WO2016119856A1 - Autophagy-inducing molecules for increasing insulin release - Google Patents

Abstract

The present invention relates to the use of autophagy-inducing molecules for increasing release or secretion of insulin by pancreatic β cells. It is useful for treating hyperglycemia, associated or not with diabetes.

Description

AUTOPHAGY-INDUCING MOLECULES FOR INCREASING INSULIN

RELEASE

FIELD OF THE INVENTION

The present invention relates to the field of medicine, in particular to the treatment of hyperglycemia and diabetes or any conditions necessitating an increase in insulin secretion.

BACKGROUND OF THE INVENTION

The epidemic of obesity and type 2 diabetes poses a challenge to our health care system and an enormous burden of morbidity and early mortality that is predicted to affect up to 20% of Americans by 2030. In analyses carried out for the World Health Report 2002, approximately 60% of diabetes and 20% of ischemic heart disease globally are attributable to obesity. Central obesity involves deposition of fat around the trunk and in visceral adipose tissue. Both obesity and abdominal obesity increased in men, and abdominal obesity increased in women between 1999 and 2008 and carries a higher risk of cardiovascular disease, dyslipidemia and type 2 diabetes. In obese individuals, the increase in adiposity results from an increase in the number and size of adipocytes, while the degree of hypertrophy relative to hyperplasia influences the level of body fat and the metabolic consequences of obesity.

Hyperglycemia is another metabolic disorder that poses a challenge to the health care system. The liver is the primary organ for glucose and lipid metabolism. During fasting, blood glucose is maintained by the breakdown of hepatic glycogen followed by gluconeogenesis. In the postprandial state, the insulin action in the liver turns off both glycogenolysis and gluconeogenesis and activates glycogenesis and lipogenesis to store the excess glucose. Tight insulin-mediated regulation of hepatic glucose production is essential to maintain normoglycemia, and dysregulation caused by insulin resistance is the most prevalent metabolic abnormality in the developed world.

For instance, among cardiac patients and critically ill populations, hyperglycemia is associated with increased mortality. Following cardiac surgery, wound infections and other morbidities are increased among patients having diabetes or uncontrolled hyperglycemia. Strict glycemic control during the perioperative time period can thus reduce morbidities and mortality.

Current treatments consist in insulin administration for type 1 diabetes and/or glucose-lowering medications or insulin sensitizers for type 2 diabetes. Insulin is a hormone involved in glucose homeostasis. Insulin is produced by pancreatic beta-cells (β-cells) located in the islets of Langerhans, and they secrete it in response to rising levels of blood glucose. Glucose is taken up from the blood by hepatocytes, muscle cells, and adipocytes used either as energy source or for storage as glycogen and triglycerides. Insulin also inhibits lipolysis, preventing fatty acid release from the fat tissue. On the contrary, low blood glucose levels result both in a reduced production and release of insulin. Together with glucagon action, it results in glucose release into blood stream. In pathological situations, either insulin production by β-cells is not sufficient (type 1 diabetes) and/or cells poorly respond to it (insulin resistance; type 2 diabetes), leading to persistent high levels of blood glucose. However, precise mechanisms involved in these pathologies are not yet completely understood.

Conclusively, there is a constant need of new drugs for controlling hyperglycemia.

SUMMARY OF THE INVENTION

The inventors surprisingly established that insulin secretion by pancreatic β-cells can be increased by using autophagy- inducing molecules on pancreatic β-cells. Indeed, they showed that an autophagy-inducing molecule is able to dramatically increase the insulin release independently of glucose concentrations.

Accordingly, the present invention relates to an autophagy-inducing molecule for use to increase insulin release or secretion. It also relates to the use of an autophagy-inducing molecule for the manufacture of a medicament for increasing insulin release or secretion. It further relates to a method for increasing insulin release or secretion in a subject in need thereof, comprising administering a therapeutically efficient amount of an autophagy-inducing molecule, thereby increasing insulin release or secretion.

Preferably, the increase of insulin release or secretion is aiming to treat hyperglycemia. More particularly, hyperglycemia is caused by or associated to a pre-diabetic condition, diabetes, preferably type 2 diabetes, hyperglycemia associated to medications, to acute stress such as stroke or myocardial infarction, to dysfunction of the thyroid, adrenal and pituitary glands, to pancreas disease such as pancreatitis and pancreas cancer, or to intracranial diseases.

In a preferred embodiment, the molecule inducing autophagy is a peptide derived from Beclin- 1 protein. Preferably, the peptide comprises Beclin 1 residues 269-283 (VFNATFHr HSGQF, SEQ ID No 1), more preferably comprises the sequence TNVFNATFH VHSGQFGT, SEQ ID No 2), wherein up to six of said residues may be substituted. More preferably, the peptide comprises or consists on a peptide comprising the amino acid sequence V-F-N-A-T-F-E/H-I- W-H-D/S (SEQ ID No 3), T-N-V-F-N-A-T-F-E/H-I-W-H-D/S-G-Q/E-F-G-T (SEQ ID No 4) or the D-retro-inverso sequence thereof, wherein up to 6 of said residues may be substituted. Still more preferably, the peptide is selected in the group consisting of YGRKKRRQRRRGGTNVFNATFHIWHSGQFGT (SEQ ID No 12); YGRKKRRQRRRGGTNVFNATFEIWHDGEFGT (SEQ ID No 13); and the D-retro-inverso amino acid sequence RRRQRRKKRGYGGTGFEGDHWIEFTANFVNT (SEQ ID No 14). In another embodiment, the molecule inducing autophagy is an antibody or a fragment or derivative thereof, an aptamer, a Spiegelmer or a chemical compound.

Optionally, the molecule may comprise a cell penetrating moiety or a moiety targeting the pancreatic β cell. Preferably, the moiety targeting the pancreatic β cell is selected from the group consisting of glucagon- like peptide- 1 (GLP-1), glucagon- like peptide-2 (GLP-2), peptide YY (PYY), neuropeptide Y (NPY), pancreatic peptide (PPY), exendin-4, naphthylalanine, and naphthylalanine derivatives, , or is a moiety that that selectively binds a protein selected from the group consisting of DiGeorge syndrome critical region gene 2 (DGCR2), golgi brefeldin A resistant guanine nucleotide exchange factor 1 (GBFl), orphan G protein-coupled receptor GPR44 (GPR44), SerpinBlO (PI- 10), FXYD domain containing ion transport regulator 2 (FXYD2), Tetraspanin-7 (TSPAN7), gap junction protein, delta 2, 36kDa (GJD2), solute carrier family 18 (vesicular monoamine), member 2 (SLC18A2), prokineticin receptor 1 (PROKR1), glutamate receptor, metabotropic 5 (GRM5), neuropeptide Y receptor Y2 (NPY2R), glucagon- like peptide 1 receptor (GLP1R), and transmembrane protein 27 (TMEM27).

Optionally, the moiety targeting the pancreatic β cell is an antibody or a fragment thereof, an aptamer, or a ligand. Alternatively, the moiety targeting the pancreatic β cell is a combination of several moieties targeting the pancreatic β cell which have different binding partners.

Optionally, the molecule inducing autophagy is used in combination with an additional active drug, preferably an anti-diabetic drug, a hypo-glycemic drug or an anti-hyperglycemic drug. Another aspect of the disclosure relates to a product containing a molecule inducing autophagy according to the invention and an additional active drug, preferably an anti-diabetic drug, an hypo-glycemic drug or an anti-hyperglycemic drug, as a combined preparation for simultaneous, separate or sequential use, in particular for treating, delaying or preventing hyperglycemia. More specifically, this product is for use in the treatment of a pre-diabetic condition, diabetes, preferably type 2 diabetes, hyperglycemia associated to medications, to acute stress such as stroke or myocardial infarction, to dysfunction of the thyroid, adrenal and pituitary glands, to pancreas disease such as pancreatitis and pancreas cancer, or to intracranial diseases. BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1. Nutrient depletion suppresses autophagy in β cells. (FIG 1A) Immunofluorescence (IF) of LC3B-GFP puncta (white arrows) in INSl cells under growing conditions (GC), without amino acids and fetal calf serum (no AA/FCS) or glucose (no Glc/FCS) for 2 and 6 hours. Quantification of LC3B-GFP puncta per cell (mean + s. e. m.). **P<0.01. (FIG IB) LC3B- GFP puncta in iNSl^{LC3B GFPendo} cells under GC and no AA/FCS for 1 hour in absence and presence of Bafilomycin Al (BafAl), 10 nM. (FIG 1C) CLEM of ptfLC3 expressing INSl cells under GC and no AA/FCS for 2 hours. EM, corresponding fluorescent and merged pictures are shown. Regions of interest (ROI) are indicated with labelled dashed squares. Yellow and black arrows indicate autophagosomes and autolysosomes, respectively. (FIG ID) Western blot of LC3B and p62 using soluble and insoluble fractions of lysates of INSl cells under GC and no AA/FCS for 1.5 hours, non-treated or treated with BafAl, 1 nM for last 1 hour of incubation. GAPDH served as a loading control. (FIG IE) IF of LC3B-GFP puncta (white arrows) and insulin (red) in β cells in islets of fed and fasted LC3B-GFP expressing mice. The nucleus was stained with DAPI. Quantification of LC3B-GFP puncta per 1600 μm²(mean + s. e. m.). **P<0.01.

