject, preferably a human.
  • the present invention concerns peptides capable of binding to AdipoRl and AdipoR2 and their (therapeutic) use.
  • a compartment refers to one or more than one compartment.
  • the value to which the modifier "about” refers is itself also specifically disclosed.
  • % by weight refers to the relative weight of the respective component based on the overall weight of the formulation.
  • the current invention provides a peptide capable of binding to adiponectin receptors 1 or 2.
  • peptide refers to any compound containing two or more amino acid residues joined by an amide bond formed from the carboxyl group of one amino acid residue and the amino group of the adjacent amino acid residue.
  • the amino acid residues may have the L-form as well as the D-form, and may be naturally occurring or synthetic, linear as well as cyclic.
  • polypeptides and peptide dimers which can be peptides linked C-terminus to N- terminus (tandem repeats) or peptides linked C-terminus to C-terminus or N- terminus to N-terminus (parallel repeats).
  • said peptide is a synthetic peptide.
  • synthetic peptide it is understood to refer to a peptide which has been artificially synthesized. Such synthesis methodologies are readily known in the art.
  • therapeutic peptide denotes a bioactive peptide that has therapeutic utility.
  • Mimetics are peptide-like molecules which mimic elements of protein or peptide secondary structure.
  • the underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions.
  • a peptide mimetic is expected to permit molecular interactions similar to the natural molecule.
  • the peptide according to the embodiments of the current invention may be purified.
  • purified will refer to a protein or peptide composition which has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity.
  • substantially purified will refer to a composition in which the peptide forms the major component of the composition, such as constituting about 50% or more of the peptides in the composition.
  • Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is appreciated that a cation-exchange column chromatography performed utilizing an HPLC apparatus will generally result in a greater - fold purification than the same technique utilizing a low pressure chromatography system. Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein.
  • the peptides will have a length of 6 to 25 amino acids, more preferably between 10 to 15 amino acids, even more preferably twelve amino acids.
  • said peptides according to the current invention share at least 95%, more preferably at least 99% sequence identity to an amino acid sequence chosen from SEQ:ID n° 1 to 12, preferably SEQ:ID n° 4, 5 or SEQ:ID n°12.
  • said peptides have a sequence which differs maximally 3, more preferably maximally 2, even more preferably maximally 1 amino acid from one of the sequences chosen from SEQ:ID n° 1 to 12, preferably SEQ:ID n° 4, 5 or SEQ:ID n°12.
  • said peptides have an amino acid sequence which incorporates one of the amino acid sequences chosen from SEQ:ID n° 1 to 12, or a sequence which has at least 95%, more preferably 99% sequence identity with the amino acid sequences chosen from SEQ:ID n° 1 to 12, preferably SEQ:ID n° 4, 5 or SEQ:ID n°12.
  • sequence identity refers to the extent that sequences are identical on an amino acid-by-amino acid basis over a window of comparison.
  • a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical amino acid occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • a gap i.e., a position in an alignment where a residue is present in one sequence but not in the other is regarded as a position with non-identical residues.
  • Determining the percentage of sequence identity can be done manually, or by making use of computer programs that are available in the art. Examples of useful algorithms are PILEUP. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).
  • Amino acid sequence variants of a peptide contemplated herein may be substitutional, insertional or deletion variants.
  • Deletion variants lack one or more residues of the peptide which may not be critical for function.
  • Substitutional variants typically contain an alternative amino acid at one or more sites within the peptide and may be designed to modulate one or more properties of the polypeptide such as stability against proteolytic cleavage. Substitutions preferably are conservative, that is, one amino acid is replaced with one of similar size and side chain or functional group.
  • Conservative substitutions are well known in the art and include, for example, the changes of: alanine to glycine, valine or leucine; arginine to lysine; asparagine to glutamine; aspartate to glutamate; cysteine to methionine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to glutamine, tyrosine, arginine, lysine, asparagine or cysteine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to phenylalanine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.
  • said peptides have an amino acid sequence which differs by maximal 3, maximal 2, more preferably maximal 1 amino acid from one of the amino acid sequences chosen from SEQ:ID n° 1 to 12.
  • Said peptides have a half-life of between 1 and 30 hours, more preferably between 2 and 20 hours.
  • the current invention equally relates to a synthetic sequence encoding for a peptide according to SEQ ID n° 1 - 12 and according to one of the embodiment above.
  • AdipoRl and AdipoR2 present 66.41% sequence identity and are composed of 7 transmembrane domains with a membrane topology that is reversed when compared to the G-protein-coupled receptors (GPCRs), meaning that their N- terminus is internal, whereas the C-terminus is external.
  • GPCRs G-protein-coupled receptors
  • AdipoR-12C 12-amino acid sequence within the C-terminal domain of adiponectin receptors (AdipoR), a 12-amino acid sequence (AdipoR-12C) was identified that is homologous in AdipoRl (Q96A54) and AdipoR2 (Q86V24), both in humans and mice.
  • the peptides as described above are able to bind to a (C-terminal) region of adiponectin receptors 1 or 2, wherein said region comprises an amino acid sequence according to SEQ ID 13.
  • said peptides are able to bind to the amino acid sequence as given in SEQ ID n° 13 and which is part of the adiponectin receptors 1 or 2.
  • the current invention is thus also directed to the sequence given in SEQ ID n° 13 which is conserved in both AdipoRl and AdipoR2 and to molecules, including peptides (such as the peptides as identified above), proteins such as antibodies or compounds such as small molecules able to bind to this conserved region.
  • the term "small molecule” is defined as a usually low molecular weight ( ⁇ 900 daltons) organic compound that may regulate a biological process, with a size on the order of 1 nm. Said small molecule is a molecule able to bind specific biological macromolecules (in the current case AdipoRl and AdipoR2) and acts as an effector, altering the activity or function of the target.
  • binding molecules may be able to block (antagonist or inhibitors) or activate (agonists) the adiponectin receptor signaling.
  • said binding molecules are agonists of AdipoRl and AdipoR2 and as such are able to activate AdipoRl and/or AdipoR2 signaling upon binding and thus able to modulate the lipid and glucose metabolism of a subject, preferably a mammal, such as a human.
  • Suitable molecules can be identified via ligand binding assays, phage display, computational modeling or other high throughput screenings.
  • the present invention also pertains to a composition, preferably a pharmaceutical composition comprising one or more peptides or molecules able to bind to SEQ ID n° 13 according the embodiments as described above.
  • Embodiments herein provide for administration of compositions to subjects in a biologically compatible form suitable for pharmaceutical administration in vivo.
  • biologically compatible form suitable for administration in vivo is meant a form of the active agent (e.g., pharmaceutical chemical, protein, gene, antibody, aptamers etc. of the embodiments) to be administered in which any toxic effects are outweighed by the diagnostic or therapeutic effects of the active agent.
  • Administration of an active amount of the compositions according to the current invention is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result, whether it may be for imaging or therapeutic reasons.
  • an active amount of a compound may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the peptides to elicit a desired response in the individual. Dosage regimen may be adjusted to provide the optimum therapeutic response.
  • compositions containing a peptide of the current invention, or analog thereof, or a functional derivative thereof (e.g., a mimetic of said peptides) or a molecule able to bind to SEQ ID n° 13 may be administered to a subject, for example by subcutaneous, intravenous, peritoneal, intracardiac, intracoronary, intramuscular, by oral administration (formulated as solutions, liquids, (lyophilized) powders, capsules, tablets, liposomes, and the like), inhalation, transdermal application, intravaginal application, topical application, intranasal or rectal administration.
  • the active compound may be coated in a material to protect the active agent from the degradation by enzymes, acids and other natural conditions that may inactivate the compound.
  • the composition may be administered intravenously.
  • the active agent in the current case the peptide may be administered to a subject in an appropriate carrier or diluent, co-administered with enzyme inhibitors or in an appropriate carrier such as liposomes.
  • pharmaceutically acceptable carrier as used herein is intended to include diluents such as saline and aqueous buffer solutions. It may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation.
  • the active agent may also be administered parenterally or intraperitoneally. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils.
  • the peptides could be loaded in polymeric microparticles of chitosan, dextran, alginate, PLGA etc., in colon -targeted microparticles, hydrogel-based microparticles, or in microcapsules or microspheres often composed of polymers. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.
  • compositions suitable for injectable use may be administered by means known in the art.
  • sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion may be used.
  • Sterile injectable solutions can be prepared by incorporating the active agent in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • Aqueous compositions can include an effective amount of the active agent, being one or more peptides according to the current invention dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.
  • Compounds and biological materials disclosed herein can be purified by means known in the art. Solutions of the active compounds as free-base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • said composition may be formulated to be administered via a medical device such as an insulin pump. Said composition could be either separately administered or in combination with insulin. Other medical devices are insulin syringes or insulin pens.
  • Active agents may be formulated within a mixture to comprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 5.0 mg per dose or even about 1 to 10 grams per dose, depending on the specific reasons for use (e.g. therapeutic or dietary). If the peptide or molecule is used for therapeutic reasons the chosen dose may lie between 1 mg/dose to 15 g/dose, more preferably 1 g to 10 g/dose.
  • compositions When the composition is used for therapeutic purpose, a single dose or multiple doses can also be administered on an appropriate schedule for a predetermined condition such as daily, bi-weekly, weekly, bimonthly etc.
  • Pharmaceutical compositions are administered in an amount, and with a frequency, that is effective to modulate side effects. The precise dosage and duration of treatment may be determined empirically using known testing protocols or by testing the compositions in model systems known in the art and extrapolating therefrom. Dosages may also vary with the severity of the condition. In certain embodiments, the composition range can be between 10 and 75 mg/kg introduced daily or weekly to a subject.
  • a therapeutically effective amount can be also measured in molar concentrations and can range between about 10 nmol to about 100 pmol of peptide/molecule per kg body weight of said subject, more preferably between 20 nmol to 50 pmol of peptide/molecule per kg body weight of said subject, even more preferably between 1 pmol and 40 pmol of peptide/molecule per kg body weight of said subject.
  • Therapeutic application of the peptides, molecules and compositions according to the current invention comprising them can be accomplished by any suitable therapeutic method and technique presently or prospectively known to those skilled in the art. Further, the peptides or molecules of the invention can be used as starting materials or intermediates for the preparation of other useful compounds and compositions.
  • the peptides, molecules or compositions as described above can be used for therapeutic use or as a food supplement, preferably a dietary food supplement.
  • adiponectin signaling pathways via AdipoRl and AdipoR2 regulate glucose and lipid metabolism, showing a relevance for T2D and obesity associated pathologies.
  • the peptides according to the current invention and other molecules able to bind to the sequence given in SEQ ID n°13 of AdipoRl and AdipoR2 are shown to be able to influence the clinical picture of diseases or syndromes linked to adiponectin signaling.
