WO2016209659A1 - Reversal of persistent ros generation after transient hyperglycemia - Google Patents

Reversal of persistent ros generation after transient hyperglycemia Download PDF

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WO2016209659A1
WO2016209659A1 PCT/US2016/037334 US2016037334W WO2016209659A1 WO 2016209659 A1 WO2016209659 A1 WO 2016209659A1 US 2016037334 W US2016037334 W US 2016037334W WO 2016209659 A1 WO2016209659 A1 WO 2016209659A1
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agent
subject
hyperglycemia
ros
production
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PCT/US2016/037334
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French (fr)
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Michael Alan BROWNLEE
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Albert Einstein College Of Medicine, Inc.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 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
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/26—Glucagons
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5064—Endothelial cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/84—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving inorganic compounds or pH

Definitions

  • Diabetic and pre-diabetic hyperglycemia continue to be a major cause of blindness, renal failure, heart attacks, and lower limb amputation.
  • Hyperglycemia-induced overproduction of mitochondrial reactive oxygen species (ROS) initiates many of the complex series of molecular events that result in diabetic tissue damage.
  • ROS mitochondrial reactive oxygen species
  • the assumption underlying current diabetes treatment is that lowering the level of time-averaged glucose concentrations, measured as HbAlc, prevents microvascular complications.
  • recent analyses by the DCCT/EDIC study group showed that 89% of variation in risk of retinopathy, microalbuminuria or albuminuria is due to elements of glycemia not captured by mean HbAlc values.
  • HbAlc time-averaged glucose concentrations
  • technologies targeting diabetic complications mostly target HbAlc values by providing improved insulin secretion or action.
  • Developing technologies include iPSC differentiation, mechanical insulin or insulin-glucagon devices, and a variety of monomelic peptides engineered to be agonists for multiple peptide-hormone receptors. Small molecular weight compounds and peptides that increase tissue sensitivity to insulin action are also in development. None, to the inventor's knowledge, address the long-term effects of brief spikes of hyperglycemia.
  • ROS reactive oxygen species
  • a method for obtaining an agent which reduces hyperglycemia- induced ongoing or persistent or post-hyperglycemic spike reactive oxygen species (ROS) production in a cell in a subject, wherein the agent inhibits an ROS production feedback loop,
  • ROS reactive oxygen species
  • a method for obtaining an agent which reduces hyperglycemia- induced ongoing reactive oxygen species (ROS) production in a cell in a subject, wherein the agent inhibits an ROS production feedback loop,
  • ROS reactive oxygen species
  • ROS hyperglycemia-induced ongoing reactive oxygen species
  • ROS reactive oxygen species
  • the method further comprises physically recovering the candidate agent identified as reducing ROS production so as to obtain an agent which reduces reactive oxygen species (ROS).
  • ROS reactive oxygen species
  • ROS reactive oxygen species
  • Also provided is a method of treating diabetes complication in a subject comprising administering to the subject an amount of an agent effective to inhibit reactive oxygen species production by a cell of a subject.
  • ROS reactive oxygen species
  • Also provided is a method for treating diabetes complication in a subject comprising administering to the subject an amount of an agent effective to activate Aktl, to inhibit PP2A, to inhibit GSK-3beta, to inhibit phosphorylation of VDAC threonine, or to increase association of VDAC with HK-II.
  • Also provided is a method for treating diabetes or prediabetic hyperglycemia in a subject comprising administering to the subject an amount of an agent effective to the subject an amount of an agent effective to activate Aktl, to inhibit PP2A, to inhibit GSK- 3beta, to inhibit phosphorylation of VDAC threonine, or to increase association of VDAC with HK-II.
  • FIG. 1A-1D Transient exposure to high glucose induces persistent mitochondrial ROS production by shifting the glucose concentration-ROS curve to the left.
  • IB ROS levels in HAEC exposed to 25mM glucose for varying times.
  • FIG. 2A-2H Persistent mitochondrial ROS production after transient exposure to high glucose is maintained by activation of a multi-component feedback loop.
  • Upper panel Representative photomicrographs of increased JC-1 red fluorescence indicating increased mitochondrial membrane potential.
  • Lower panel ROS production measured by CM-H2DCFDA.
  • (2B-G) HAEC were exposed to 25 mM glucose for the indicated times with (red bars) or without (green bars) subsequent incubation in 5 mM glucose for 2 days. Cells exposed to 5mM glucose were used as controls (blue bars).
  • Lower panel Quantitation of IP-WB data from upper panel.
  • Free iron in HAEC was measured by Electron Paramagnetic Spectroscopy (EPR).
  • EPR Electron Paramagnetic Spectroscopy
  • HAEC were exposed to 5mM glucose for 6 hrs after infection with adenoviral vectors expressing either Akt-DN, GSK-3 -CA, or vector alone.
  • Upper panel Representative photomicrographs of increased JC-1 red fluorescence indicating increased mitochondrial membrane potential.
  • Lower panel ROS production measured by CM- H2DCFDA.