Fig. 2. Nutrient depletion induces SINGD in β cells. (FIG 2A) Left: Immunofluorescence (IF) of Phogrin and Lampl in INSl cells under growing conditions (GC) or without amino acids and fetal calf serum (no AA/FCS) for 2 hours. Regions of interest (ROI) are indicated with dashed squares. White arrows indicate co-localization of Phogrin with Lampl. A Golgi marker (pGolgi-CFP) was used. Quantification of co-localization of Phogrin and Lampl per cell (mean + s. e. m.). **P<0.01. Right: Increased lysosomal degradation of secretory granules in INSl cells upon starvation. (Upper panel) Quantification of co-localized Lampl- and Phogrin- positive puncta in INSl cells under growing conditions (GC) and without amino acids and fetal calf serum (no AA/FCS) for 2 hours in presence and absence of lysosomal inhibitors (LI) (mean + s.e.m.). *P<0.05, **P<0.01. (Lower panel) Quantification of areas of Lampl/Phogrin- positive puncta in INSl cells under GC and no AA/FCS for 2 hours in presence and absence of lysosomal inhibitors (LI) (mean + s.e.m.). Values are normalized to areas in INSl cells under GC without LI. *P<0.05, ns: not significant. (FIG 2B) CLEM of INSl cells co-expressing Phogrin-GFP and Lampl-RFP under GC and no AA/FCS for 2 hours. EM, corresponding fluorescent and merged pictures are shown. Regions of interest (ROI) are indicated with dashed squares. White arrows indicate co-localization of Phogrin-GFP with Lampl-RFP. Yellow arrows indicate lysosomes correlated to co-localized Phogrin-GFP and Lampl-RFP. (FIG 2C) Western blot of Lamp2, Phogrin and LC3B using lysates of indicated fractions from INSl cells under GC and no AA/FCS for 30 minutes. Right: EM of GCLs in shifted fractions (red dashed boxes). (FIG 2D) Western blot of proinsulin using lysates of INSl cells treated or not with lysosomal inhibitors (LI) under GC and no AA/FCS for 6 hours. GAPDH was used as a loading control. (FIG 2E) Left: EM of Golgi areas in primary murine islets under GC and no AA/FCS for 2 hours. Yellow arrows indicate granule-containing lysosomes (GCLs). Right: Quantification of GCLs (N per cell view) in Golgi areas of β cells in primary murine islets under GC and no AA/FCS for 2 hours (mean + s.e.m.). **P<0.01. (FIG 2F) Left: IF of proinsulin/insulin ((Pro)insulin) and Lamp2 (left panel) and (Pro)insulin and LC3B-GFP in β cells in islets of fed and fasted LC3B-GFP expressing mice. The nuclei were stained with DAPL; Right: Enhanced GCL formation in β cells in fasted as compared to fed mice. (Upper panel) Quantification of co-localizing Lamp2 and proinsulin/insulin ((Pro)insulin) puncta in β cells in islets isolated from fed and fasted LC3B-GFP expressing mice (mean + s.e.m.). **P<0.01. (Lower panel) Quantification of co-localizing LC3B-GFP and (Pro)insulin) puncta in β cells in islets isolated from fed and fasted LC3B-GFP expressing mice (mean + s.e.m.). ns: not significant.

Fig. 3. (FIG 3A) Granule containing lysosomes (GCLs) increase in INSl cells upon starvation. Electron microscopy of Golgi areas in INSl cells under growing culture (GC) conditions and without AA and fetal calf serum (no AA/FCS) for 2 hours. Regions of interest (ROI) highlighted with white dashed squares. Yellow asterisks indicate GCLs. Quantification of GCLs (mean + s.e.m.). **P<0.01. (FIG 3B) Identification of granule containing lysosomes (GCLs) in starved LNSl cells by immunogold-labeling. Immunogold-labeling of AA/FCS-deprived LNSl cells for proinsulin/insulin ((Pro)insulin, 12 nm gold, yellow arrows)(left panel). Double immunogold- labeling for (Pro)insulin (12 nm gold, yellow arrows) and Lampl (6nm gold, black arrows) (right panel).

Fig. 4. Co-localization of LC3B-GFP and Phogrin did not increase in INSl cells upon starvation. LC3B-GFP and immunofluorescence of Phogrin in INSl cells endogenously expressing LC3B-GFP (INS lLC3B-GFPendo cells) under growing conditions (GC) and without amino acids and fetal calf serum (no AA/FCS) for 1 hour. DAPI was used to stain nuclei. Merged, LC3B-GFP and Phogrin signals are shown separately. Quantification of Phogrin/LC3B-GFP puncta per cell view (mean + s.e.m.). ns: not significant.

Fig. 5. SINGD is induced upon starvation independent of autophagy. (FIG 5A) Western blotting of Beclinl or ATG5 using lysates of INSl cells transfected with non-silencing (NS) siRNA, siRNA against Beclinl (siBeclinl) or siRNA against ATG5 (siATG5). GAPDH was used as a loading control. (FIG 5B) Immunofluorescence of Lampl and Phogrin in INSl cells in media without amino acids and fetal calf serum for 2 hours. Cells have been either transfected with NS siRNA, siATG5 or siBeclinl (lower panel) or treated or not with the autophagy inhibitor 3-methyladenine (3-MA). DAPI was used to stain nuclei. White arrows point at yellow puncta indicating co-localization of Phogrin with Lampl. Quantifications of Phogrin/Lampl puncta per cell view (mean + s.e.m.). ns: not significant.

Fig. 6. SINGD suppresses autophagy in an mTOR-dependent manner. (FIG 6A) IF of LC3B- GFP puncta (white arrows) in INSl cells under growing conditions (GC) or without amino acids and fetal calf serum (no AA/FCS) for 2 hours treated or not with rapamycin, 100 nM or torin-1, 250 nM as indicated. Quantification of LC3B-GFP puncta per cell (mean + s. e. m.). **P<0.01. (FIG 6B) Immunofluorescence (IF) of mTOR in INSl cells co-expressing Phogrin- GFP and Lampl-RFP under GC and no AA/FCS for 2 hours. White arrows in regions of interest (ROI) highlighted with dashed squares indicate co-localization of Phogrin-GFP with mTOR and Phogrin-GFP with Lampl-RFP. A Golgi marker (Giantin) was used. (FIG 6C) Western blot of indicated proteins using lysates of INSl cells treated or not with rapamycin, 100 nM under GC, no AA/FCS or without glucose/FCS (no Glc/FCS) for 2 hours. GAPDH was used as a loading control. (FIG 6D) Upper panel: IF of phosphorylated ULK1 (S757-ULK1) in INSl cells co-expressing Phogrin-GFP and Lampl-RFP under GC and no AA/FCS for 2 hours. ROI are indicated with dashed squares. White arrows in ROI indicate co-localization of Phogrin- GFP with Lampl-RFP and S757-ULK1. A Golgi marker (Giantin) was used. Lower panel: Starvation led to formation of large bright S757-ULK1 puncta in INSl cells. Quantification of the areas of the largest phospho-S757 ULK1 puncta in INSl cells under growing conditions (GC) or without amino acids and fetal calf serum (no AA/FCS) normalized to areas of INSl cells under GC conditions (mean + s.e.m.). ***P<0.001. (FIG 6E) Insulin in supernatants of INSl cells treated with non-stimulatory 2.8 mM Glc in presence of DMSO or rapamycin. Insulin concentrations are expressed as a percentage of DMSO control (mean + s. e. m.).(FIG 6F) Insulin in supernatants of human islets treated as indicated. Insulin concentrations are expressed as a percentage of insulin upon 16.7 mM stimulatory Glc (mean + s. e. m.). ***P<0.05.

Fig. 7. Induction of autophagy by tat-beclinl in murine primary islets triggers insulin release. (FIG 7A) Electron microscopy of β cells in isolated murine islets treated either with scrambled (scr.) tat-beclinl or tat-beclinl. The yellow asterisk indicates an autophagosome. Quantification of autophagic compartments (AC) per 100 μιη2 cytoplasm area (mean + s.e.m.). ***P<0.001. (FIG 7B) Viability of islets treated as indicated expressed as a percentage of viability of islets in 2.8 mM glucose (Glc) (mean + s.e.m.). ns: not significant. (FIG 7C) Insulin of supernatants of islets treated as indicated. Insulin concentrations are expressed as a percentage of insulin upon 16.7 mM stimulatory Glc. (FIG 7D) Insulin in supernatants of islets treated as indicated normalized to insulin concentrations in supernatants of islets treated with DMSO (mean + s.e.m.). ***P<0.001.

Fig. 8. Induction of autophagy by tat-beclinl in human primary islets triggers insulin release. (FIG 8A) Electron microscopy of β cells in isolated murine islets treated either with scrambled (scr.) tat-beclinl or tat-beclinl. The yellow asterisk indicates an autophagosome. Quantification of autophagic compartments (AC) per 100 μητ2 cytoplasm area (mean + s.e.m.). *P<0.05. (FIG 8B) Viability of islets treated as indicated expressed as a percentage of viability of islets in 2.8 mM glucose (Glc) (mean + s.e.m.). ns: not significant.