  • the current invention provides thus a method for treating or controlling syndromes or diseases linked to adiponectin signaling through AdipoRl and AdipoR2. Examples are for instance pathologies such as obesity, MS, cardiovascular diseases (CVD), fatty liver disease, liver fibrosis, Type 2 Diabetes or systemic disease. This will be further elaborated on in the experimental data section.
  • Figure 1 shows the location of the targeted AdipoR-12C (white frame) at the end of the 7 th transmembrane domain (TMD7) that precedes the C-terminal domain (CTD) in AdipoRl (PDB id : 3WXV) (A, B) and AdipoR2 (PDB id : 3WXW) (C, D).
  • the specific affinity (expressed as target/BSA) of the phage pools recovered after three rounds of panning was assessed against AdipoR-12C (E) and AdipoRl (F), whereas that of the 50 phage clones isolated from the 3 rd round phage pool was evaluated against AdipoR-12C (G); the gray horizontal line represents the mean specific affinity.
  • Figure 2 shows the amino acid frequency in the sequence of 12 identified peptides (A) and titration curves with K d values of the three selected peptide clones (B).
  • Figure 3 shows the three-dimensional structure (A) and spatial conformation (B) of peptides P16, P17 and P18, compared to AdipoRl-12C and AdipoR2-12C.
  • the three- dimensional structure of peptides was drawn with ACD/ChemSketch 2.0 software.
  • the three-dimensional structure of AdipoRl-12C and AdipoR2-12C and the spatial conformations of all molecules were obtained with MarvinSketch 5.11.5 software (2013, http://www.chemaxon.com ' ) ⁇
  • Figure 4 shows colocalization of peptides P16, P17 and P18 with mouse AdipoRl in muscle (A) and AdipoR2 in liver (B) as observed by immunofluorescence.
  • AdipoRl and AdipoR2 are detected with fluorescein, peptides are evidenced with Texas Red, whereas nuclei are revealed with DAPI.
  • the Manders' Colocalization Coefficients Ml (overlap of channel 1 over channel 2) and M2 (overlap of channel 2 over channel 1) were evaluated using the JACoP plugin of ImageJ software.
  • Figure 5 shows colocalization of peptides P16, P17 and P18 with AdipoRl (A) and insulin (B) in human pancreas as observed by immunofluorescence.
  • AdipoRl is detected with fluorescein, insulin observed with Texas Red, whereas nuclei are revealed with DAPI.
  • Peptides are stained with Texas Red in (A) and with fluorescein in (B).
  • the Manders' Colocalization Coefficients Ml (overlap of channel 1 over channel 2) and M2 (overlap of channel 2 over channel 1) were evaluated using the JACoP plugin of ImageJ software.
  • Figure 6 shows colocalization of slow myosin with AdipoRl (A) and peptides P16, P17 and P18 (B) in mouse muscle as observed by immunofluorescence.
  • Slow myosin is stained with Texas Red, peptides are revealed with fluorescein, whereas nuclei are detected with DAPI.
  • Figure 7 shows expression of AdipoR2 by HepaRG cells (A) and of AdipoRl by differentiated C2C12 cells (B) observed by immunofluorescence with fluorescein; nuclei are stained with DAPI.
  • the cells were induced for 15, 60 and 135 minutes by incubation in a culture medium enriched in glucose (Glc, 25 mM for HepaRG; 39 mM for C2C12) or glucose and free fatty acids and cholesterol (FFAC).
  • the relative ratio of fluorescent labeling (RRFL, normalized to cell number and the background) of AdipoR2 (left) and AdipoRl (right) was evaluated with ImageJ software and represented graphically in (C). The results are expressed as means ⁇ SD.
  • Figure 8 shows the effect of peptides and of a commercial AdipoR agonist (AgoAdipoR) on AMPK activation by T172 phosphorylation (AMPKa-pT172) in HepaRG (A, B, C, D) and C2C12 cells (E, F, G, H).
  • the cells were incubated for 15, 60 and 135 minutes in a culture medium containing a glucose supplement (A, C, E, G) or the basal glucose concentration (B, D, F, H).
  • the medium was supplemented (C, D, G, H) or not (A, B, E, F) with a mixture of free fatty acids and cholesterol (FFAC).
  • the results are expressed as means ⁇ SD.
  • the statistical significance of the results was calculated by ANOVA, using the SigmaPlot 11.0 software, by comparing each test group to the control group, incubated in the same culture media as the test groups excepting the peptides and AgoAdipoR.
  • NS stands for non-significant.
  • the peptides that present a significant effect are framed with the same color as their graphical representation.
  • Figure 9 shows the effect of peptide P17 on SDHA and GK expression in HepaRG (A) and differentiated C2C12 (B) cells incubated for 60 minutes in a culture medium enriched with glucose (Glc, 25 mM for HepaRG; 39 mM for C2C12) or glucose and free fatty acids and cholesterol (FFAC).
  • SDHA was detected with Texas Red
  • GK was observed with fluorescein
  • nuclei were stained with DAPI.
  • the relative ratio of fluorescent labeling (RRFL, normalized to cell number and the background) was evaluated with ImageJ software and represented graphically in (C). The results are expressed as means ⁇ SD.
  • the statistical differences were calculated by ANOVA, using the SigmaPlot 11.0 software.
  • Figure 10 shows the relative ratio of fluorescent labeling (RRFL, normalized to cell number and the background) of the microphotographs of AdipoRl colocalization with lysosomes and caveolae (examples shown in Figures 11 and 12) was evaluated with ImageJ software and represented graphically. The results are expressed as means ⁇ SD. The statistical differences were calculated by ANOVA, using the SigmaPlot 11.0 software.
  • Figure 11 shows colocalization of AdipoRl with lysosomes (observed with anti- LAMP1 antibody) (A) and caveolae (observed with anti-caveolin-1 antibody) (B) in differentiated C2C12 cells incubated for 60 minutes in a culture medium enriched with glucose (Glc, 39 mM) and which were stimulated or not with peptide P17; plain control cells were incubated in basal culture medium.
  • AdipoRl was detected with Dylight 594, lysosomes and caveolae were observed with Dylight 488, while nuclei were stained with DAPI.
  • the Manders' Colocalization Coefficients Ml (overlap of channel 1 over channel 2) and M2 (overlap of channel 2 over channel 1) were evaluated using the JACoP plugin of ImageJ software.
  • Figure 12 shows colocalization of AdipoRl with lysosomes (observed with anti- LAMP1 antibody) (A) and caveolae (observed with anti-caveolin-1 antibody) (B) in differentiated C2C12 cells incubated for 60 minutes in a culture medium enriched with glucose (Glc, 39 mM) and free fatty acids and cholesterol (FFAC) and which were stimulated or not with peptide P17; plain control cells were incubated in basal culture medium.
  • AdipoRl was detected with Dylight 594, lysosomes and caveolae were observed with Dylight 488, while nuclei were stained with DAPI.
  • the Manders' Colocalization Coefficients Ml (overlap of channel 1 over channel 2) and M2 (overlap of channel 2 over channel 1) were evaluated using the JACoP plugin of ImageJ software.
  • Figure 13 shows the effect of peptide P17 on the body weight of db/db mice fed on a Western high-fat diet containing 60 kcal % or on a standard chow diet containing 10 kcal %.
  • the mice were treated with P17 each evening for a duration of 4 days and were compared to control db/db mice treated with PBS and fed on the same diet or with healthy NMRI mice fed on a standard chow diet, which did not receive any treatment.
  • the results are shown in box-and-whisker plots and the statistical differences were calculated by ANOVA, using the SigmaPlot 11.0 software.
  • Figure 14 shows the effect of peptide P17 on glycemia (A), plasma triglycerides and adiponectin (B) of db/db mice fed on a Western high-fat diet containing 60 kcal % or on a standard chow diet containing 10 kcal %.
  • the mice were treated with P17 each evening for a duration of 4 days and were compared to control db/db mice treated with PBS and fed on the same diet or with healthy NMRI mice fed on a standard chow diet, which did not receive any treatment.
  • the results of glycemia are shown in box-and-whisker plots. Plasma triglycerides and adiponectin are expressed as means ⁇ SD. The statistical differences were calculated by ANOVA, using the SigmaPlot 11.0 software.
  • Figure 15 shows the effect of peptide P17 on the expression of AdipoRl in skeletal muscle (A) and of AdipoR2 in liver (B) of db/db mice fed on a Western high-fat diet containing 60 kcal % or on a standard chow diet containing 10 kcal %.
  • Phosphorylated AMPK (AMPK-p) in skeletal muscle (C) and liver (D) and phosphorylated PPARa (PPARa-p) in liver (E) were also analyzed on tissue samples of the same mice.
  • mice were treated with P17 each evening for a duration of 4 days and were compared to control db/db mice treated with PBS and fed on the same diet or with healthy NMRI mice (F) fed on a standard chow diet, which did not receive any treatment.
  • the different biomarkers are observed by immunofluorescence with fluorescein (AdipoRl, AdipoR2, PPARa) or with Dylight 488 (AMPK-p); nuclei are stained with DAPI.
  • Figure 16 shows the relative ratio of fluorescent labeling (RRFL, normalized to cell number and the background) evaluated on microphotographs of the same experimental conditions as in Figure 15 using ImageJ software and the results were represented graphically in (A) for AdipoRl in muscle, in (B) for AdipoR2 in liver, in (C) for AMPK-pT172 in muscle, in (D) for AMPK-pT172 in liver, and in (E) for PPARa- pS12 in liver. The results are expressed as means ⁇ SD. The statistical differences were calculated by ANOVA, using the SigmaPlot 11.0 software.
  • Figure 17 shows the effect of peptide P17 on liver apoptosis of db/db mice fed on a Western high-fat diet containing 60 kcal % or on a standard chow diet containing 10 kcal %.
  • the mice were treated with P17 each evening for a duration of 4 days and were compared to control db/db mice treated with PBS and fed on the same diet or with healthy NMRI mice fed on a standard chow diet, which did not receive any treatment.
  • Apoptotic livers from Balb/c mice treated with anti-Fas antibody were used as positive controls.
  • Apoptosis was observed by the immunofluorescent detection of activated caspase-3, stained with Dylight 488; nuclei are stained with DAPI (A).
  • the relative ratio of fluorescent labeling (RRFL, normalized to cell number and the background) was evaluated on microphotographs and the results were represented graphically in (B). The results are expressed as means ⁇ SD. The statistical differences were calculated by ANOVA, using the SigmaPlot 11.0 software.
  • Figure 18 shows the Masson's trichrome staining of liver tissue from db/db mice fed on a Western high-fat diet containing 60 kcal % or on a standard chow diet containing 10 kcal %. The mice were treated with P17 each evening for a duration of 4 days and were compared to control db/db mice treated with PBS and fed on the same diet or with healthy NMRI mice fed on a standard chow diet, which did not receive any treatment. Apoptotic livers from Balb/c mice treated with anti-Fas antibody were used as positive controls.