  • ROS data are the mean +/- S.E.M from 3 independent experiments with at least 8 technical replicates. All data shown are the mean +/- S.D. from 5 independent experiments. * p ⁇ 0.05.
  • FIG. 3 Schematic representation of the multi-component feedback loop which maintains persistently increased ROS production after transient exposure to high glucose.
  • Transient exposure to high glucose induces a transient increase in ETC flux increasing ⁇ and thereby increasing mitochondrial superoxide production.
  • this superoxide causes release of Fe 2+ from ferritin and iron sulfur cluster-containing proteins.
  • This released free iron reacts with diffused superoxide-derived hydrogen peroxide to form hydroxyl radicals, which increase PP2A activity.
  • Activated PP2A then dephosphorylates Aktl, decreasing its activity. Decreased Aktl activity increases GSK-3 activity, which then increases VDAC threonine phosphorylation.
  • Glucose concentration-ROS dose response curve from HAEC exposed to 12mM for 6 hrs followed by 2 days of 5mM glucose, and then transiently treated with a peptide GSK-3 inhibitor (gold curve). This curve is shown in comparison to the glucose concentration-ROS curves from cells exposed to 5mM glucose for 2 days (blue curve), and the curve from cells exposed to 6 hrs 12mM glucose followed by 5mM glucose for 2 days (red curve) from Figure ID. ROS levels were measured by CM- H2DCFDA. ROS data are the mean +/- S.E.M from 3 independent experiments with at least 8 technical replicates. * p ⁇ 0.05. (4B-4E) Effect of GLP-1 (9-36)amide on feedback loop components.
  • HAEC were exposed to 12 mM glucose for 6 hrs with or without 100 pM GLP-1 (9-36)amide. Cells exposed to 5mM glucose were used as controls (blue bars).
  • GLP-1 (9-36)amide reverses the persistent reduction of prostacyclin synthase caused by transient hyperglycemia in vivo.
  • Upper panel Experiment schematic.
  • IP glucose
  • a micro-osmotic pump with GLP-1 (9-36)amide 300 ⁇ g/ml was inserted in the mice for 24 hours and then removed.
  • ROS overproduction during hyperglycemia involves increased glucose metabolism causing increased mitochondrial electron leak, which can also activate other ROS -generating proteins such as NADPH oxidases and monomeric ENOS.
  • glucose levels are normal, there is no increased glucose metabolism in target cells of complications.
  • a method for obtaining an agent which reduces hyperglycemia- induced ongoing reactive oxygen species (ROS) production in a cell in a subject, wherein the agent inhibits an ROS production feedback loop,
  • ROS reactive oxygen species
  • ROS reactive oxygen
  • the cell is a vascular cell.
  • candidate agents are selected from a peptide library.
  • candidate agents are selected from a peptide library wherein the peptiodes are from 5 to 40 amino acids in length.
  • Hyperglycemic glucose and normoglycemic glucose are easily determined by the species of mammal the cell being tested.
  • the cells are human cells, and the hyperglycemic glucose and normoglycemic glucose are hyperglycemic glucose for a human and normoglycemic glucose for a human.
  • the cells are previously exposed to hyperglycemic glucose for 5.75 - 6.25 hours.
  • the cells are previously exposed to hyperglycemic glucose for 6 hours.
  • the cells are exposed to normoglycemic glucose for over 1.5 days with the candidate agent.
  • the cells are exposed to normoglycemic glucose for 2.0 days with the candidate agent.
  • a method for obtaining an agent which reduces hyperglycemia- induced ongoing reactive oxygen species (ROS) production in a cell in a subject, wherein the agent inhibits an ROS production feedback loop,
  • ROS reactive oxygen species
  • the cell is a vascular cell.
  • the VDAC, a GSK-3beta or an Akt is contacted with the candidate agent.
  • the cell is
  • the method further comprises physically recovering the candidate agent identified as reducing ROS production so as to obtain an agent which reduces hyperglycemia-induced ongoing ROS production in a vascular cell or an agent which reduces ROS production in a vascular cell, respectively.
  • VDAC phosphorylation, GSK-3beta activity or Akt activity are experimentally quantified using Western blot and/or immunoprecipitation.
  • the candidate agent is a peptide. In an embodiment of the methods, the candidate agent is a peptide of less than 20 amino acid residues. In an embodiment of the methods, the candidate agent is a peptide of less than 10 amino acid residues.
  • the candidate agent is a small organic molecule of 1,500 daltons or less.
  • the hyperglycemia of the hyperglycemia- induced ongoing ROS production or the hyperglycemia associated with the ROS production is transient. In an embodiment of the methods, the hyperglycemia is from about 4 to 6 hours. In an embodiment of the methods, the hyperglycemia is undetectable using HbAlc quantification. In an embodiment of the methods, the hyperglycemia does not increase HbAlc production in the subject.
  • the candidate the agent does not affect glycemia in the subject. In an embodiment of the methods, the candidate the agent does not affect insulin production or affect insulin activity in a subject.