Fig. 9. PKD 1 controls SINGD. (FIG 9A) Left: Western blot of proinsulin using lysates of INS 1 cells treated with CID755673 for indicated times. GAPDH was used as a loading control. Right: PKDl knockdown in INSl cells does not alter proinsulin biosynthesis. Autoradiography of immunoprecipitated proinsulin using lysates of INSl cells transfected with short hairpin RNA against PKDl (shPKDl) compared to cells transfected with non-silencing shRNA (NS) and incubated with indicated concentrations of glucose in presence of [35S] -methionine. Autoradiography of total input was used as a loading control. (FIG 9B) Left: EM of Golgi areas in non-silenced and PKDl-depleted (shPKDl) INSl cells. Yellow arrows indicate secretory granules-containing lysosomes (GCLs). Right: Increased granule containing lysosomes (GCLs) in INSl cells depleted of PKDl. Quantification of GCLs in INSl cells transfected with short hairpin RNA against PKDl (shPKDl) compared to cells transfected with non- silencing shRNA (NS) (mean + s.e.m.). ***P<0.001. (FIG 9C) Immunofluorescence (IF) of mTOR and Lamp2 in non-silenced and PKDl-depleted (shPKDl) INSl cells. White arrows indicate co- localization of mTOR with Lamp2. The nucleus was stained with DAPI. (FIG 9D) Western blot of indicated proteins using lysates of non-silenced and PKDl-depleted (shPKDl) INS l cells. GAPDH was used as a loading control. (FIG 9E) LC3B-GFP puncta in iNSl^{LC3B GFPendo} cells treated with CID755673 or DMSO for 4 hours in presence and absence of BafAl, 10 nM.(FIG 9F) Live-cell FRET assay in INSl cells expressing G-PKDrep-live under growing culture (GC) conditions, without amino acids and fetal calf serum (no AA/FCS) or without glucose/FCS (no Glc/FCS). FRET was measured and expressed as normalized YFP-C/CFP ratios during a time course as indicated. (FIG 9G) Left: Upper panel : EM of Golgi areas of fasted β cells in primary islets of ρ38δ^+,+ and ρ38δ^~'^~ mice. Yellow arrows indicate secretory GCLs. Lower panel : EM of cytoplasm of fasted β cells in primary islets of ρ38δ^+,+ and ρ38δ^~'^~ mice. The yellow arrow indicates an autophagosome. Right: Granule containing lysosomes (GCLs) decrease and autophagic compartments (AC) increase in β cells in fasted primary murine islets of ρ38δ-/- mice. Quantification of GCLs (Upper panel) in Golgi areas and of ACs (Lower panel) in cytoplasm areas per 100 μηΐ2 θί β cells in islets of mice of indicated genotypes (mean + s.e.m.). **P<0.01. (FIG 9H) Model highlighting links between SINGD, secretion and autophagy. SG : secretory granule, PM : plasma membrane.

Fig. 10. PKD1 knockdown in INSl cells decreases accumulation of newly formed insulin. Autoradiography of immunoprecipitated proinsulin/insulin using lysates of INSl cells transfected with short hairpin RNA against PKD1 (shPKDl) compared to cells transfected with non-silencing shRNA (NS) and incubated for 30 minutes with [35S] -methionine (pulse) followed by chase for indicated times. Autoradiography of total input was used as a loading control.

Fig. 11. Identification of granule containing lysosomes (GCLs) in PKDl-depleted INSl cells by immunogold-labeling. Immunogold-labeling of INSl cells expressing a short hairpin RNA (shRNA) against PKD1 (shPKDl) for proinsulin/insulin ((Pro)insulin, 12 nm gold, yellow arrows)(left panel). Double immunogold-labeling for (Pro)insulin (12 nm gold, yellow arrows) and Lampl (6nm gold, black arrows)(right panel).

Fig. 12. Identification of GCLs in lysosomal fractions. Western blotting of Lamp2, Phogrin and LC3B using lysates of indicated density gradient fractions from top to bottom from INS 1 cells expressing non-silencing (NS) short hairpin RNA (shRNA) or PKD1 -silencing shRNA (shPKDl) as indicated. Electron microscopy of GCLs in shifted fractions 7 to 9 (red dashed boxes) (right panel).

Fig. 13. Inhibition of PKD markedly increased Phogrin and Lampl co-localization in INSl cells. From left to right: Immunofluorescence of LAMP1 and Phogrin of INS 1 cells treated with DMSO or the PKD inhibitor CID755673 for 4 hours in presence and absence of lysosomal inhibitors (LI). DAPI was used to stain nuclei. Quantification of co-localized Lampl- and Phogrin-positive puncta in INSl cells treated with DMSO or CID755673 in presence and absence of LI (mean + s.e.m.). **P<0.01. Quantification of areas of Lampl/Phogrin-positive puncta in INSl cells treated with DMSO or CID755673 in presence and absence of LI (mean + s.e.m.). Values are normalized to areas in INSl cells treated with DMSO without LI. *P<0.05, ns: not significant.

Fig. 14. Co-localization of Phogrin with LC3B-GFP remained unchanged in PKD-inhibited INSl cells. LC3B-GFP puncta in INSl cells endogenously expressing LC3B-GFP (INS1^{LC3B~ GFPendo}cells) either exposed to DMSO or the PKD inhibitor CID755673 for 4 hours. DAPI was used to stain nuclei. Merged, LC3B-GFP and Phogrin signals are shown separately. Fig. 15. Reduced autophagic flux in INSl cells depleted for PKD1. Western blotting of LC3B using lysates of non-silenced (NS) and PKDl-depleted (shPKDl) INS l cells treated with BafAl at indicated concentrations for indicated times. GAPDH was used as a loading control. Fig. 16. SINGD was compromised in β cells of fasted ρ38δ-/- mice. Immunofluorescence of proinsulin or insulin (Pro)insulin and Lamp2 in β cells of islets from mice with indicated genotypes. Merged, (Pro)insulin and Lamp2 signals are shown separately. DAPI was used to stain nuclei. Quantification of (Pro)insulin/Lamp2 co-localizing puncta per cell view (mean + s.e.m.). *P<0.05. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an autophagy-inducing molecule for increasing insulin release or secretion, in particular by pancreatic β cells. Indeed, the inventors surprisingly observed that a molecule inducing pancreatic β cell autophagy directly increases the insulin release/secretion, even at high blood glucose concentration.

Therapeutic indications

The present invention relates to an autophagy-inducing molecule for increasing insulin release or secretion, in particular by pancreatic β cells.

Conditions needed an increase of insulin are mainly hyperglycemia. Hyperglycemia is a condition in which an excessive amount of glucose circulates in the blood plasma. Generally, it is a glucose level higher than 11.1 mmol/1. A subject with a consistent range between 5.6 a,d 7 mmol/1 is considered as hyperglycemic, even if symptoms may not start to become noticeable until 15-20 mmol/1. Chronic levels exceeding 7 mmol/1 can produce organ damage such as kidney damage, neurological damage, cardiovascular damage, retinal damage and feet and leg damage. Acute hyperglycemia involving glucose levels that are extremely high is a medical emergency. Ketoacidosis, a life-threatening condition, may occur with untreated hyperglycemia.

In this context, the molecule inducing autophagy is useful for controlling blood glucose levels in hyperglycemic conditions. Hyperglycemia can be due to different conditions. It can be caused by a pre-diabetic condition or diabetes. Indeed, hyperglycemia is caused by low insulin levels in the context of Type I diabetes or by resistance at the cellular level for type II diabetes. In a particular aspect, any stage of Type II diabetes can be of interest, i.e. stage 1, stage 2, stage 3, stage 4 and stage 5. Preferably, treatment of prediabetic and late diabetic stage is contemplated. In a particular aspect, the molecule is suitable for treating or delaying the progression or onset of diabetes mellitus, insulin resistance, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, insulin resistance, and obesity. Indeed, decreasing hyperglycemia by increasing insulin release or secretion could treat or delay the progression or onset of these diseases.

Hyperglycemia can be due to certain medications including corticosteroids, octreotide, beta blockers, epinephrine, thiazide diuretics, niacin, pentamidine, protease inhibitors, L- asparaginase and some antipsychotic agents.

An acute stress such as stroke, or myocardial infarction may lead to hyperglycemia, even in the absence of diabetes. Other conditions can cause hyperglycemia, in particular in the absence of diabetes. They include dysfunction of the thyroid, adrenal and pituitary glands, especially hyperthyroidism and Cushing's syndrome; numerous diseases of the pancreas such as pancreatitis and a pancreatic cancer; sepsis and certain infections; intracranial diseases such as encephalitis, brain tumors (especially those located near the pituitary gland), brain bleeds and meningitis; convulsions and terminal stages of many diseases.

As a standard, an initial oral drug monotherapy with insulin-sensitizing metformin is used to treat hyperglycemia in type 2 diabetes. In many cases this monotherapy is not sufficient in long- term. Therefore, this treatment is followed by two- or three-drug combinations. In two-drug combinations, metformin is either combined with sulfonylureas or meglitinides targeting ATP- dependent potassium channels of the β cell. The latter drugs can provoke hypoglycemia and cause a weight gain. In long term, they can lead to β cell failure. Alternatively, insulin- sensitizing thiazolidinediones (TDZs) are suggested to be used in combination with metformin. They are, however, very expensive, also provoke a weight gain, and most importantly come along with severe side effects, such as cardiac failure, skeletal fractures and peripheral edema. For pioglitazone, bladder cancer has been reported. Therefore, in many countries, TDZs have been withdrawn from the market. More recently, very promising drugs targeting the incretin axis, such as glucagon-like peptide 1 receptor agonists (GLP-1RA) and dipeptidyl peptidase-4 (DPP-IV) inhibitors are in clinical use. These drugs seem to have good effects in controlling glycemia, lead to body weight reduction and do not trigger hypoglycemia. Moreover, they evoke favorable effects on cardiovascular risk factors and biomarkers. However, these agents seem to induce unfavorable gastrointestinal side effects such as nausea. It is controversially discussed whether GLP-1 -based therapeutics will cause inflammation of the pancreas (pancreatitis) or even pancreatic cancer. Strikingly, postmortem histology revealed that the use of GLP-1RA or DPP-IV inhibitors was associated with pancreatic exocrine cell dysplasia accompanied by hyperplasia of glucagon-secreting a-cells. These drugs entail thus quite effective glucose-lowering effects, but their long-term effects on beta cell function, safety and beta cell mass expansion remains to be investigated.

As a matter of fact, insulin supplementation regimens in combination or not with the above drugs will become the therapy of choice if β cell failure is progressing. Given above caveats and obvious uncertainties on long-term effects, there is a clear demand on new drugs acting through distinct mechanisms that offer an overall broader therapeutic flexibility in type 2 diabetes. Increasing autophagy in pancreatic β cells as described herein is yet another way to increase insulin secretion in type 2 diabetes that should be explored in the future. Importantly, as for incretins, autophagy has also been reported to protect against β cell failure in rodents. As the long-term effects of incretin-related therapies in humans may not be sufficient, targeting autophagy might represent an additional potential strategy.