  • Figure 19 shows the effect of peptide P17 on pancreatic islet cell apoptosis of db/db mice fed on a Western high-fat diet containing 60 kcal % (A) or on a standard chow diet containing 10 kcal % (B).
  • the mice were treated with P17 each evening for a duration of 4 days and were compared to control db/db mice treated with PBS and fed on the same diet or with healthy NMRI mice fed on a standard chow diet, which did not receive any treatment (C).
  • Apoptosis was observed by the immunofluorescent detection of activated caspase-3, stained with Dylight 488.
  • Pancreatic beta cells were detected by the immunofluorescent staining of insulin, observed red with Texas Red; nuclei were stained with DAPI.
  • Figure 20 shows the colocalisation of activated caspase-3 with pancreatic alpha cells detected by the immunostaining of glucagon. Apoptosis was observed by the immunofluorescent detection of activated caspase-3, stained with Dylight 488.
  • Pancreatic alpha cells were observed with Texas Red; nuclei were stained with DAPI (A).
  • the percentage of beta cells and alpha cells was related to the total number of beta and alpha cells per pancreatic islet; the total number of beta cells per pancreatic islet is also represented (B).
  • the results are expressed as means ⁇ SD. The statistical differences were calculated by ANOVA, using the SigmaPlot 11.0 software.
  • Cys was coupled to the N-terminus of AdipoR-12C via a polyethylene glycol (PEG) spacer, whereas its C-terminus was amidated. Cys was used to immobilize AdipoR-12C on the surface of magnetic beads (Dynabeads ® M280 Tosylactivated, Life Technologies, Gent, Belgium) according to the manufacturer instructions. AdipoR-12C was synthesized by the PolyPeptide company (Strasbourg, France) and presented the following composition: Cys-8-amino-3,6-dioxanoctanoyl-His-Phe-Tyr-Gly-Val-Ser-Asn- Leu-Gln-Glu-Phe-Arg-CONH2.
  • AdipoR-12C was screened with a combinatorial linear 12-mer peptide library fused to the minor coat protein (pill) of M 13 bacteriophage (PhD-12, New England BioLabs Inc., Bioke, Leiden, The Netherlands).
  • the Escherichia coli host ER2738 (E. coli K12 ER2738, F+, tetracycline-resistant strain; New England BioLabs) was employed for phage amplification and clone isolation.
  • Bovine serum albumin was used as a control protein during the preselection steps of the panning rounds to exclude non-specific phages.
  • BSA Bovine serum albumin
  • TCEP Tris [2- carboxyethyl] phosphine hydrochloride
  • Both AdipoR-12C and BSA coupled Dynabeads were blocked for lh with the blocking buffer (0.5% BSA in sodium Phosphate Buffered Saline (NaPBS, for 1L): 0.262 g NaH 2 P0 -H 2 0, 2.901 g Na 2 HPO - 10 H 2 0, 0.88 g NaCI, pH 7.4).
  • the incubation time with the target (at an estimated concentration of 102 mM) was reduced stepwise during the 3 panning rounds (120, 90 and 60 min); the incubation times with BSA were increased stepwise (60, 90, 120 min), while the Tween-20 concentration was increased at each panning round from 0.1% to 0.5% in the incubation and rinsing buffer.
  • the DNA of the selected phage clones was isolated and purified by phenol extraction - ethanol precipitation, and it was sequenced by the company Beckman Coulter Genomics (Grenoble, France). The DNA sequences and the encoded peptides were read with JaMBW 1.1 software (http://bioinformatics.org/JaMBW/). Peptide sequences were aligned with pertinent proteins by BLAST (The Basic Local Alignment Search Tool).
  • this last one was immobilized (50 pg/mL in NaPBS containing 10 mM EDTA, pH 7.2; 150 pL/well) in the wells of a Pierce® maleimide activated 96-well ELISA plate (Thermo Fisher Scientific) according to the manufacturer instructions.
  • the control wells were coated with BSA (50 pg/mL), with disulfide bonds reduced as described above.
  • the plate was rinsed with NaPBS pH 7.2 (0.05% Tween-20) and then blocked with 10 pg/mL of cysteine-HCI (Thermo Fisher Scientific) prepared in the same buffer as AdipoR-12C and incubated (200 pL/well) for lh at room temperature (RT). After rinsing, the phage samples (5xl0 n /120 pL of NaPBS pH 7.2, 0.05% Tween-20) were incubated with AdipoR-12C or BSA coated wells for lh at RT.
  • cysteine-HCI Thermo Fisher Scientific
  • the plate was then rinsed, and bound phages were detected with HRP-conjugated anti- M IS antibody (Amersham Pharmacia Biotech Benelux, Roosendaal, The Netherlands) diluted 1 : 5000 in NaPBS pH 7.2, containing 5 mg BSA/mL.
  • the staining reaction was developed with ABTS [2,2 ' -Azino-bis(3-Ethylbenzothiazoline-6- sulfonic acid), diamonium salt (Sigma-Aldrich, Bornem, Belgium)] solution completed with 0.05% H 2 0 2 .
  • the OD os was measured using a microplate reader (StatFax-2100, Awarness Technology, Fisher Bioblock Scientific, Tournai, Belgium).
  • the binding to human AdipoRl was evaluated after protein immobilization (10 pg/mL, NaHCCh 0.1 M, pH 8.6; 100 pL/well; 4°C overnight) in the wells of a medium binding Microlon® ELISA plate (Greiner Bi-One, Wemmel, Belgium).
  • the wells were rinsed (rinsing/incubation buffer: NaPBS pH 7.2, 0.5% Tween-20) and then incubated (2h, RT, mild agitation at 350 rpm) with phages diluted in the rinsing/incubation buffer at 5xlO n virions/100 pL, or a range of concentrations (10 12 to 2xl0 9 virions/100 pl_) for the estimation of the apparent dissociation constant (K*d) : the phage binding to AdipoRl (test wells) and to BSA (control wells) was assessed concomitantly. The wells were then rinsed again, and the bound phages were detected as described above.
  • rinsing/incubation buffer NaPBS pH 7.2, 0.5% Tween-20
  • the peptides were synthesized (PolyPeptide Laboratories) as biotinylated or not biotinylated 8-amino-3,6-dioxaoctanoyl derivatives.
  • tissue sections were then dewaxed and rehydrated before blocking the endogenous biotin with a blocking kit (Vector Labconsult, Brussels, Belgium), followed by the blockage of non-specific epitopes with 1% BSA in potassium PBS (KPBS, for 1L: 0.2 g KCI, 0.2 g KH 2 P0 , 2.31 g NaH 2 P0 4 ⁇ 12H 2 0, 8 g NaCI, pH 7.4).
  • KPBS potassium PBS
  • the bound peptides were revealed by incubation (lh, RT) with 10 pg/mL of anti-biotin antibody made in goat and with 10 pg/mL of fluorescein anti-goat IgG made in rabbit (both from Vector Labconsult) both diluted in phosphate buffer (for 1L: 0.305 g
  • AdipoRl skeletal muscle and pancreas
  • AdipoR2 liver binding
  • peptides were co-incubated with AdipoRl/R2-specific antibodies on the same tissue sections, which were submitted to the same pretreatment as described above.
  • the slices were co-incubated (overnight, 4°C) with 20 mM of biotinylated peptides and 4 pg/mL of goat anti-AdipoRl or anti-AdipoR2 IgG (Santa Cruz Biotechnology, Heidelberg, Germany).
  • the human pancreas sections were treated with 10 mM sodium citrate (pH 6.0; 0.05% Tween-20) to unmask the epitopes.
  • the endogenous biotin and non-specific epitopes were blocked as described above, followed by overnight coincubation at 4°C with 20 pM of biotinylated peptides and 2 pg/mL of mouse anti-human insulin IgG (Abeam, Cambridge, GB).
  • the sections were incubated (lh, RT) with 10 pg/mL of goat anti-biotin antibody (Vector Labconsult) diluted in phosphate buffer pH 7.8, followed (lh, RT) by a coincubation with 20 pg/mL of hoarse anti-mouse IgG conjugated to Texas Red and 20 pg/mL of rabbit anti-goat IgG conjugated to fluorescein (both from Vector Labconsult) both diluted in phosphate buffer pH 7.8 completed with 0.05% Tween-20 and 0.5% BSA.
  • the samples were mounted and observed as explained above.
  • tissue sections were pre-treated identically (but biotin blocking was included) to those used for AdipoRl/R2 colocalization with slow myosin. Then, tissue sections were co incubated (overnight, 4°C) with 20 mM of biotinylated peptides and 20 pg/mL of mouse anti-slow myosin IgG (Abeam) diluted in phosphate buffer pH 7.4 containing 0.1% Tween-20.
  • HepaRG cells were cultivated (37 °C, 5% CO2) in Williams' E medium supplemented with 10% Fetal Bovine Serum (FBS), 1% glutaMAX and 13% Thaw, Plate & General Purpose Supplement (TPGPS).
  • FBS Fetal Bovine Serum
  • TPGPS Plate & General Purpose Supplement
  • HepaRG cells were grown in this culture medium for 96 hours (renewed after 48h). Then, the cells were grown for 72 hours in the culture medium comprising the Maintenance/Metabolism Medium Supplement at the place of TPGPS, according to the manufacturer instructions. Finally, the cells were incubated in the culture medium comprising the Induction Medium Supplement (all from Life Technologies) instead of TPGPS, in addition to the tested compounds as described below.
  • C2C12 cells were grown (37 °C, 5% CO2) in DMEM medium containing high glucose concentration (4.5 g/L) and glutamine, and supplemented with 10% FBS and 1% penicillin/streptomycin (all from Life Technologies).
  • C2C12 cells were grown in this culture medium for 5 days, when differentiation was induced by three days incubation in the same medium comprising 2% horse serum instead of FBS. After 72h, 33% of the current differentiation medium was removed and completed with an equal amount of fresh differentiation medium comprising various compounds as described below.
  • HepaRG and C2C12 cells were prepared for experimental procedures, they were induced for 15, 60 and 135 min in the final culture medium comprising a supplement of 14 mM glucose (25 mM total glucose for HepaRG and 39 mM total glucose for C2C12) or a solution of free fatty acids and cholesterol (FFAC, Chemically Defined Lipid Concentrate, Life Technologies) or both.
  • the FFAC solution was diluted 100 times in the induction medium to obtain 1.18 mM saturated FFA, 1.53 mM unsaturated FFA and 5.69 mM cholesterol.
  • the induction medium furthermore included 40 mM of one of the assessed non- biotinylated peptides (P16, P17, P18) or a commercial AdipoR agonist (AgoAdipoR; compound 112254, Santa Cruz Biotechnology) at a concentration of 2 mM.