By "treat" or "treatment" is intended that the disease or condition is cured, alleviated or delayed. It includes the preventive or curative treatment. The term treatment designates in particular the correction, retardation, or reduction of an impaired glucose homeostasis. The term "treatment" also designates an improvement in the insulin secretion or release. Within the context of the invention, the terms "controlling the blood glucose level" or "the control of blood glucose level" refer to the normalization or the regulation of the blood or plasma glucose level in a mammalian subject having abnormal levels (i.e., levels that are below or above a known reference, median, or average value for a corresponding mammalian subject with a normal glucose homeostasis). The present invention relates to the pharmaceutical or veterinary use of the molecule. Accordingly, the subject may be any mammal, preferably a human subject, such as an adult or a child. In a particular embodiment, the subject may be a subject suffering of obesity. Optionally, the subject has no detectable anti-islet antibodies, and ultrasonography revealed no pancreatic abnormalities. In the context of a veterinary application, the subject can be an animal, preferably a mammal, in particular a pet animal such as a dog, a cat or a horse.

Autophagy -inducing molecules

Several molecules inducing or promoting autophagy are available to the person skilled in the art. By "induce", or "promote" autophagy is intended to refer to the occurrence of autophagy in a cell population increased by at least 10, 20, 30, 40, 50, 60, 70, 80 or 90 % when compared to the autophagy measured in absence of the molecule in the same conditions. The method suitable for measuring autophagy is detailed below and in the example section. In a preferred aspect, the capacity to induce or promote autophagy is determined by the method disclosed in Orvedahl et al (2011, Nature, 480, 113-117, the disclosure of which being incorporated herein by reference).

In a preferred embodiment, the autophagy-inducing molecule is a peptide derived from Beclin- 1 protein.

In a first embodiment, the peptide derived from Beclin- 1 protein is a variant of human Beclin- 1 in which the threonine at position 119 is substituted with a phospho-mimicking residue, preferably a glutamic acid or an aspartic acid as disclosed in WO 10/064250 (the disclosure of which being incorporated herein by reference). Alternatively, the peptide derived from Beclin- 1 protein is a variant of human Beclin- 1 in which Ser234, Ser295, Tyr229, Tyr233 or Try352 is substituted with a phospho-silencing residue, preferably an alanine as disclosed in WO14/046966 (the disclosure of which being incorporated herein by reference). Preferably, the molecule is a polynucleotide encoding such a variant of Beclin- 1.

In a preferred embodiment, the autophagy-inducing molecule is a peptide comprising Beclin 1 fragment as disclosed in WO2013/119377 and US8, 802,633 (the disclosure of which being incorporated herein by reference).

In a first embodiment, the autophagy-inducing peptide comprises Beclin 1 residues 269-283 (VFNATFHIWHSGQF, SEQ ID No 1), more preferably comprises the sequence TNVFNATFHIWHSGQFGT, SEQ ID No 2), wherein up to six of said residues may be substituted. Preferably, the peptide comprises at least one of F270, F274 and W277. Optionally, the peptide comprises at least one, two or three of substitutions: H275E, S279D and Q281E. In a preferred embodiment, the autophagy-inducing peptide comprises or consists on a peptide comprising the amino acid sequence V-F-N-A-T-F-E/H-I-W-H-D/S (SEQ ID No 3), T-N-V-F- N-A-T-F-E/H-I-W-H-D/S-G-Q/E-F-G-T (SEQ ID No 4) or the D-retro-inverso sequence thereof, wherein up to 6 of said residues may be substituted. Optionally, there is no substitution in the sequence according any of SEQ ID Nos 1-4. Preferably, the F residues are not substituted. More preferably, the peptide comprises the amino acid sequence VFNATFEr HD (SEQ ID No 5) or TNVFNATFEIWHDGEFGT (SEQ ID No 6) or the D-retro-inverso sequence thereof. In a most preferred embodiment, the peptide comprises the D-retro-inverso amino acid sequence TGFEGDHWIEFTANFVNT (SEQ ID No 7).

Optionally, the peptide can be linked to additional moiety, optionally through a linker or spacer (e.g., diglycine). The additional moiety can be a moiety facilitating its cellular uptake or entry, in particular a PTD (protein transduction domain); a homing peptide; a stabilizing agent such as PEG (polyethyleneglycol), oligo-N-methoxy-ethylglycine (NMEG), albumin, an albumin- binding protein or an immunoglobulin Fc domain; an affinity tag such as an immune-tag, biotin, lectin, or chelator; a detectable label such as an optical tag, a chelated lanthamide, a fluorescent dye, or a FRET acceptor/donor; a targeting moiety or a combination thereof. Optionally, the peptide can be part of a protein fusion. PTD generally comprises a certain amino acid sequence of 10 to 20 amino acids (Matsushita and Matsui, (2005), J Mol Med 83, 324-328; Vives et al, Biochimic et Biophysica Acta, 2008, 1786, 126-138). PTD is mainly composed of basic amino acids such as arginine or lysine, and representative examples of the PTD include arginine rich peptides such as poly R₈ (RRRRRRRR, SEQ ID No 8) or (RRPRRPRRPRRPRRP, SEQ ID No 9), antennapedia or penetratin peptide such as (RQIKIWFQNRRMKWKK, SEQ ID No 10) or HIV-Tat (YGRKKRRQRRR, SEQ ID No 11). In particular, Tat-Beclin 1 peptides have been disclosed as powerful inducer of autophagy (Shoji-Kawata et al, 2013, Nature, 494, 201-206; US 8,802,633). Examples of such Tat-Beclin 1 peptides are the followings: the wild-type Tat- Beclin 1 peptide of sequence YGRKKRRQRRRGGTNVFNATFITiWHSGQFGT (SEQ ID No 12); the mutated Tat-Beclin 1 peptide of sequence

YGRKKRRQRRRGGTNVFNATFEr HDGEFGT (SEQ ID No 13); and the D-retro-inverso amino acid sequence RRRQRRKKRGYGGTGFEGDHWIEFTANFVNT (SEQ ID No 14). The diglycine linker in these peptides could be replaced by another amino acid linkers. Optionally, up to 6, 5, 4, 3, 2 or 1 residue substitutions can be introduced. Optionally, one or both F residues can be substituted or linked by other moieties as disclosed in US 8,802,633 (the disclosure of which being incorporated herein by reference), for instance crosslinkable moieties, by homocysteines for preparing cyclic peptides.

The peptide can comprise one or more D-amino acids, L-P-homo amino acids, D-P-homo amino acids, or N-methylated amino acids. The peptide can be cyclized. It can be acetylated, acylated, formylated, amidated, phosphorylated, sulfated or glycosylated. In particular, it can comprise an N-terminal acetyl, formyl, myristoyl, palmitoyl, carboxyl or 2-furosyl group, and/or a C- terminal hydroxyl, amide, ester or thioester group. Modifications in some or all peptide bounds are also contemplated in order to increase the proteolysis resistance, in particular by (-CO-NH- ) by (-CH2-NH-), (-NH-CO-), (-CH₂-0-), (-CH2-S-), (-CH2-CH2-), (-CO-CH2-), (-CHOH-CH2- ), (-N=N-), and/or (-CH=CH-). Optionally, the peptide can comprise the D-retro-inverso amino acid sequence of one peptide described above.

Preferably, the peptide may have between 5 and 50 amino acids. More preferably, it has between 5 and 20 amino acids.

Other molecules inducing or promoting autophagy are available for use according to the present disclosure. For instance, the following molecules can be cited: Telmisartan (2-(4-{ [4-methyl-6-(l-methyl-lH-l,3-benzodiazol-2-yl)-2-propyl-lH- 1,3-benzodiazol-l-yl] methyl} phenyl) benzoic acid;

with R being selected from the group consisting of dihydroxyacetone, glucose, galactose, glyceraldehyde, threose, xylose, mannose, ribose, ribulose, tagatose, psicose, fructose, sorbose, rhamnose, erythrose, erthrulose, arabinose, lyxose, allose, altrose, gulose, idose, talose, sucrose, lactose, maltose, lactulose, trehalose, cellobose, isomaltotriose, nigerotriose, maltotriose, melezitose, maltotriulose, raffinose, kestose and a combination thereof (US 2014296175);

A Ganoderma lucidum extract as disclosed in WO14/081439 (the disclosure of which being incorporated herein by reference);

as defined in WO14/055644 (the disclosure of which being incorporated herein by reference);

A human Beclin 1 protein wherein Ser234, Ser295, Tyr229, Tyr233 or Try352 is substituted with a phospho-silencing residue as disclosed in WO14/046966, preferably by an alanine (the disclosure of which being incorporated herein by reference);

Urolithin, preferably selected from the group consisting of urolithin A, urolithin B, urolithin C, urolithin D, and any combination thereof as disclosed in WO14/004902 (the disclosure of which being incorporated herein by reference);

carbomezepine, tamoxifen, minoxidil, erapumil, or clonidine ;

an agent that suppresses GST-pi as disclosed in US2014315975 (the disclosure of which being incorporated herein by reference);

wherein Ri R₂, R₃, R4, R5, R6, Rn, Ri₂ and Ri₃ are each independently selected from hydrogen, hydroxyl, halogen, Cl-6 alkyl and Cl-6 haloalkyl; R₇ and Rs are each independently selected from methoxyl and hydroxyl; R9 and Rio are each independently selected from hydrogen, hydroxyl, halogen, Cl-6 alkyl as disclosed in WO12126390 (the disclosure of which being incorporated herein by reference);

a ceramide such as N-acylsphingosine or as disclosed in WO09076598 (the disclosure of which being incorporated herein by reference); an mTOR inhibitor such as rapamycin (sirolimus), rapamycin derivatives, CI-779, everolimus (Certican™), ABT-578, tacrolimus (FK 506), ABT- 578, AP-23675, BEZ-235, OSI-027, QLT-0447, ABI-009, BC-210, salirasib, TAFA-93, deforolimus (AP-23573), AP-23841, and temsirolimus (Torisel™); temozolomide; arsenic trioxide; an Akt inhibitor such as perifosine or an Akt inhibitor disclosed in WO12/087336; a lithium salt such as lithium chloride, lithium carbonate, lithium citrate, lithium sulfate, lithium aspartate, lithium orotate; a BH3 mimetic; loperamide; amiodarone; niguldipine; pimozide; nicardipine; penitrem A; fluspirilene; trifluoperazine; or any combination thereof;

spermidine, or promethazine ;

a compound as disclosed in WOl 1/143444 (the disclosure of which being incorporated herein by reference),

a salt of a l-methyl-2- [3-(l-methyl-l,2-dihydroquinolin-2-yliden)prop-l-enyl]quinolinium cation;

a compound as disclosed in US8039502, US8445483 and US8741944 (the disclosure of which being incorporated herein by reference);

a compound as disclosed in WO08/113100 (the disclosure of which being incorporated herein by reference);

a compound as disclosed in WO08/099175 (the disclosure of which being incorporated herein by reference);

a calpain inhibitor such as calpastatin, ALLM, calpeptin, leupeptin, a-dicarbonyls, quinolinecarboxamides, sulfonium methyl ketones, diazomethyl ketones, Leu-Abu- CONHEt (AK275), 27-mer calpastatin peptide, Cbz-Val-Phe-H (MDL28170), calpeptin (Z-Leu-Nle-H), a- mercaptoacrylic acids, phosphorus derivatives, epoxysuccinates, acyloxymethyl ketones, halomethylketones and E64 as disclosed in WO07/003941 (the disclosure of which being incorporated herein by reference);

X = H, CN, CI. OMe. or NMe₂ e, or NMe₂

! X = 0, CH₂, or NH II X = 0, CH₂, or H

Y = NH₂, H, or OH Y = NH_2l H. or OH

III O, CH₂, or NH

VI X = O, CH₂, or NH VII X = O, CH₂, or N H

Y = NH₂, H . or OH Y = NH₂, H, or OH a compound is selected from dodecahydro-l,4,7,9b-tetraazaphenalene, l-(4-chlorophenyl)- 3-(4-nitrophenyl)-2,3-di(piperidin-l-yl)propan-l-one, (3E)-3-[(5-chloro-2- hydroxyphenyl)hydrazinyleidene]-5-hydroxy-4-oxonapthalene-2,7-disulfonic acid or 3- (l,3,3a,4,5,6,7,7a-octahydroisoindol-2-yl)-N,N-dimethylpropan-l-amine, SM-hexamethyl-1 SJ 1-tetrazacyclotetradecane, NSC21689 or combinations thereof as disclosed in WOl 1/019636 (the disclosure of which being incorporated herein by reference).

Finally, new molecules inducing or promoting autophagy, especially in β cells, can be identified by a screening method available to the person skilled in the art.

These molecules might be selected from the group consisting of peptides, polypeptides, peptide mimetics, antibodies, fragments or derivatives thereof, aptamers, Spiegelmers, and chemical compounds.

In an alternative embodiment, the molecule is an antibody, a fragment thereof or a derivative thereof. As used herein, the terms "antibody" and "immunoglobulin" have the same meaning and are used indifferently in the present invention. The term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that immune-specifically binds an antigen. Antibodies include any kind of antibodies, preferably monoclonal. They can be for instance IgG (immunoglobulin G) or VHH (heavy chain variable domain antibody from camelids). Antibodies fragments or derivatives thereof include Fab, Fab', F(ab')2, scFv, (scFv)2, dAb, complementarity determining region (CDR) fragments, linear antibodies, single- chain antibody molecules, minibodies, nanobodies, diabodies, and multispecific antibodies formed from antibody fragments.

For aptamers and Spiegelmers, similar methods can be used in order to select aptamers and Spiegelmers. These methods are well-known by the person skilled in the art. As used here, the term "aptamer" means a molecule of nucleic acid or a peptide. It refers to a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., Science, 1990, 249(4968):505-10. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., Clin. Chem., 1999, 45(9): 1628-50. Peptide aptamers consist of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., Nature, 1996,380, 548-50).

Spiegelmers have been disclosed for instance in WO 98/08856. They are molecules similar to aptamers. However, spiegelmers consist either completely or mostly of L-nucleotides rather than D-nucleotides in contrast to aptamers. Otherwise, particularly with regard to possible lengths of spiegelmers, the same applies to spiegelmers as outlined in connection with aptamers. Chemical compounds refers to a molecule of less than about 1500 Daltons, 1000 Daltons, 800 Daltons, or even less than about 500 Daltons, in particular organic or inorganic compounds. Structural design in chemistry should help to find such a molecule. The molecule may have been identified by a screening method disclosed in the present invention.

Synthetic compound libraries are commercially available from a number of companies including Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, N. J.), Brandon Associates (Merrimack, N.H.), and Microsource (New Milford, Conn.). Combinatorial libraries are available or can be prepared according to known synthetic techniques. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available from e.g., Pan Laboratories (Bothell, Wash.) and MycoSearch (NC), or are readily producible by methods well known in the art. Additionally, natural and synthetically produced libraries and compounds can be further modified through conventional chemical and biochemical techniques.

A screening method refers to an in vitro and in vivo method allowing to measure or assess the effect of a candidate molecule on autophagy, and especially autophagy on β cells. For example, the in vitro method can comprise the use of insulin secreting cell lines, comprising HIT, RIN, HIT, beta TC, MIN6, INS-1 and INS-2 cells lines. Alternatively, the in vivo method can comprise the use of animal model as Obese mouse, Diabetic mouse, Sand mouse [Psammomys obesus], Spiny mouse [Acomys cahirinus], BB rats, KK mouse, Yellow mouse, Yellow KK mouse, New Zealand obese mouse, Tuco-tuco [clenomys talarum], Chinese hamster [Cricetulus griseus], NOD mouse, Japanese wistar rat [Goto rat] and C57BL/6 mice. Optionally, the in vivo method may comprise the use of chemical agents capable of inducing diabetes, as Alloxan, Steptozocin, Diphenyl thiocarbazine, Oxine-9- hydroxyquinolone, Vacor, 6- aminonicotinamide, 1-asparginase, Azide, Cyanide, Cyproheptadine, Phenyloin, Thiazides, Malonates, Anti insulin antibodies Somatostatins, Catecholamines, Glucocorticoids or Glucagon.

In a preferred embodiment, the screening method can comprise the measure of RNA expression level or protein level expression and/or localization of beclin-1, ATG1 (also known as ULK1), lysosome-associated membrane proteins 2 (LAMP2) and cathepsin B, D, F, H, Atg8, Atg7 and micro tubule-associated proteinl light chain 3 (LC3). Methods like quantitative RT-PCR or by Western blotting or immunocytochemistry can be used to determine the accumulation of autophagic vacuoles and autophagosomes in cells. In other embodiment, the screening method comprise the next steps (1) treating insulin secreting cell lines with a test substance; (2) measuring the amount of the accumulation of autophagic vacuoles and autophagosomes ; (3) optionally, measuring the amount of the insulin release or secretion ; (4) selecting the test substance if it increases the amount of the accumulation of autophagic vacuoles and autophagosomes and/or, optionally if it increases the insulin release.

In a preferred aspect, the capacity to induce or promote autophagy is determined by the method disclosed in Orvedahl et al (2011, Nature, 480, 113-117, the disclosure of which being incorporated herein by reference). ρβ cells targeting.

Autophagy-inducing molecules can be linked, covalently or not, to a moiety targeting the pancreatic tissue and more specifically β cells. Several targeting molecules have already been disclosed in the art.

In some embodiments, the pancreatic β cells targeting moiety can comprise an antibody, receptor, receptor ligand and the like, which preferentially and/or specifically bind to pancreatic β cells. One or more targeting moieties can be attached to the autophagy inducing molecule to provide a composition that is capable targeting the composition to pancreatic β cells. In various embodiments, targeting moieties include, but are not limited to peptides that preferentially bind pancreatic β cells, antibodies that bind pancreatic β cells, and receptor ligands that bind pancreatic β cells. For example, aptamers are good affinity tools for cell surface proteins. Alternatives that can be coupled to autophagy-inducing moieties include antibody fragments (scFvs) or Nanobodies (VHHs).

In one embodiment, the pancreatic β cells specific targeting moiety comprises one or more peptides or other moieties that preferentially bind pancreatic β cells, selected from the group consisting of glucagon- like peptide- 1 (GLP-1), glucagon- like peptide-2 (GLP-2), peptide YY (PYY), neuropeptide Y (NPY), pancreatic peptide (PPY), exendin-4, naphthylalanine, naphthylalanine derivatives, and a combination thereof. Alternatively, the pancreatic β cells specific targeting moiety can be a moiety that selectively binds a protein selected from the group consisting of DiGeorge syndrome critical region gene 2 (DGCR2), golgi brefeldin A resistant guanine nucleotide exchange factor 1 (GBF1), orphan G protein-coupled receptor GPR44 (GPR44), SerpinBlO (PI- 10), FXYD domain containing ion transport regulator 2 (FXYD2), Tetraspanin-7 (TSPAN7), gap junction protein, delta 2, 36kDa (GJD2), solute carrier family 18 (vesicular monoamine), member 2 (SLC18A2), prokineticin receptor 1 (PROKR1), glutamate receptor, metabotropic 5 (GRM5), neuropeptide Y receptor Y2 (NPY2R), glucagon- like peptide 1 receptor (GLP1R), transmembrane protein 27 (TMEM27) and a combination thereof. The moiety that selectively binds a protein as listed can be a ligand of the protein, an antibody specific for the protein or an aptamer specific for the protein. Details of such targeting strategies are disclosed in WO14140113, the disclosure of which being incorporated herein by reference. For instance, a molecule which binds the transmembrane protein 27 (TMEM27) which is a surface N-glycoprotein that is highly expressed on β cells could be used, e.g. an antibody or fragment thereof or an aptamer. More specifically, an antibody 8/9-mAb is disclosed in Vats et al (2012, Diabetologia, 55, 2407-2416, the disclosure of which being incorporated herein by reference). Alternatively, Hart et al (2010, FASEB J., 24, 816.9; 2014, Chembiochem. 15_^ 135- 145) discloses a ligand composed of Glucagon-like Peptide 1 and Glibenclamide, this ligand displaying a high specificity for β cells.