  • the cells were seeded (2xl0 5 HepaRG cells/200 pL; 8xl0 4 C2C12 cells/200 pL) on microscope coverslips pre-coated with 200 pL of 200 pg collagen/mL (type I collagen from rat tail, Sigma-Aldrich, Diegem, Belgium) placed in 6-well culture plates (CellStarTM, Greiner BioOne).
  • collagen/mL type I collagen from rat tail, Sigma-Aldrich, Diegem, Belgium
  • CellStarTM 6-well culture plates
  • the cells were seeded (10 5 HepaRG cells/120 pL/well; 5xl0 4 C2C12 cells/120 pL/well) in 96-well culture plates (Greiner BioOne). Then, the cells were grown and induced as described above.
  • the cell samples were fixed with 4% formaline (15 min, RT) followed by rinsing with 1 mL/well of KPBS. Then, they were blocked (lh, RT) with 1% BSA in KPBS before incubation (overnight, 4°C) with 4pg/mL of goat anti-AdipoRl (for C2C12) or anti-AdipoR2 (HepaRG) IgG (both from Santa Cruz Biotechnology). The bound primary antibodies were detected (lh, RT) with 10 pg/mL of fluorescein anti-goat IgG made in rabbit prepared in phosphate buffer pH 7.8 containing 0.5% BSA. The cell samples were mounted with Vectashield Mounting Medium with DAPI before observing at microscope. 2.4.3. Quantification of phosphorylated AMPKa [pT172]
  • AMPKa phosphorylated on Thrl72 [pT172] was quantified on cell samples using a sandwich ELISA kit (Life Technologies), comprising two anti-AMPKa-pT172 antibodies, one for the antigen capture and the second one for detection.
  • the cells were cultured and induced as described at point 2.4.1, and then total proteins were extracted with an extraction buffer (Life Technologies) containing a protease inhibitor cocktail (Sigma-Aldrich) and the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF, Sigma-Aldrich), according to the manufacturer instructions.
  • the buffer was incubated (50 pL/well, 30 min, 4°C) with cells, the culture plate being agitated vigorously every 10 min. The cells were furthermore removed from each well surface by vigorous individual pipetting. Finally, the plate was centrifuged for 5 min at 3000 rpm and 4°C, and the supernatant was transferred in Eppendorf tubes to be again centrifuged for 10 min at 13.000 rpm and 4°C.
  • the cell lysates were stored at -80°C before their use for AMPKa-pT172 quantification, which was performed according to the supplier's instructions, using a calibration curve to obtain AMPKa-pT172 concentration in U/mL. The results were normalized to the protein concentration of each sample, as estimated with the PierceTM BCA Protein Assay kit (Thermo Fisher Scientific).
  • Glucokinase (GK) and succinate dehydrogenase (SDHA) were detected concomitantly by immunofluorescence on cell samples cultured and induced as explained above (point 2.4.1), with the sole difference that cells were induced for only 60 min. Subsequently, the cell samples were fixed (-20°C, 10 min) with 2 mL/well of 100% methanol, followed by rinsing with 1 mL/well of KPBS and blockage/permeabilization (lh, RT) with 1% BSA and 0.3% Triton-X100 in KPBS.
  • GK was detected with 5 pg/mL of rabbit anti-GK antibody, while SDHA was labeled with 5 pg/mL of mouse anti-SDHA antibody (both from Abeam), both diluted in KPBS. After overnight incubation at 4°C, the bound primary antibodies were detected by co-incubation of cells (lh, RT) with 20 pg/mL of Texas red conjugated anti-mouse IgG developed in horse and 20 pg/mL of fluorescein conjugated anti rabbit IgG developed in goat (both from Vector Labconsult) diluted in phosphate buffer pH 7.8 containing 0.5% BSA. Finally, the cell samples were mounted and observed at microscope as described above. 2.4.5. Fluorescent colocalization of AdipoRl with lysosomes and caveolae on C2C12 cells
  • AdipoRl was colocalized with lysosomes and caveolae on differentiated C2C12 cells cultured and induced as described at point 2.4.1, excepting the induction time that was limited to 60 min.
  • the cells were fixed and blocked as explained at point 2.4.5, and then they were co-incubated (overnight, 4°C) with 4pg/ml_ of goat anti-AdipoRl IgG and 4 pg/mL of either rabbit anti-LAMP-1 IgG or rabbit anti-caveolin-1 IgG, prepared in phosphate buffer pH 7.8 containing 0.5% BSA.
  • the bound primary antibodies were detected by co-incubation (lh, RT) with 10 pg/mL of Dylight 594 conjugated horse anti-goat IgG and 20 pg/mL of Dylight 488 conjugated horse anti-rabbit IgG (both from Vector Labconsult).
  • the cell samples were finally mounted and observed at microscope as described above.
  • mice aged of 6 weeks at the beginning of treatment injected with peptide P17 and fed on a Western high-fat diet containing 60 kcal % fat (D12492, Rodent Diet with 60 kcal % fat, Research Diets Inc., New Brunswick, USA);
  • mice aged of 6 weeks at the beginning of treatment injected with PBS and fed on a Western high-fat diet containing 60 kcal % fat;
  • P17 was injected i.p. each evening at a dose of 40 pmol/kg b.w. (60 pL/30 g of b.w.) for a duration of 4 days.
  • the control mice received PBS that was injected at the same volume and manner as P17.
  • a supplementary group of 4 healthy NMRI mice (Janvier Labs) was used as a witness group of subjects that received a standard chow diet and no substance administration.
  • mice The body weight and glycemia of mice were evaluated regularly during the experimental period.
  • the mice were euthanized (injection of 500 mg/kg b.w. of Nembutal and of 0.05 mg/kg b.w. of buprenorphine) the fifth day after the beginning of treatment, when the blood plasma and several tissues and organs (liver, skeletal muscle, pancreas) were collected for supplemental analysis.
  • the triglycerides and adiponectin were measured in blood plasma, while specific biomarkers of adiponectin signaling pathway were analyzed on histologic samples of the collected tissues and organs (fixed in 4% paraformaldehyde and paraffin embedded).
  • AdipoRl skeletal muscle
  • AdipoR2 liver
  • AMPKa-pT172 skeletal muscle and liver
  • PPARa-pS12 liver
  • Insulin and glucagon were detected in pancreas samples and were colocalized with activated caspase-3 as a biomarker of apoptosis, aiming to evaluate the reported beta cell depletion in db/db mice.
  • the presence of apoptotic cells was also investigated in liver samples, where the characteristic hepatic steatosis in db/db mice could increase the hepatocyte cell death by apoptosis.
  • the general tissue integrity was observed in liver by the Masson's trichrome staining.
  • Blood glycemia was measured in a drop of blood taken from the caudal vein, using the OneTouch® Verio Blood Glucose Meter (Johnson & Johnson Company).
  • the blood was collected on heparin from mice after euthanasia and blood plasma was separated by centrifugation (30 min, 7000 rpm).
  • the plasma triglycerides were quantified using the triglyceride dosing kit from BioAssay Systems (Gentaur BVBA, Kampenhout, Belgium).
  • Mouse adiponectin was measured in plasma using an ELISA kit from Invitrogen (Life Technologies). Both biomarkers were quantified according to the manufacturers' protocols.
  • tissue sections liver, skeletal muscle and pancreas
  • TBS Protein-Free Blocking Buffer
  • AdipoRl and AdipoR2 were detected with 4 pg/mL of goat anti-AdipoRl or anti-AdipoR2 IgG (Santa Cruz Biotechnology) incubated overnight at 4°C. Next day, the sections were incubated (lh, RT) with 10 pg/mL of horse anti-goat IgG conjugated to fluorescein (Vector Labconsult) diluted in phosphate buffer supplemented with 0.05% Tween-20 and 0.5% BSA.
  • tissue sections were incubated (overnight, 4°C) with 2 pg/mL of rabbit anti- AMPKa-pT172 antibody (Santa Cruz Biotechnology), followed by 15 pg/mL of Dylight 488 conjugated anti-rabbit IgG developed in horse diluted in phosphate buffer comprising 0.05% Tween-20 and 0.5% BSA.
  • PPARa-pS12 was observed by incubating (overnight, 4°C) tissue sections with 5 pg/mL of rabbit anti-PPARa-pS12 (Thermo Fisher Scientific), followed by 0.02 pg/mL of horse anti-rabbit IgG coupled to fluorescein (Vector Labconsult) diluted in phosphate buffer containing 0.5% BSA.
  • Activated caspase-3 was stained on liver sections by incubation (overnight, 4°C) with 5 pg/mL of rabbit anti-activated caspase-3 antibody (Thermo Fisher Scientific).
  • Apoptotic livers from Balb/c mice injected i.v. with 1 mg/kg b.w. of anti- Fas antibody (clone Jo2, isotype L2, BD Biosciences Pharmingen, Erembodegen, Belgium) were used as positive controls.
  • sections were incubated (lh, RT) with 15 pg/mL of Dylight 488 conjugated anti-rabbit IgG made in horse, diluted phosphate buffer containing 0.5% BSA.
  • activated caspase-3 was co-localized with insulin or glucagon by co-incubation (overnight, 4°C) with 5 pg/mL of rabbit anti-activated caspase-3 antibody (Thermo Fisher Scientific) and 0.2 pg/mL of anti-insulin antibody (clone E2E3, Abeam) or with 2 pg/mL of anti-glucagon antibody (clone K79bB10, Sigma-Aldrich), both produced in mouse.
  • rabbit anti-activated caspase-3 antibody Thermo Fisher Scientific
  • anti-insulin antibody clone E2E3, Abeam
  • anti-glucagon antibody clone K79bB10, Sigma-Aldrich
  • liver morphology and integrity were evaluated by staining tissue sections with Masson's Trichrome stain (Accustain® kit, Sigma-Aldrich) performed according to the manufacturer's protocol. Briefly, the nuclei were stained in black with Weigert's iron hematoxylin, while cytoplasm was stained in red with Beibrich scarlet- acid fuchsine. The collagen is stained in blue with aniline blue after treating sections with phosphotungstic and phosphomolybdic acid. The tissue sections were then rinsed in acetic acid and distilled water and mounted in a permanent medium after dehydration.
  • results are expressed as means ⁇ standard deviation (SD).
  • SD standard deviation
  • AdipoR-12C The homologous amino acid sequence (AdipoR-12C) identified within the C- terminal domain of AdipoRs are 351 HFYGVSNLQEFR 361 in AdipoRl and 362 HFHGVSNLQEFR 373 in AdipoR2, respectively, the only difference between them being thus at the third amino acid position.
  • the C- terminal extracellular region of AdipoRl started at L358, whereas that in AdipoR2 had L294 as the starting residue.
  • AdipoR-12C belonged to the end of the 7 th transmembrane domain (TMD7) ( Figure 1A-D).
  • the three other extracellular loops of AdipoRs present either a lower degree of homology or a sequence length shorter than 12 amino acid residues.