Methods of treatment and drugs combination

The molecules according to the invention can be used in combination with one or more additional active drugs, preferably anti-diabetic or hypo-glycemic/anti-hyperglycemic drugs, in particular for treating, delaying or preventing hyperglycemia and associated disorders and diseases.

Therefore, the present invention also relates to a pharmaceutical composition comprising a molecule according to the present invention and one or more additional active drugs, preferably an anti-diabetic drug, a hypo-glycemic drug or an anti-hyperglycemic drug.

It further relates to a product or kit containing a molecule according to the invention and one or more additional active drugs, preferably an anti-diabetic drug, a hypo-glycemic drug or an anti- hyperglycemic drug, as a combined preparation for simultaneous, separate or sequential use, or a combined preparation which comprises a molecule according to the invention and one or more additional active drugs, preferably anti-diabetic drug, an hypo-glycemic drug or an anti- hyperglycemic drug, for simultaneous, separate or sequential use, in particular for treating, delaying or preventing hyperglycemia and associated disorders and diseases.

It relates to a molecule according to the invention for use for treating, delaying or preventing hyperglycemia and associated disorders and diseases in combination with one or more additional active drugs, preferably an anti-diabetic drug, a hypo-glycemic drug or an anti- hyperglycemic drug.

It further relates to the use of a molecule according to the invention and one or more additional active drugs, preferably an anti-diabetic drug, a hypo-glycemic drug or an anti-hyperglycemic drug, for the manufacture of a medicament, in particular for treating, delaying or preventing hyperglycemia and associated disorders and diseases.

Finally, it relates to a method for treating, delaying or preventing hyperglycemia and associated disorders and diseases, wherein a therapeutic effective amount of a molecule according to the invention is administered in combination with a therapeutic or sub -therapeutic effective amount of one or more additional active drugs, preferably an anti-diabetic drug, a hypo-glycemic drug or an anti-hyperglycemic drug. By "sub-therapeutic" is intended to refer to an amount can be for instance 90, 80, 70, 60, 50, 40, 30, 20 or 10 % of the conventional therapeutic dosage (in particular for the same indication and the same administration route). In particular, the additional active drug is a drug used for treating or delaying the progression or onset of diabetes mellitus, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, and hyperglycemia. For instance, the additional drug can be an anti-diabetic drug such as a hypoglycemic agent or an antihyperglycemic agent. It may be selected in the non-exhaustive list comprising insulin, metformin, sulfonylureas such as tolbutamide, acetohexamide, tolazamide, chlorpropamide, glyburide (also called glibenclamide), glimepiride, glipizide, glicazide, glycopyramide and gliquidone, alpha-glucosidase inhibitors such as acarbose, miglitol and voglibose, thiazolidinediones such as pioglitazone and rosiglitazone, a meglitinide such as repaglinide and nateglinide, incretin mimetics, glucagon-like peptide analogs and agonists such as exenotide, taspoglutide and liraglutide, dipeptidyl peptidase-4 inhibitors such as vildagliptin, sitagliptin, saxagliptin, linagliptin, allogliptin, and septagliptin, amylin analogs such as pamlintide, glycourics such as canagliflozin and dapagliflozin, or any combination thereof. The form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc.

The pharmaceutical or therapeutic compositions of the invention can be formulated for a topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous or intraocular administration and the like.

The molecule used in the pharmaceutical composition of the invention is present in a therapeutically effective amount. The term "therapeutically effective amount" as used in the present application is intended to an amount of therapeutic agent, administered to a patient that is sufficient to constitute a treatment of diabetes mellitus, diabetic retinopathy, diabetic neuropathy, diabetic nephropathy, hyperglycemia, as defined above.

The pharmaceutical composition comprising the molecule is formulated in accordance with standard pharmaceutical practice (Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York) known by a person skilled in the art.

For oral administration, the composition can be formulated into conventional oral dosage forms such as tablets, capsules, powders, granules and liquid preparations such as syrups, elixirs, and concentrated drops. Non-toxic solid carriers or diluents may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, magnesium, carbonate, and the like. For compressed tablets, binders, which are agents which impart cohesive qualities to powdered materials, are also necessary. For example, starch, gelatine, sugars such as lactose or dextrose, and natural or synthetic gums can be used as binders. Disintegrants are also necessary in the tablets to facilitate break-up of the tablet. Disintegrants include starches, clays, celluloses, algins, gums and crosslinked polymers. Moreover, lubricants and glidants are also included in the tablets to prevent adhesion to the tablet material to surfaces in the manufacturing process and to improve the flow characteristics of the powder material during manufacture. Colloidal silicon dioxide is most commonly used as a glidant and compounds such as talc or stearic acids are most commonly used as lubricants.

For transdermal administration, the composition can be formulated into ointment, cream or gel form and appropriate penetrants or detergents could be used to facilitate permeation, such as dimethyl sulfoxide, dimethyl acetamide and dimethylformamide.

For transmucosal administration, nasal sprays, rectal or vaginal suppositories can be used. The active compound can be incorporated into any of the known suppository bases by methods known in the art. Examples of such bases include cocoa butter, polyethylene glycols (carbowaxes), polyethylene sorbitan monostearate, and mixtures of these with other compatible materials to modify the melting point or dissolution rate.

Pharmaceutical compositions according to the invention may be formulated to release the active drug substantially immediately upon administration or at any predetermined time or time period after administration.

Pharmaceutical compositions according to the invention can comprise one or more molecule of the present invention associated with pharmaceutically acceptable excipients and/or carriers. These excipients and/or carriers are chosen according to the form of administration as described above.

In a particular embodiment, the pharmaceutical composition according to the invention comprises 0.001 mg to 10 g of the molecule of the invention. Preferably, pharmaceutical composition according to the invention comprises 0.01 mg to 1 g of the molecule of the invention. Further aspects and advantages of this invention are disclosed in the following experimental section, which should be regarded as illustrative and not limiting the scope of this application.

EXAMPLES Pancreatic β cells lower insulin release in response to nutrient depletion. Whether nutrient- deprived β cells induce macroautophagy (hereafter referred to as "autophagy"), a predominant cellular mechanism maintaining energy homeostasis upon starvation, remains poorly explored. The inventors demonstrated that, in contrast to many other mammalian cells, autophagy in β cells is suppressed upon nutrient deprivation. Instead of autophagy, starved β cells induce lysosomal degradation of nascent secretory insulin granules in the vicinity of the Golgi. Starvation-Induced Nascent Granule Degradation (SINGD) is controlled by Protein Kinase 1 (PKDl), a key player in Secretory Granule (SG) biogenesis. SINGD in turn triggers lysosomal recruitment and activation of mechanistic Target Of Rapamycin (mTOR) that suppresses autophagy. Switching from autophagy to SINGD is important to keep insulin secretion low under nutrient-poor conditions. Hence, β cells employ a PKDl -dependent mechanism to adapt to nutrient availability coupling autophagy flux to secretory function.

Proper nutrient sensing is crucial in all organisms. In mammals upon feeding, the pancreatic β cell catabolizes nutrients to secrete insulin that acts to induce anabolic pathways. Inversely, upon fasting insulin secretion has to be maintained at a low level. Shortage of nutrients is also expected to induce autophagy. During autophagy, cellular components are sequestered into double-membrane autophagosomes, which fuse with lysosomes (autolysosomes), where degradation occurs. Resulting catabolites maintain cells metabolically active ensuring cell survival. In fact, catabolism of nutrients also triggers insulin release, which should be avoided during fasting. Therefore, autophagy might be a bad strategy for a β cell to cope with nutrient deprivation. Intriguingly, both induction and no induction of autophagy in fasted β cells depending on the models and protocols used were reported (C. Ebato et al. , Cell Metab. 8, 325- 32 (2008); R. Singh, A. M. Cuervo, Cell Metab. 13, 495-504 (2011)). All this challenged the importance of autophagy in fasted β cells and prompted the inventors to address this conundrum.

During autophagy, microtubule- associated protein 1 light chain 3 B (LC3B) incorporates into membranes of autophagosomes appearing as punctate structures. INS1 cells (a rat insulinoma- derived β cell line) exogenously and transiently expressing LC3B tagged with GFP (LC3B- GFP) showed abundant LC3B-GFP puncta under nutrient-rich conditions. Unexpectedly, INS1 cells deprived of serum and amino acids (AA) or glucose (Glc) dramatically decreased LC3B- GFP puncta (Fig. 1A). Deprivation from serum alone did not change the amount of LC3B-GFP puncta. In strong contrast and as previously reported EMBO J. 19, 5720-8 (2000)), starved human embryonic kidney 293 (HEK-293) cells markedly increased LC3B-GFP puncta as compared to non-starved cells. The inventors next generated INS 1 cells endogenously expressing LC3B-GFP (INS 1 ^LC3B-^GFPendo cells). These cells also reduced the amount of LC3B-GFP puncta upon AA/serum deprivation. Importantly, reduction of LC3B-GFP puncta was not due to increased lysosomal disposal of LC3B-GFP. Indeed, treatment with Bafilomycin Al (BafAl), that blocks autolysosomal degradation of LC3B, also revealed decreased LC3B-GFP puncta in AA/serum deprived jj^g jLcsB-GFPendo _cevj_{s as com}p_areci to non-starved cells (Fig. IB). To further corroborate reduced autophagy flux, the inventors used RFP-GFP tandem fluorescence-tagged LC3 (ptfLC3), in which the GFP tag is acid-sensitive and the RFP tag is acid-insensitive, allowing for discrimination of yellow fluorescent autophagosomes and red fluorescent autolysosomes. Time-lapse fluorescent microscopy of INS1 cells expressing ptf-LC3 revealed multiple RFP- GFP puncta that were converted into RFP only puncta upon onset of AA/serum or Glc/serum deprivation indicating the delivery of RFP-GFP-positive autophagosomes to autolysosomes. Subsequently, no reappearance of GFP-RFP puncta was observed and the number of RFP puncta was reduced. Correlative Light and Electron Microscopy (CLEM) confirmed the autophagic origin of RFP-GFP and RFP-only puncta (Fig. 1C).

The inventors next assessed endogenous autophagy flux by Western Blotting (WB). They observed decreased accumulation of lipidated autophagosomal LC3B (LC3B-II) in AA/serum- starved INS 1 cells non-treated or treated with Baf A 1 as compared to non-starved cells (Fig ID). In contrast, starvation markedly increased LC3B-II in HEK293 cells. p62 binds polyubiquitinylated substrates for subsequent targeting to autophagosomes and was therefore used as another autophagy marker. Total p62 levels were moderately increased during starvation independent of BafAl (Fig. ID). Immunofluorescence (IF) revealed that p62 puncta were abundant under nutrient-rich conditions and remained equally high during starvation. Interestingly, the size of p62 puncta increased and they clustered in cytoplasm of starved cells suggesting decreased p62-dependent substrate degradation. Accordingly, co-localization between p62 and LC3B-GFP was decreased in starved iNSi ^LC3B-^GFPendo _cen_s indicating suppressed autophagy-dependent clearance of p62. To address early steps of autophagosome formation, the inventors next performed IF of ATG16L1 that binds to pre-autophagosomes before LC3B incorporation. ATG16L1- and ATG16Ll/LC3B-GFP-positive puncta moderately decreased upon AA/serum deprivation of iNSi ^LC3B-^GFPendo cells. To expand their findings to primary β cells, the inventors next performed Quantitative EM (QEM) in β cells of non-starved and AA/serum-deprived murine primary islets. QEM confirmed reduced autophagic compartments in starved as compared to non- starved primary β cells. The inventors next used LC3B-GFP expressing mice et al, I Mol Biol Cell. 15, 1101-11 (2004)) to address autophagy levels in β cells in vivo. Strikingly, they found reduced LC3B-GFP puncta in β cells of fasted as compared to fed mice (Fig. IE). Overall, these data strongly suggest that autophagy is suppressed in fasted β cells.

What about other secretory cells, in which nutrients do not act as secretagogues? Plasma cells derived from antigen- stimulated B lymphocytes secrete immunoglobulins (Igs). In contrast to β cells, primary murine plasma cells induced autophagy in response to nutrient deprivation, while secretion of Ig G was reduced. Hence, the above effect on autophagy does not occur in all secretory cells. Collectively, the present data suggest that nutrient sensing β cells utilize a distinct mechanism to overcome shortage of nutrients.

Direct targeting of secretory granules (SGs) to lysosomes and their degradation might be an alternative strategy to produce energy metabolites. Therefore, the inventors assessed cellular localization of lysosomal Lampl and SG protein Phogrin. Phogrin- and Lamp 1 -positive puncta markedly increased in INSl cells upon AA/serum withdrawal. Lampl and Phogrin preferentially co-localized close to the Golgi. The increase was more prominent upon treatment with lysosomal protease inhibitors confirming lysosomal degradation of secretory granules. In addition, lysosomal protease inhibitors enlarged the size of Lampl/Phogrin positive puncta (Fig. 2A). QEM of starved INSl cells confirmed abundant lysosomes containing electron-dense SGs in proximity of the Golgi (Fig. 3A). CLEM and immuno-gold labelling corroborated that the latter structures corresponded to granule-containing lysosomes (GCLs) (Fig. 2B and Fig. 3B). The inventors next used density gradients to purify lysosomes from non-starved and starved INSl cells. They detected phogrin- and Lampl -positive fractions indicating the presence of SGs and lysosomes. Notably, double positive signals shifted to heavier fractions in AA/serum-deprived INSl cells as compared to non-starved cells. Subsequent TEM analysis of shifted fractions confirmed presence of GCLs. Importantly, the signal for LC3B in shifted fractions was almost not detectable suggesting independence of GCLs from autophagy (Fig. 2C). In line with this, AA/serum deprivation did not increase co-localization between LC3B- GFP and Phogrin in iNSl^LC3B"GFPendo cells (Fig. 4). Moreover, the amount of GCLs in AA/serum deprived INSl cells depleted from key autophagy genes, beclin-1 and ATG5 as well as treated with the autophagy inhibitor 3-methyladenine (3-MA) was not changed as compared to starved control cells (Fig. 5 A and B).

SGs are generated at the trans-Golgi network (TGN). The inventors thus wondered whether nascent granules are specifically targeted to lysosomes. Readily formed SGs contain proinsulin, which is subsequently converted into insulin. A 6-hour depletion from AA/serum led to an almost complete loss of proinsulin (Fig. 2D). The decrease in proinsulin was partially restored by lysosomal protease inhibitors suggesting that nutrient deprivation leads to lysosomal degradation of de novo synthesized SGs in β cells. Abundant GCLs upon starvation were confirmed ex vivo by QEM of primary murine islets (Fig. 2E). To underpin autophago some- independent GCLs, the inventors next explored them in vivo in fed and fasted GFP-LC3B expressing mice. They found a marked increase in co-localization between Lamp2 and proinsulin/insulin (hereafter referred to as "(Pro)insulin"). Importantly, there was no increase in co-localized LC3B-GFP and (Pro)insulin (Fig. 2F). Hence, β cells employ Starvation- Induced Nascent Granule Degradation (SINGD) instead of autophagy to counteract shortage of nutrients.

Lysosomal AAs derived from degraded proteins induce translocation of mechanistic Target Of Rapamycin Complex 1 (mTORCl) to lysosomal membranes, mTORCl activation and subsequent suppression of autophagy. Do lysosomes involved in SINGD suppress autophagy through mTORCl? Strikingly, the mTOR inhibitors rapamycin and torin 1 increased the number of LC3B-GFP puncta in AA/serum-depleted INSl cells as compared to control treated cells (Fig. 6A). This indicates that mTORCl activity is required for suppression of autophagy upon starvation. Moreover, depletion of AA/serum from INSl cells resulted in translocation of mTOR to Phogrin/Lamp 1 -positive puncta close to the Golgi area (Fig. 6B). mTORCl was shown to suppress starvation-induced autophagy through inhibitory phosphorylation of Unc- 51-like kinase 1 (ULK1) on Serine 757 (ULK1-S757). Thus, starvation is expected to decrease ULK1-S757 phosphorylation. Western blotting revealed that ULK1-S757 phosphorylation levels remained high in starved as compared to non-starved INSl cells (Fig. 6C). Importantly, phosphorylation of ULK1-S757 was markedly decreased when mTORCl was inhibited by rapamycin (Fig. 6C). Interestingly, while under nutrient-rich conditions S757-ULK1 phosphorylation appeared as discrete fluorescent dots throughout the cytoplasm, AA/serum deprivation led to formation of large bright S757-ULK1 puncta that co-localized with both Lampl and Phogrin close to the Golgi (Fig. 6D). Collectively, this implies that mTORCl- mediated ULK1-S757 phosphorylation in the SINGD compartment is important in autophagy suppression. Levels of ULK1-S757 phosphorylation did not increase under starving as compared to growing conditions suggesting that observed basal autophagy in non-starved cells is largely independent of ULK1-S757 phosphorylation. Phosphorylation of the mTORCl substrate S6K1 on threonine 389 (T389), involved in regulation of protein translation, almost completely disappeared during starvation (Fig. 6C). This is in line with a previous report showing that S6K1 phosphorylation requires higher mTOR activity than ULK1 phosphorylation. Thus, the present data indicate that nutrient depletion leads to induction of SINGD that locally activates mTOR to suppress autophagy.

Fasting suppresses both insulin secretion and autophagy. The inventors therefore wondered whether insulin secretion remains low if autophagy is induced upon starvation. To address this, they treated INS1 cells with rapamycin at non- stimulatory Glc concentrations, corresponding to physiologic fasting Glc levels, and measured insulin release. Keeping autophagy high in β cells at non-stimulatory Glc levels precluded suppression of insulin secretion (Fig. 6E). To fortify and expand this finding, the inventors next used more specific means to trigger autophagy in β cells of primary murine islets. To this end, they used the recently developed autophagy-inducing peptide, tat-beclinl (S. Shoji-Kawata et ah, Nature. 494, 201-206 (2013)). Treatment with tat-beclinl but not with scrambled tat-beclinl markedly increased autophagy in ex vivo fasted murine islets and did not increase cell death (Fig. 7A and B). Strikingly, this treatment resulted in increased insulin secretion comparable to levels observed upon stimulatory Glc (Fig. 7C). Importantly, the increase in insulin release was abolished by the insulin secretion inhibitor diazoxide (Fig. 7D). To address the relevance of our findings in a human context, the inventors assessed effects of forced autophagy on insulin secretion from β cells in isolated primary human islets. Activation of autophagy with tat-beclinl led to a remarkable increase in insulin secretion from ex vivo fasted human islets without affecting cell survival (Fig. 6F and Fig. 8A and B). Thus, SINGD-mediated suppression of autophagy is important to keep insulin secretion low during fasting.