  • the homology we preferred to simultaneously target AdipoRl and AdipoR2 with the goal to concomitantly modulate both glucose (mainly regulated by AdipoRl) and lipid (mainly regulated by AdipoR2) metabolism, simulating in this way the physiological activity of adiponectin.
  • Concerning the length of the targeted protein fragment we have observed during our previous studies that fragments shorter than 12 amino acid residues lead to the selection of peptide candidates exposed in duplicate on the phage capside and are probably meant to equilibrate the molecular interaction.
  • AdipoR-12C has been screened by phage display using a linear 12-mer random peptide library.
  • the affinity for AdipoR-12C of the phage pools has increased from 1.79 to 2.53 times over that for BSA ( Figure IE), demonstrating an increased specificity.
  • the specific binding to AdipoRl was even better, the ratio over BSA increasing from 0.95 to 7.08 (the 2 nd round) and 6.58 (the 3 rd round) ( Figure IF).
  • 50 clones were isolated from the 3 rd round of panning and their binding to AdipoR-12C was assessed (Figure 1G). Among them, 20 clones presented a ratio AdipoR-12C/BSA superior to the mean (>1.6) and were selected for supplemental characterization. 2.
  • the 20 lead phage clones express 12 different peptides (Table 1). They are generally expressed by one clone each, excepting peptides 3, 9 and 10 that are associated to 3 or 4 clones. Several amino acids (G, A, P, K, R, H, S, T, W) are more frequent ( Figure 2A) and some of them form consensus motifs (i.e., SWR, GS, RTS) repeated in different clones (Table 1).
  • amino acids are either basic (K, R, H), uncharged polar (S, T) or hydrophobic (G, A, P, W), which is quite similar to the amino acid composition of AdipoR-12C (16.67% basic, 41.67% hydrophobic, 33.33% uncharged polar) (Table 2), meaning that their interaction could occur via hydrogen bonds, hydrophobic attraction and saline bridges. The presence of hydrophobic amino acids could also promote a closer attraction to the cell membrane.
  • aliphatic index the relative volume occupied by aliphatic side chains; half-life was theoretically estimated in mammalian reticulocytes in vitro according to the N-end rule.
  • ExPASy proteomics server was used to estimate pi, A. I. and half-life.
  • ACD/ChemSketch 2.0 software was used to calculate LogP.
  • P17 and P18 present a more hydrophobic character than P16, being closer to AdipoR-12C and predicting their ability to interact with both the target and cell membrane.
  • P17 is characterized by the best theoretical half-life, namely 20 hours, which highlights it as a promising pharmacological candidate for in vivo applications.
  • P16 presents homologies with proteins involved in membrane translocation of phospholipids, protein and lipid phosphatases involved in insulin signaling and secretion, or an enzyme playing a role in glycogen accumulation (Table 3).
  • the analysis of peptide sequence of P17 reveals homologies with proteins involved in insulin sensitivity, cell proliferation, the defense against cell stress and control of protein folding.
  • this one shows sequence homologies with several proteins playing a role in cell cycle, DNA repair, intracellular trafficking of proteins and signal transduction.
  • Table 4 Sequence alignment of peptide P17 with relevant human protein sequences as identified with BLAST of the NCBI proteomics server using the UniProtKB/Swiss-
  • AdipoRl is known to be mainly expressed in skeletal muscle, where it activates the signaling pathway of AMPK, while AdipoR2 is predominantly expressed in liver, where it activates the pathway of PPARa.
  • the AMPK activation by phosphorylation (AMPK-p) increases the fatty acid b-oxidation and glucose uptake via the membrane translocation of glucose transporter GLUT4 (in muscle) or GLUT2 (in liver) and inhibits gluconeogenesis (in liver).
  • PPARa plays major roles in lipid and glucose metabolism and exerts anti-inflammatory effects.
  • pancreatic beta cells In pancreatic beta cells, the level of AdipoRl and AdipoR2 expression is comparable to that in liver and greater than in muscle, but AdipoRl isoform is predominant at least in mouse islets. However, it seems that AMPK pathway is not activated in pancreatic beta cells, where Akt protein kinase and extracellular signal-regulated kinase (ERK) are involved in adpiponectin signaling to protect against apoptosis and stimulate insulin expression and secretion.
  • Akt protein kinase and extracellular signal-regulated kinase ERK
  • Adiponectin receptors were observed at the level of cell membranes, but also in the cytoplasm, mainly in the skeletal muscle. According to literature, AdipoRl and AdipoR2 can be detected at the level of cellular organelles when cells are permeabilized, which is the case of our tissue samples.
  • ER endoplasmic reticulum
  • ERp46 ER protein 46
  • AdipoRl adaptor protein containing pleckstrin homology domain (APPL1) and adenylate cyclase within a signaling complex requiring caveolin-3, and in clathrin-coated endosomes that regulate AdipoR recycling and degradation.
  • adiponectin receptors are predominantly located at plasma membrane, but also in the intracellular compartment.
  • AdipoRl is mainly expressed by fast-twitch type II fibers
  • AdipoR2 is expressed by both fiber types, although its distribution in slow- twitch type I fibers is more heterogenous.
  • P17 and P18 seem to bind both AdipoRl and AdipoR2 in type I and type II muscle fibers
  • P16 is more specific to AdipoRl in fast-twitch type II fibers.
  • P16 and P17 bind with high efficacy to AdipoR2 in liver and are able to concentrate in human pancreatic islets, where they could probably recognize both AdipoRl and AdipoR2.
  • AdipoRl (but not AdipoR2) expression is significantly enhanced after 48 hours of starvation, while a high-fat meal diminishes AdipoRl expression, AdipoR2 being less sensitive to dietary fat.
  • AdipoRl (but not AdipoR2) promoter activity is repressed (in C2C12 myoblasts) by insulin via the PI3K/Foxol pathway.
  • AdipoRl expression was decreased by hyperinsulinemia and hyperglycemia, whereas AdipoR2 expression was stimulated by hyperinsulinemia.
  • AdipoRl promoter harbors a responsive element recognized by the ER stress-inducible activating transcription factor 3 (ATF3) able to downregulate AdipoRl expression in C2C12 and HepG2 cells, which may be responsible of impaired AdipoRl signaling in obese and diabetic patients.
  • ATF3 ER stress-inducible activating transcription factor 3
  • AdipoR2 expression was significantly inhibited after 60 min of incubation with 25 mM glucose (p ⁇ 0.01 vs. FFAC at 60 min, and vs. glucose at 15 and 135 min).
  • AdipoR2 expression was amplified in HepaRG cells induced with both glucose and FFAC (p ⁇ 0.01 vs. glucose at 60 min, and vs. glucose & FFAC at 15 and 135 min).
  • AdipoRl presented higher levels of expression at all incubation times, but principally at 60 min, when the cells were incubated with both glucose and FFAC (p ⁇ 0.01 vs. all experimental conditions in C2C12 cells). Glucose also induced significant AdipoRl expression after 60 min of induction (p ⁇ 0.01 vs. glucose at 15 and 135 min).
  • AMPK liver kinase B1
  • CaMKK2 Ca 2+ /calmodulin-dependent protein kinase kinase 2
  • the same residue is also phosphorylated by CaMKK2, but only when intracellular Ca 2+ concentration is elevated following the APPLl-induced opening of Ca 2+ channels during AdipoRl activation by adiponectin.
  • the two pathways of AMPK activation can operate simultaneously when both AMP and Ca 2+ concentrations are increased intracellularly. Once activated, AMPK is involved in the fatty acid oxidation and glucose uptake by peripheral tissues.
  • the ability of our three candidate peptides to bind AdipoRs and activate AMPK phosphorylation was evaluated on HepaRG and differentiated C2C12 cell lines.
  • the cells were incubated with peptides or with the positive control compound Ago- AdipoR for 15, 60 and 135 minutes in two types of culture media. The first one was supplemented with 14 mM glucose (total glucose concentration in the culture medium was of 25 mM for HepaRG and 39 mM for C2C12) and was complemented or not with a mixture of free fatty acids and cholesterol (FFAC).
  • FFAC free fatty acids and cholesterol
  • the second one comprised basal glucose concentration of the culture media (11 mM for HepaRG; 25 mM for C2C12), which was supplemented or not with FFAC.
  • the control group was incubated in the same culture media as the test groups by excluding the peptides and AgoAdipoR.
  • the phosphorylated AMPK (AMPKa- pT172) was quantified by ELISA on protein extracts obtained from cell lysates.
  • P16 presented the weaker effect on AMPK activation and at short incubation times of HepaRG cells, namely at 15 minutes (Figure 8D) and 60 minutes ( Figure 8A). This effect was observed in culture media supplemented either with glucose ( Figure 8A) or with FFAC ( Figure 8D). In C2C12 cells induced with FFAC, P16 even induced a significant inhibition (p ⁇ 0.05) of AMPK phosphorylation after 15 minutes of incubation.
  • Peptide P17 induced a significant increase of AMPKa-pT172 concentration in almost all experimental conditions and particularly on HepaRG cells.
  • the higher effect has been observed when HepaRG cells were challenged with both 25 mM glucose and FFAC (Figure 8C).
  • AMPKa-pT172 concentration increased three times ( ⁇ 123 U/mg protein) as compared to HepaRG cells incubated with 25 mM glucose alone ( ⁇ 40 U/mg protein) (Figure 8A) after 60 and 135 minutes of induction with P17.
  • Peptide P18 manifested an optimal effect on AMPK activation in HepaRG cells incubated in culture media supplemented with glucose (Figure 8A), glucose and FFAC (Figure 8C) or FFAC ( Figure 8D), demonstrating its agonist activity on AdipoR2 expressing cells.
  • P18 triggered AMPK activation only when cells were challenged for 60 minutes with a supplement of glucose and FFAC ( Figure 8G).
  • the positive control compound, AgoAdipoR induced a significant increase of AMPKa-pT172 concentration when culture media were supplemented with both glucose and FFAC ( Figure 8C) or with FFAC alone ( Figure 8D).
  • C2C12 cells were stimulated by AgoAdipoR to activate AMPK by phosphorylation when culture media were supplemented with glucose ( Figure 8E) or with glucose and FFAC ( Figure 8G).
  • Activated AMPK regulates the lipid metabolism by different mechanisms, such as the activation of peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGCla, stimulates mitochondrial biogenesis), of carnitine palmitoyltransferase-1 (CPT-1, responsible of FFA b-oxidation) and of PPARa (stimulates the expression of genes involved in FFA b-oxidation), and the inhibition of 5-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase, involved in cholesterol production).
  • PDCla peroxisome proliferator-activated receptor-gamma coactivator 1 alpha
  • CPT-1 carnitine palmitoyltransferase-1
  • PPARa PPARa
  • HMG-CoA reductase 5-hydroxy-3-methylglutaryl-coenzyme A reductase
  • Glucose metabolism is also regulated by AMPK at different levels, such as the activation of glucose uptake via the membrane translocation of glucose transporter 4 (GLUT4) and 1 (GLUT1), the inhibition of glucose efflux via GLUT2, the activation of 6-phosphofructo-2-kinase (PFK2) to stimulate glycolysis, and the inhibition of gluconeogenesis via the downregulation of specific gene expression.