How is SINGD controlled? Degradation of nascent SGs in vicinity of the Golgi is likely to rely on molecular mechanisms involved in SG biogenesis. The inventors identified Protein Kinase Dl as a key regulator of early steps of insulin SG biogenesis at the TGN. Thus, PKD1 inactivation could have an effect on the turnover of de novo formed SGs. Indeed, treatment of INS1 cells with the PKD inhibitor CID755673 markedly decreased the amount of proinsulin under nutrient-rich conditions (Fig. 9A). The rate of proinsulin biosynthesis was unchanged in INS1 cells stably expressing shRNA against PKD1 as compared to non-silenced cells (Fig. 9A). However, accumulation of newly formed insulin was markedly decreased in PKD1 knockdown cells, suggesting enhanced degradation of de novo synthesized insulin (Fig. 10). QEM and immunogold analyses of cells lacking PKD1 revealed abundant GCLs in the Golgi area (Fig 9B, and immunogold analysis Fig. 11). Moreover, the inventors detected shifted Phogrin- and Lamp 1 -positive and LC3B-negative fractions containing GCLs in INS1 cells depleted of PKD 1 (Fig. 12). Inhibition of PKD markedly increased Phogrin and Lampl co-localization, which was more prominent upon treatment with lysosomal inhibitors (Fig. 13), while co-localization of Phogrin with LC3B-GFP remained unchanged (Fig. 14). Would inactivation of PKDl lead to mTOR-dependent suppression of autophagy, as SINGD does? Strikingly, mTOR largely co- localized with lysosomal Lampl in cells depleted of PKDl (Fig. 9C). Moreover, mTOR- mediated ULK1-S757 phosphorylation was markedly increased (Fig. 9D). PKDl knockdown led to decreased accumulation of LC3-II in presence of BafAl (Fig. 15). In addition, PKD inhibition considerably decreased LC3B-GFP puncta in presence and absence of BafAl (Fig. 9E). Hence, inactivation of PKD is sufficient to trigger SINGD and to attenuate autophagy even under nutrient-rich conditions.

If PKD controls SINGD, its activity should be regulated by nutrients. The inventors therefore used a TGN-localized Fluorescence Resonance Energy Transfer (FRET) reporter of PKDl activity (G-PKDrep-live), and performed time-lapse FRET measurements upon nutrient deprivation in INS1 and in MIN6B1 cells (mouse insulinoma-derived β cell line). The FRET signals rapidly and markedly decreased during nutrient deprivation (Fig. 9F). They previously found that loss of the MAPK ρ38δ leads to enhanced PKDl activity in β cells. Importantly, ρ38δ^~'^~ mice displayed higher fasting insulin release than control mice. This might be due to decreased SINGD-dependent suppression of autophagy. To test whether high PKD activity prevents SINGD, the inventors performed QEM of β cells of fasted islets isolated from ρ38δ knockout (ρ38δ^~'^~) and control (ρ38δ^+,+) mice. Fasted ρ38δ^+,+ β cells displayed abundant GCLs, which were markedly decreased in ρ38δ^~'^~ cells indicating reduced SINGD (Fig. 9G, upper panel). Accordingly, (Pro)insulin and Lamp2 co-localized to a lesser extend in β cells of in vivo fasted ρ38δ^~'^~ mice (Fig. 16). In contrast, autophagic compartments were markedly increased in fasted ρ38δ^~'^~ β cells indicating that SINGD-dependent suppression of autophagy was diminished (Fig. 9G). The present data thus suggest that PKDl is a major regulator of SINGD in β cells.

Altogether, rapid inactivation of PKDl evokes SINGD, localized activation of mTOR and suppression of autophagy, which is critical to prevent insulin release during fasting. On the other hand, PKD 1 activity is required for proper SG biogenesis and subsequent insulin secretion (Fig. 9H). As the nascent SGs were shown to be preferentially secreted, SINGD-mediated suppression of autophagy is an optimal strategy to counteract insulin secretion, at the same time providing sufficient nutrients. Since SINGD-mediated suppression of autophagy depends on an abundant nascent SG pool, its depletion will likely derepress autophagy without increasing insulin release. Timing when depletion occurs in the course of starvation may vary substantially depending on the models and protocols used and may thus explain previously described inconsistencies (C. Ebato et ah, supra). Accordingly, prolonged fasting of mice was reported to induce autophagy in β cells (K. Fujimoto et ah, J. Biol. Chem. 284, 27664-73 (2009)). The inventors' observations point at the fact that triggering autophagy results in increased secretion of insulin. While this should be avoided during fasting, it may entail beneficial effects in conditions, where insulin demands are high, for example after a meal or in a diabetic state. The observed positive correlation between autophagy and insulin secretion may suggest an involvement of autophagy in postprandial insulin release probably going beyond the widely established housekeeping role of autophagy. This is indeed a very interesting subject to be pursued in the near future. The identified positive correlation between autophagy and insulin secretion as well as the discovery of SINGD-mediated suppression of autophagy thus offers interesting therapeutic perspectives in diabetes.

Claims

1- A molecule inducing autophagy for use for increasing release or secretion of insulin by pancreatic β cells.

2- The molecule for use according to claim 1 for use for treating hyperglycemia.

3- The molecule for use according to claim 1 or 2 for the treatment of a pre-diabetic condition, diabetes, preferably type 2 diabetes, hyperglycemia associated to medications, to acute stress such as stroke or myocardial infarction, to dysfunction of the thyroid, adrenal and pituitary glands, to pancreas disease such as pancreatitis and pancreas cancer, or to intracranial diseases.

4- The molecule for use according to any one of claims 1 to 3, wherein the molecule inducing autophagy is a peptide derived from Beclin-1 protein.

5- The molecule for use according to claim 4, wherein the peptide comprises Beclin 1 residues 269-283 (VFNATFHIWHSGQF, SEQ ID No 1), more preferably comprises the sequence TNVFNATFHIWHSGQFGT, SEQ ID No 2), wherein up to six of said residues may be substituted.

6- The molecule for use according to claim 4 or 5, the peptide comprises or consists on a peptide comprising the amino acid sequence V-F-N-A-T-F-E/H-I-W-H-D/S (SEQ ID No 3), T-N-V-F-N-A-T-F-E/H-I-W-H-D/S-G-Q/E-F-G-T (SEQ ID No 4) or the D- retro-in verso sequence thereof, wherein up to 6 of said residues may be substituted.

7- The molecule for use according to claim 6, wherein the peptide is selected in the group consisting of YGRKKRRQRRRGGTNVFNATFHIWHSGQFGT (SEQ ID No 12); YGRKKRRQRRRGGTNVFNATFEIWHDGEFGT (SEQ ID No 13); and the D-retro- inverso amino acid sequence RRRQRRKKRGYGGTGFEGDHWIEFTANFVNT (SEQ ID No 14).

8- The molecule for use according to any one of claims 1 to 3, wherein the molecule inducing autophagy is an antibody or a fragment or derivative thereof, an aptamer, a Spiegelmer or a chemical compound.

9- The molecule for use according to any one of claims 1 to 8, wherein the molecule comprises a cell penetrating moiety or a moiety targeting the pancreatic β cell.

10- The molecule for use according to claim 9, wherein the moiety targeting the pancreatic β cell is selected from the group consisting of glucagon- like peptide- 1 (GLP-1), glucagon- like peptide-2 (GLP-2), peptide YY (PYY), neuropeptide Y (NPY), pancreatic peptide (PPY), exendin-4, naphthylalanine, and naphthylalanine derivatives, , or is a moiety that that selectively binds a protein selected from the group consisting of DiGeorge syndrome critical region gene 2 (DGCR2), golgi brefeldin A resistant guanine nucleotide exchange factor 1 (GBF1), orphan G protein-coupled receptor GPR44 (GPR44), SerpinBlO (PI- 10), FXYD domain containing ion transport regulator 2 (FXYD2), Tetraspanin-7 (TSPAN7), gap junction protein, delta 2, 36kDa (GJD2), solute carrier family 18 (vesicular monoamine), member 2 (SLC18A2), prokineticin receptor 1 (PROKR1), glutamate receptor, metabotropic 5 (GRM5), neuropeptide Y receptor Y2 (NPY2R), glucagon- like peptide 1 receptor (GLP1R), and transmembrane protein 27 (TMEM27).

11- The molecule for use according to claim 9 or 10, wherein the moiety targeting the pancreatic β cell is an antibody or a fragment thereof, an aptamer, or a ligand.

12- The molecule for use according to claim 9 or 10, wherein the moiety targeting the pancreatic β cell is a combination of several moieties targeting the pancreatic β cell which have different binding partners.

13- The molecule for use according to any one of claims 1-12, wherein it is used in combination with an additional active drug, preferably an anti-diabetic drug, a hypoglycemic drug or an anti-hyperglycemic drug.

14- A product containing a molecule according to any one of claims 1-12 and an additional active drug, preferably an anti-diabetic drug, an hypo-glycemic drug or an anti- hyperglycemic drug, as a combined preparation for simultaneous, separate or sequential use, in particular for treating, delaying or preventing hyperglycemia.

15- The product according to claim 14 for use in the treatment of a pre-diabetic condition, diabetes, preferably type 2 diabetes, hyperglycemia associated to medications, to acute stress such as stroke or myocardial infarction, to dysfunction of the thyroid, adrenal and pituitary glands, to pancreas disease such as pancreatitis and pancreas cancer, or to intracranial diseases.