  • AMPK 6-phosphofructo-2-kinase
  • peptide P17 has been selected as a lead compound among the three candidate peptides. Its putative effects on GK and SDHA expression and eventual activation have been assessed on HepaRG and differentiated C2C12 cells incubated in the same culture conditions as for the study of AMPK modulation. GK and SDHA were simultaneously detected by immunofluorescence on the same cell samples, and the relative ratio of fluorescent labeling (RRFL) was semi- quantitatively evaluated using the ImageJ software ( Figure 9).
  • GK binds to its regulatory protein (GKRP) in hepatocytes and moves to the nucleus, where GK is sequestered and inactivated.
  • GKRP regulatory protein
  • High glucose concentration releases GK from GKRP and returns to cytoplasm to participate in glucose metabolism.
  • AMPK increases the FFA uptake (via the plasma membrane translocation of FFA transporter) and their b-oxidation (increases CPT-1 activity and FFA transport into the mitochondria), induces the expression of proteins involved in oxidative phosphorylation (i.e., PGCla) and the activities of mitochondrial enzymes (i.e., SDHA), and activates PFK2 by phosphorylation which produces F2,6P 2 , an inducer of GK expression and activity.
  • PGCla proteins involved in oxidative phosphorylation
  • SDHA mitochondrial enzymes
  • AdipoRl and AdipoR2 can self-associate to form homomers or heteromers at the plasma membrane.
  • FLAdipoQ full-length adiponectin
  • AdipoRs redistribute to early endosomes within the first 5 minutes after stimulation and disappear 30 minutes later.
  • the globular fragment of adiponectin (GAdipoQ) induces AdipoRs endocytosis after 30 minutes of stimulation. After endocytosis, AdipoRs can return to the membrane or be degraded within lysosomes to down-regulate adiponectin signaling.
  • AdipoRl and caveolin-1 associate at the plasma membrane of endothelial cells, this association being critical for adiponectin transmembrane signaling.
  • AdipoRl interaction with caveolin-1 is downregulated majorly because of the reduced caveolin-1 expression.
  • peptide P17 may modulate AdipoRl endocytosis via lysosomes (detected with anti-LAMP-1 antibody) or caveolae (detected with anti-caveolin-1 antibody) in differentiated C2C12 cells incubated for 60 minutes in a culture medium supplemented with glucose or glucose and FFAC.
  • AdipoRl, LAMP-1 and caveolin-1 were observed by immunofluorescence, and the level of immunostaining was semi-quantitatively measured with ImageJ software on the acquired microphotographs.
  • caveolin-1 expression was stimulated by glucose supplement in C2C12 cells and its colocalization with AdipoRl may assist the signaling cascade of this receptor, we could conclude that lysosomal degradation of AdipoRl may contribute to its downregulation.
  • adiponectin plays multiple roles in protecting against metabolic syndrome, type 2 diabetes (T2D) and cardiovascular disease (CVD).
  • Adiponectin regulates the food intake and body weight, the lipid and glucose metabolism, has an antiatherogenic activity, it improves the insulin sensitivity, and is inversely correlated with CVD risk factors such as blood pressure, low-density lipoproteins (LDL) and triglycerides.
  • Adiponectin expression and secretion are decreased in subjects with obesity, hypertension, T2D or presenting other characteristics that define the metabolic syndrome. Its expression and secretion seem to be regulated by TNFa, probably via an increased production of IL-6.
  • peptide P17 has been selected as a potential agonist candidate of adiponectin receptors that may be able to regulate the metabolic state of obese and diabetic subjects. Aiming to evaluate its agonist ability in pathologic conditions, P17 has been assessed on db/db mice, which develop obesity and hyperglycemia starting by 4-5 weeks of age, but also glycosuria, polyuria, polydipsia, polyphagia and insulin resistance. Therefore, this mouse strain is widely used as a model of T2D and obesity.
  • mice treated with P17 have the tendency to stabilize their weight as opposed to db/db mice treated with PBS.
  • the mice in 60 kcal group presented a weight gain of 8.04% after 4 days of P17 treatment, whereas those in 10 kcal % group gained 5.78% to their weight after 4 days of P17 treatment, being similar to the weight evolution of healthy NMRI that presented a weight gain of 6.19% at the end of the experimental period.
  • db/db mice treated with PBS have shown a weight gain of 13.11% in 60 kcal % group and of 11.23% in 10 kcal % group.
  • P17 has also induced a significant decrease of glycemia in 60 kcal group after 66h (p ⁇ 0.01; -37.9% vs. 24h of treatment) and 90h (p ⁇ 0.05; - 34.92% vs. 24h of treatment) of treatment (Figure 4A).
  • P17 induced a significant decrease of glycemia (p ⁇ 0.05; -24.84% vs. 24h of treatment) after 90h of treatment.
  • adiponectin reduces plasma triglycerides by increasing the expression and activity of lipoprotein lipase (LPL) in skeletal muscle, as well as the expression of VLDL receptor (VLDLr), enhancing in this way the VLDL-triglyceride catabolism.
  • LPL lipoprotein lipase
  • VLDLr VLDL receptor
  • FLAdipoQ and GAdipoQ interact with AdipoRl/R2 and stimulate the APMK and PPARa activation, which are involved in the FFA oxidation and glucose uptake by the targeted cells. Consequently, the Thrl72 residue of AMPK is phosphorylated by LKB1 and CaMKK2, which fully activate AMPK.
  • PPARa is a nuclear receptor that is indirectly activated by AMPK via the activation of p38 mitogen-activated protein kinase (p38 MAPK), which in turn phosphorylates several serine residues (S6, S12 and S21) of PPARa.
  • the tissues that obtain most of their energy from FFA oxidation are characterized by high levels of PPARa expression. It has been shown that serum levels of adiponectin are lower in subjects with T2D and obesity, while adiponectin administration decreases the plasma levels of FFA and triglycerides, improving thus the insulin sensitivity. Moreover, the mRNAs of AdipoRl/R2 were found to be significantly decreased in skeletal muscle and adipose tissue of ob/ob mice, whereas they were increased in the liver of insulin resistant obese patients, where the high AdipoRs expression was proposed to contribute as a compensatory mechanism for reduced plasma adiponectin.
  • db/db mice present a significantly high expression (p ⁇ 0.01) of AdipoRl in muscle ( Figures 15A and 15A) and of AdipoR2 in liver ( Figures 15B and 15B) when compared to healthy NMRI mice ( Figures 15F and 16A/B).
  • This high AdipoRl/R2 expression was independent of the diet composition (i.e., 60 kcal % or 10 kcal %), but it was relatively weaker in 10 kcal % fed mice and more variable in the liver of the same mice.
  • AdipoRl/R2 expression in the skeletal muscle and liver of db/db mice could be related to their known insulin resistance and non-alcoholic steatohepatitis (NASH), the same as in T2D and obese patients.
  • NASH non-alcoholic steatohepatitis
  • AdipoRl but not AdipoR2 promoter activity seems to be enhanced by insulin via the PI3K/Foxol pathway.
  • our in vivo study confirms the in vitro results, which have shown that AdipoRl and AdipoR2 expression is stimulated in HepaRG and C2C12 cells, when challenged with glucose and FFAC (Figure 7C).
  • AdipoRl was homogenously distributed in the skeletal muscle fibers independently of the experimental group, including the healthy NMRI mice.
  • AdipoR2 was homogenously distributed in the hepatocytes' cytoplasm and membrane of the db/db mice fed on a 60 kcal % diet, while AdipoR2 in mice fed on a 10 kcal % diet presented a predominant plasma membrane localization.
  • P17 administration induced a more restrictive localization of AdipoR2 at the level of plasma membrane, which was similar to that of healthy NMRI mice mainly in the case of db/db mice fed on a 60 kcal % diet.
  • AdipoRl/R2 The cytoplasmic distribution of AdipoRl/R2 could be related to their recycling and/or degradation in lysosomes after endocytosis, which is one of the mechanisms involved in the regulation of adiponectin signaling. It is thus possible that the high AdipoRl/R2 expression in db/db mice could be associated with an enhanced lysosome degradation and adiponectin signaling downregulation. As observed above on C2C12 cells, the glucose and FFAC supplement induced a striking increase of AdipoRl expression, but also of the lysosome content, which could confirm the in vivo studies.
  • AdipoRl and caveolin-1 associate at the plasma membrane, phenomenon that promotes AdipoRl endocytosis within caveolae and adiponectin transmembrane signaling. This interaction is downregulated by high glucose and lipids concentrations, mainly because of the reduced caveolin-1 expression.
  • AdipoR2 the sequence analysis revealed a potential caveolin-binding motif in its transmembrane domain, which may be responsible of adiponectin signalosome formation.
  • the cellular localization of AMPK-pT172 in skeletal muscle and liver presented a similar distribution with that of AdipoRl/R2, meaning that it was predominantly cytoplasmic in skeletal muscle and concentrated to the cell membrane in the case of liver, including in healthy NRMI mice.
  • the predominant cytoplasmic localization of AMPK-pT172 in the liver of db/db mice fed on a 60 kcal % diet was similar to that of AdipoR2 in the same tissue.
  • the catalytic a subunit of AMPK comprises an activating phosphorylation site (T172) and occurs in two isoforms, al and a2.
  • the AMPKa2 presents a nuclear and non-nuclear localization, while AMPKal is only found in non-nuclear fractions.
  • Our results confirm thus the non nuclear localization of AMPKa l, which is detected by the antibody employed in our study.
  • the kinase LKB1 responsible of AMPK phosphorylation, is translocated from the nucleus to the cytosol when adiponectin binds to its receptors. This binding stimulates the interaction of the intracellular region of AdipoRl/R2 with the adaptor protein APPL1, which in turn immobilizes LKB1 that becomes able in this way to phosphorylate AMPK.
  • AMPK is recruited in the proximity of cell membrane, where the complex APPL1/LKB1 is located.
  • the second kinase responsible of AMPK phosphorylation, the CaMKK2 is indirectly activated by APPL1, which activates phospholipase C (PLC) that induces the release of Ca 2+ from the endoplasmic reticulum subsequent to the production of inositol 1,4,5- trisphosphate (IP3).
  • PLC phospholipase C
  • IP3 inositol 1,4,5- trisphosphate
  • AMPK phosphorylates several downstream targets, the global effect being to inhibit the ATP consuming pathways (i.e., protein and fatty acid synthesis), whereas the ATP producing pathways (i.e., glycolysis and FFA oxidation) are upregulated.
  • AMPK was significantly activated in the skeletal muscle and liver of db/db mice independently of the diet composition, although it was higher in mice fed on a 10 kcal % diet ( Figures 16C and 16D). Concomitantly, this activation was more variable in the liver of the same mice.
  • the lower level of AMPK activation in mice fed on a 60 kcal % diet could be associated to insulin resistance, as explained above for AdipoRl/R2.
  • the adiponectin resistance could also contribute to lower AMPK activation. It is known that obese subjects present a reduced AMPK activation that is not related to a reduced expression of AdipoR, which tends to be higher.
  • a 60% saturated fat diet can induce adiponectin resistance, as demonstrated by the failure of GAdipoQ to inactivate acetyl coenzyme A carboxylase (ACC) and stimulate FFA oxidation.
  • Adiponectin resistance could thus explain the high plasma triglyceride concentration observed in db/db mice ( Figure 14B), which is certainly the consequence of a reduced FFA oxidation.
  • peptide P17 induced a significant decrease of AMPK activation (p ⁇ 0.05) in skeletal muscle and liver of db/db mice fed on a 60 kcal % diet as compared to the control group.
  • AMPK activation was not significantly different in db/db mice fed on a 10 kcal % diet when compared to the control group, the general tendency was to reduce AMPK phosphorylation at a level closer to the healthy NMRI mice, which could be a sign of metabolic improvement.
  • AMPK-pT172 in liver was restricted to the cell membrane of hepatocytes in db/db mice treated with P17, which could be related to its activation by LKB1.
  • the significant decrease of plasma triglyceride concentration in both groups of mice treated with P17 ( Figure 14B) could confirm the AMPK activation and FFA oxidation.
  • these results seem to confirm the striking AMPK activation produced by P17 in HepaRG cells incubated with a supplement of FFAC and glucose, effect that could be explained by the allosteric modulation of AMPK in the presence of FFA, which potentiates its phosphorylation by LKB1.
  • PPARa activation in liver followed the same evolution as that of AMPK phosphorylation among the experimental groups, meaning that db/db mice presented a higher level of PPARa phosphorylation independently of the diet composition ( Figures 15E, 15F and 16E), although those fed on a 10 kcal % diet were characterized by the highest level of PPARa activation, at least from the viewpoint of its phosphorylation.
  • PPARa-pS12 was distributed both in the nuclei and cytoplasm of hepatocytes. This distribution was mostly observed in the case of db/db mice fed on a 10 kcal % diet, but also in those fed on a 60 kcal % diet.
  • PPARa is a transcription factor that regulates the expression of genes involved in FFA oxidation, ketogenesis, lipid transport and gluconeogenesis. Its dysregulation has been involved in the etiology and pathogenesis of diabetes, obesity, hyperlipidemia, atherosclerosis, cancer, inflammation etc. In three obese mouse models (i.e., ob/ob, db/db, 5-HT2cR), the PPARa mRNA expression was increased by 2- to 3-fold as compared to healthy lean mice. It has also been demonstrated that PPARa and PPARy are dynamically shuttled between nucleus and cytoplasm, although they are present constitutively and predominantly in the nucleus.
  • mice with peptide P17 induced a significant decrease of PPARa activation (and probably expression) in db/db mice fed on a 60 kcal % diet (p ⁇ 0.01 vs. control db/db mice), its level being identical to that in healthy NMRI mice ( Figure 16E).
  • the level of PPARa-pS12 was also decreased by the treatment with P17, although the results were more variable and did not attain significance.
  • PPARa-pS12 was predominantly located in the hepatocytes' nuclei in the liver of mice treated with P17, which was comparable to its subcellular localization in the liver of healthy NMRI mice.
  • db/db mice present high levels of AdipoRl/R2 expression in skeletal muscle and liver, which might be related to their known insulin resistance and NASH, phenomena that are characteristic to T2D and obese subjects.
  • the subcellular localization of AdipoRl/R2 was homogenous in the cytoplasm and membrane of skeletal muscle fibers and liver, the cytoplasmic distribution being probably related to their lysosome degradation.
  • the treatment of db/db mice with P17 presented the tendency to restore the level of AdipoRl/R2 expression in the range of healthy NMRI mice.
  • AdipoR2 was more restricted at the level of plasma membrane of hepatocytes, where it could have been associated to caveolin and contribute to signalosome formation.
  • the AMPK-pT172 was also present in higher quantities in the skeletal muscle and liver of db/db mice as compared to healthy NMRI mice. However, AMPK activation was lower in mice fed on a 60 kcal % diet, possibly in conjunction with adiponectin resistance, characteristic to obese subjects.
  • the administration of P17 induced the return of AMPK-pT172 level in the range of healthy NMRI mice, mainly in the case of db/db mice fed on a 60 kcal % diet, probably in relationship with allosteric modulation of AMPK by FFA.
  • P17 induced the restriction of AMPK at the level of hepatocyte membrane, where it could be phosphorylated by LKB1.
  • PPARa in liver was also highly activated by phosphorylation (PPARa-pS12) independently of the diet composition, but it was located both in the nuclei and cytoplasm of db/db mice.
  • the cytoplasmic distribution could be related to its downregulation by proteolysis at the level of 26S proteasome and explain the low level of lipid oxidation as shown by the high plasma triglyceride concentration in these mice.
  • the P17 administration presented the tendency to return the level of PPARa activation in the range of healthy NMRI mice and restricted its subcellular distribution to the nucleus, where it could regulate the expression of genes involved in lipid metabolism. This regulation is furthermore confirmed by the diminution of plasma triglyceride concentration at the level of healthy NMRI mice.
  • Apoptosis is an important feature of T2D and metabolic syndrome, dealing with various cell types such as pancreatic beta and alpha cells, but also the hepatocytes.
  • the death of beta cells by apoptosis is characteristic to both T1D and T2D, where this is triggered by the signaling pathways of interleukin (IL)- 1b, nuclear factor (NF)-KB and Fas.
  • IL interleukin
  • NF nuclear factor
  • Alpha cell area is moreover reduced in obese mice fed a high fat diet, in conjunction with alpha cell hypotrophy, increased apoptosis and decreased proliferation.
  • the non-alcoholic fatty liver disease is a common complication of obesity and T2D.
  • the NAFLD can occur in two clinical presentations, the non-alcoholic fatty liver (NAFL), characterized by hepatic inflammation, and NASH, which is characterized by steatosis and hepatocyte apoptosis, and is related to insulin resistance.
  • NASH non-alcoholic fatty liver
  • the proliferative and anti-apoptotic activities of adiponectin were already demonstrated in various tissues, such as the heart, liver and pancreas.
  • Akt and ERK pathways are furthermore involved in the stimulation of insulin gene expression and secretion by pancreatic beta cells.
  • Adiponectin also exerts hepato-protective actions by inactivating ACC, which reduces lipid synthesis and enhances the FFA oxidation, and downregulates the expression of sterol regulatory element-binding protein lc (SREB-Plc), a transcription factor involved in lipid synthesis.
  • SREB-Plc sterol regulatory element-binding protein lc
  • the activation of PPARa is moreover responsible for the FFA oxidation. All these pathways contribute to an enhanced fat oxidation, reduced lipogenesis and prevention of hepatic steatosis.
  • peptide P17 could prevent cell apoptosis in pancreatic islets and liver of db/db mice if it binds correctly to AdipoRl/R2 and triggers the adiponectin pathway. Apoptotic cells were thus detected in these tissues by the immunofluorescent staining of activated caspase- 3, while NASH was observed in liver by IHC after its Masson's Trichrome staining.
  • control db/db mice presented a significantly increased (p ⁇ 0.05, p ⁇ 0.01 vs. heathy NMRI mice) staining of activated caspase-3 (Figure 17), independently of the diet composition.
  • the staining level was in the range of positive control represented by apoptotic livers from Balb/c mice injected with anti-Fas antibody and confirmed that T2D developed by db/db mice is indeed characterized by liver apoptosis probably induced by NASH as described by literature.
  • Pancreatic islets did not show any apoptotic events associated with beta cells in either of the experimental groups, as confirmed by the absence of colocalization between activated caspase-3 immunoreactive cells and cells stained for insulin (Figure 19). Conversely, alpha cells were all immunoreactive for activated caspase- 3 ( Figure 20), which perfectly co-localized with glucagon in all experimental groups including healthy NMRI mice.
  • Figure 20 we have also identified a perfect colocalization between activated caspase-3 and alpha cells in the pancreas of healthy C57BL6/J mice (data not shown), which confirms that alpha cell apoptosis is not a feature characteristic to healthy NMRI mice.
  • the literature published so far is mainly focused on beta cell apoptosis in diabetes subjects, the studies being largely performed in vitro on isolated pancreatic islets or beta cells. Consequently, the reported data cannot be used to compare or interpret our results.
  • the increased percentage of alpha cells may belong to a compensatory mechanism of adaptation, which could originate from the necessity to equilibrate glycemia under a high insulin secretion by the enlarged beta cell mass, at least at the beginning of pathological process.
  • the death of alpha cells by apoptosis may contribute to a recycling mechanism, meant to renew these cells under the challenge of metabolic stress.
  • db/db mice with peptide P17 restored the ratio of beta cells over alpha cells in the range of healthy NMRI mice, being significantly different (p ⁇ 0.05) from PBS treated db/db mice.
  • the beta cell number per pancreatic islet has also significantly increased (p ⁇ 0.01, p ⁇ 0.05) in comparison with healthy NMRI mice, probably as a mechanism intended to compensate for the increased metabolic demand, triggered by the activation of adiponectin pathway.
  • adiponectin triggers PI3K/Akt and ERK pathways, which are responsible of cell survival and proliferation.
  • liver steatosis is associated with apoptotic hepatocyte death in db/db mice, characteristic to NASH as described by literature, although leucocyte infiltration and liver fibrosis could not be observed in our animal model.
  • Treatment with P17 has significantly reduced both steatohepatitis and apoptosis, possibly in relationship with its ability to restore the physiological activity and cellular localization of AMPK and PPARa, and to enhance lipid oxidation as demonstrated by the reduced plasma triglyceride concentration.
  • pancreatic islets In the case of pancreatic islets, no apoptotic beta cells could be observed in db/db mice, confirming the published literature, which failed to evidence a significant increase of apoptotic cells in pancreatic islets of 5- to 24-week old db/db mice. However, the apoptotic death of alpha cells was identified in the present study both in db/db and healthy mice, phenomenon that deserves to be considered and investigated in future studies of diabetes research. On the other hand, the total number of beta cells per islet was larger in control db/db mice as compared to heathy NMRI mice, the same as the percentage of alpha cells per islet.
  • beta cell hyperplasia characteristic to at least early stages of T2D and obese subjects, is accompanied by a proportional increment of alpha cell number, which could represent a compensatory mechanism contributing to glycemia homeostasis upon insulin hypersecretion.
  • Alpha cells furthermore present the highest replication rate under basal conditions or mitogen stimulation in comparison to beta, delta and PP cells, which could be associated with a shorter life-span and contribute to the islet cell plasticity.
  • the ratio of beta cells over alpha cells was restored by peptide P17 to the level characteristic to healthy mice, while beta cell mass was augmented, phenomenon that could be explained by the activation of adiponectin pathways involved in metabolism, but also in cell proliferation and survival.
  • Adipokines are among the most important molecular actors in the pathophysiology of obesity-linked disorders by their ability to regulate inflammatory and metabolic processes.
  • Adiponectin is an adipokine secreted by the adipose tissue, which decreases in diabetic and obese patients. Its replenishment has an anti- diabetic effect by improving insulin sensitivity and cell survival, including that of beta cells.
  • Adiponectin has been furthermore reported to produce pleiotropic beneficial effects in various pathologies, such as obesity, MS, CVD, fatty liver disease and liver fibrosis.
  • Adiponectin has two main receptors, AdipoRl and AdipoR2, which have seven transmembrane domains that are structurally and functionally distinct from G protein-coupled receptors.
  • AdipoRl activates AMPK pathways that regulate the inhibition of gluconeogenesis, increase fatty acid oxidation and glucose uptake.
  • AdipoR2 activates PPARa pathways, which stimulate energy dissipation by increasing fatty acid oxidation and inhibit oxidative stress and inflammation. These molecular pathways contribute to an increased insulin sensitivity and to a reduced risk of incident T2D in apparently healthy individuals.
  • a peptide agonist of AdipoRl/AdipoR2 was developed that modulates adiponectin signaling pathways and regulate glucose and lipid metabolism, showing a relevance for T2D and obesity associated pathologies.
  • the selected peptides bind to a 12-mer sequence comprised in the C-terminal domain of AdipoRl/AdipoR2, which is homologous for both receptors, in humans and mice.
  • the biochemical properties of amino acid residues are identical to AdipoRl-12C and similar to AdipoR2-12C and seem to reveal an ability to bind AdipoR in the close proximity of cell membrane due to the equilibrated proportion of hydrophilic (58.4%) and hydrophobic (41.7%) residues.
  • AdipoRl/R2 The binding of the peptides to AdipoRl/R2 was confirmed by immunofluorescent colocalization on tissue sections of mouse skeletal muscle and liver and on human pancreas, which suggest that the latter could recognize both receptors.
  • AdipoRl In skeletal muscle, AdipoRl is principally expressed by fast-twitch type II fibers, while AdipoR2 is expressed by both fast and slow fibers although its distribution in slow-twitch type I fibers is more heterogenous.
  • the peptides were identified in both fiber types, while their binding in pancreas was restricted to pancreatic islets, with no interaction with exocrine pancreas. In the liver, the peptides have been noticed at the level of cell membranes.
  • AdipoRl/R2 expression and signaling we have observed that high glucose concentration stimulates AdipoRl expression in C2C12 cells, which is associated with AMPK activation by phosphorylation.
  • FFAC seem to represent an inducer of AdipoR2 expression and of AMPK activation.
  • PPARa is a transcription factor that regulates lipid metabolism and glucose uptake and is activated by AdipoR2.
  • the enhanced SDHA expression observed in these culture conditions could be related to PPARa activation.
  • P17 (seq ID n° 5) emerged as the most potent activator of AMPK phosphorylation in HepaRG and C2C12 cells.
  • AMPK phosphorylation the cells induced with P17 presented a significant activation of GK and SDHA expression that could be related to its agonist AdipoRl/R2 activity.
  • Activated AMPK increases FFA uptake and oxidation and stimulates GK expression and activity.
  • the AdipoRl/R2 agonist activity of P17 was subsequently studied in vivo on the db/db mouse model of T2D fed ad libitum on a Western high-fat diet (60 kcal; mice aged of 6 weeks) or on a standard chow diet (10 kcal; mice aged of 7 weeks).
  • Mus musculus is a strongly nocturnal species [69]
  • AdipoRl/R2 expression found in skeletal muscle and liver of db/db mice confirmed the published literature in the case of human T2D and obese subjects.
  • AdipoRl/R2 were generally homogenously distributed in the cytoplasm and plasma membrane of skeletal muscle fibers and hepatocytes, the cytoplasmic localization being probably the consequence of their downregulation via lysosome degradation.
  • the high AdipoRl/R2 expression could thus represent a homeostatic mechanism meant to compensate for lysosome degradation.
  • AMPK and PPARa were significantly activated in skeletal muscle and liver of db/db mice, the high plasma triglyceride concentration suggests that AdipoRl/R2 intracellular signaling was inactivated by different molecular mechanisms, i.e. adiponectin resistance, AMPK and PPARa turnover by ubiquitination and proteolysis. This hypothesis is furthermore sustained by the liver steatosis and apoptosis observed in these mice.
  • AdipoRl/R2 expression presented the tendency to restore the level observed in healthy NMRI mice.
  • AdipoR2 in liver was more restricted to the plasma membrane in a similar manner as in healthy NMRI mice, which could be explained by caveolin binding and signalosome formation and contribute to the striking decrease of plasma triglycerides.
  • Future studies should confirm this hypothesis by AdipoR2 colocalization with caveolin-1, which is known to play major roles in hepatic lipid and glucose metabolism [71].
  • the AMPK-pT172 and PPARa-pS12 were also reduced by the P17 treatment, presenting the tendency to return to the level and cellular distribution characteristic to healthy NMRI mice.
  • AMPK-pT172 was furthermore restricted to the cell membrane, while PPARa-pS12 was concentrated in nuclei, restoring the subcellular distribution observed in healthy NMRI mice.
  • the membrane location of AMPK-pT172 could be related to its activation by LKB1, whereas nuclear shuttling of PPARa-pS12 is associated to the expression of genes involved in lipid metabolism.
  • the regulation of adiponectin signaling pathway is moreover demonstrated by the significantly reduced steatohepatitis and liver apoptosis in db/db mice treated with P17. In the pancreas, P17 restored the ratio of beta cells over alpha cells in the range of healthy NMRI mice and increased the beta cell mass, probably via the activation of adiponectin pathways responsible of cell proliferation and survival.
  • SEQ ID n°2 Ala His Ala His Thr Asn Trp Thr Ser Trp Trp Trp Glu SEQ ID n°3 : Asp Leu Val Ser Trp Ala Gly Ser Gly Lys Lys His SEQ ID n°4: Ala Asp Trp Tyr His Trp Arg Ser His Ser Ser Ser SEQ ID n°5: lie Pro Asn Tyr Ser Met Gin Ser Arg Glu Tyr Arg SEQ ID n°6: His Tyr Arg Pro Phe Thr Gin Glu His Arg Val Thr SEQ ID n°7 : His Ser Phe Lys Gly Trp Asp Trp Pro Arg Leu Arg SEQ ID n°8 : Gly Trp Lys Ser His Glu Pro Lys Gly His Gly Ser SEQ ID n°9 : His Ser Phe Lys Trp Leu Asp Ser Pro Arg Leu Arg SEQ ID n° 10: Gly Ala Tyr Thr Ser Trp Arg Thr Ser Thr Asn Ala SEQ ID

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Abstract

La présente invention concerne un peptide pouvant se lier aux récepteurs de l'adiponectine 1 ou 3. L'invention est caractérisée en ce que ledit peptide présente au moins 95 % d'identité de séquence avec une séquence d'acides aminés choisie parmi SEQ ID 2 à 3 ou 5 à 12, ainsi que l'utilisation de tels peptides dans des compositions.
PCT/EP2019/087006 2018-12-24 2019-12-24 Agonistes peptidiques du récepteur de l'adiponectine 1 et 2 Ceased WO2020136193A1 (fr)

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EP19824371.9A EP3902555A1 (fr) 2018-12-24 2019-12-24 Agonistes peptidiques du récepteur de l'adiponectine 1 et 2

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EP18215864.2 2018-12-24
EP18215864 2018-12-24

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WO2020136193A1 true WO2020136193A1 (fr) 2020-07-02

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009070350A1 (fr) * 2007-11-30 2009-06-04 Siemens Healthcare Diagnostics Inc. Fragments de récepteur de l'adiponectine et leurs procédés d'utilisation
WO2012142142A2 (fr) 2011-04-12 2012-10-18 Temple University - Of The Commonwealth System Higher Education Agonistes des récepteurs de l'adiponectine et procédés d'utilisation
WO2016150416A1 (fr) * 2015-03-20 2016-09-29 Forschungszentrum Jülich GmbH Peptides se liant spécifiquement aux bêta-amyloïdes et leur utilisation dans le traitement et le diagnostic de la démence d'alzheimer
CN107290539A (zh) * 2017-06-07 2017-10-24 哈尔滨医科大学 用于检测自身免疫病患者血清标志物的多肽组合物及其应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009070350A1 (fr) * 2007-11-30 2009-06-04 Siemens Healthcare Diagnostics Inc. Fragments de récepteur de l'adiponectine et leurs procédés d'utilisation
WO2012142142A2 (fr) 2011-04-12 2012-10-18 Temple University - Of The Commonwealth System Higher Education Agonistes des récepteurs de l'adiponectine et procédés d'utilisation
WO2016150416A1 (fr) * 2015-03-20 2016-09-29 Forschungszentrum Jülich GmbH Peptides se liant spécifiquement aux bêta-amyloïdes et leur utilisation dans le traitement et le diagnostic de la démence d'alzheimer
CN107290539A (zh) * 2017-06-07 2017-10-24 哈尔滨医科大学 用于检测自身免疫病患者血清标志物的多肽组合物及其应用

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
LASZLO OTVOS JR ET AL: "Design and development of a peptide-based adiponectin receptor agonist for cancer treatment", BMC BIOTECHNOLOGY, BIOMED CENTRAL LTD. LONDON, GB, vol. 11, no. 1, 5 October 2011 (2011-10-05), pages 1 - 14, XP021110409, ISSN: 1472-6750, DOI: 10.1186/1472-6750-11-90 *
MIKI OKADA-IWABU ET AL: "A small-molecule AdipoR agonist for type 2 diabetes and short life in obesity", NATURE, vol. 503, no. 7477, 30 October 2013 (2013-10-30), London, pages 493 - 499, XP055343097, ISSN: 0028-0836, DOI: 10.1038/nature12656 *
MIKI OKADA-IWABU ET AL: "Perspective of Small-Molecule AdipoR Agonist for Type 2 Diabetes and Short Life in Obesity", DIABETES & METABOLISM JOURNAL, vol. 39, no. 5, 1 January 2015 (2015-01-01), pages 363, XP055587590, ISSN: 2233-6079, DOI: 10.4093/dmj.2015.39.5.363 *
SUNGHWAN KIM ET AL: "Discovery of a novel potent peptide agonist to adiponectin receptor 1", PLOS ONE, vol. 13, no. 6, 18 June 2018 (2018-06-18), pages e0199256, XP055587574, DOI: 10.1371/journal.pone.0199256 *

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