  • the method further comprises physically recovering the candidate agent identified as reducing ROS production so as to obtain an agent which reduces reactive oxygen species (ROS).
  • the cell is a vascular cell.
  • the cell is a vascular cell.
  • the HAEC has been obtained from a diabetic human. In an embodiment, the HAEC has been obtained from a non-diabetic human.
  • the elevated glucose concentration is equivalent to over 190 mg/dL blood. In an embodiment, the elevated glucose concentration is equivalent to over 210 mg/dL blood. In an embodiment, the elevated glucose concentration is equivalent over 20 mM. In an embodiment, the elevated glucose concentration is 23 - 27 mM. In an embodiment, the elevated glucose concentration is 25 mM.
  • the candidate agent is a peptide. In an embodiment, the candidate agent is a peptide of less than 20 amino acid residues. In an embodiment, the candidate agent is a peptide of less than 10 amino acid residues. In an embodiment, the candidate agent is a small organic molecule of 1,500 daltons or less. In an embodiment, the candidate agent is a small organic molecule of 1,000 daltons or less. In an embodiment, the candidate agent is a small organic molecule of 500 daltons or less.
  • ROS levels are quantified using CM-H2DCFDA. In an embodiment, ROS levels are quantified using fluorimetry.
  • ROS reactive oxygen species
  • the cell is a vascular cell.
  • ROS reactive oxygen species
  • the cell is a vascular cell.
  • a method of treating diabetes complication in a subject comprising administering to the subject an amount of an agent effective to inhibit reactive oxygen species production by a cell of a subject.
  • Normal fasting plasma glucose is generally understood to be ⁇ 100 mg/dL (5.6 mmol/L).
  • Two-hour glucose during oral glucose tolerance test (OGTT) has blood glucose ⁇ 140 mg/dL (7.8 mmol/L).
  • the subject has an increased risk for diabetes.
  • Subjects with increased risk for diabetes include those with impaired fasting glcose (IFG) - which is generally understood to be a fasting plasma glucose of between 100 and 125 mg/dL (5.6 to 6.9 mmol/L).
  • Subjects with increased risk for diabetes also include those with impaired glucose tolerance (IGT) - a two-hour plasma glucose value during a 75 g oral glucose tolerance test between 140 and 199 mg/dL (7.8 to 11.0 mmol/L).
  • ITT impaired glucose tolerance
  • the subject has diabetes type 2 diabetes or diabtes mellitus.
  • Subjects with diabetes mellitus have an FPG at or above 126 mg/dL (7.0 mmol/L), a two-hour value in an OGTT (2-h PG) at or above 200 mg/dL (11.1 mmol/L), or a random (or "casual") plasma glucose concentration >200 mg/dL (11.1 mmol/L) in the presence of symptoms.
  • the subject has near normal FPG.
  • the subject has diabetes. "Near normal" FPG in someone who already has diabetes would be 100-145 or 150 mg/dL, while 200-250 would be quite high but not unusual in many ambulatory diabetic patients.
  • the cell is a vascular cell.
  • the complication is a retinopathy, an albuminuria or a microalbuminuria.
  • the complication is a peripheral neuropathy, a non-healing diabetic foot ulcer, a cognitive decline, a diabetic cardiomyopathy, an atherosclerosis, or a post-myocardial infarction arrhythmia.
  • a method of reducing reactive oxygen species (ROS) production in a cell in a subject, wherein the ROS production is fatty acid-induced ROS comprising administering to the subject an amount of an agent effective to activate Aktl, to inhibit PP2A, to inhibit GSK-3beta, to inhibit phosphorylation of VDAC threonine, or to increase association of VDAC with HK-II.
  • ROS reactive oxygen species
  • a method of treating diabetes or prediabetic hyperglycemia in a subject comprising: a) receiving an identification of the subject as having a persistent or an ongoing fatty acid- induced ROS;
  • Also provided is a method of treating diabetes or prediabetic hyperglycemia in a subject comprising administering to the subject an amount of an agent effective to inhibit reactive oxygen species production by a cell of a subject.
  • the cell is a vascular cell.
  • Also provided is a method of preventing or reducing development of diabetes, or preventing or reducing development of prediabetic hyperglycemia, in a subject comprising administering to the subject an amount of an agent effective to inhibit reactive oxygen species production by a cell of a subject.
  • the cell is a vascular cell.
  • the method is for treating diabetes. In an embodiment, the method is for treating prediabetic hyperglycemia. In an embodiment, the agent comprises a peptide.
  • the peptide is a non-hypoglycemic peptide.
  • the agent comprises glucagon like peptide- 1 (9-36) amide. In an embodiment, the agent does not comprise glucagon like peptide-1 (9-36) amide. In an embodiment, the glucagon like peptide-1 (9-36) amide has the sequence EGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH 2 (SEQ ID NO: l).
  • the agent comprises glucagon like peptide-1 (9-37). In an embodiment, the agent does not comprise glucagon like peptide-1 (9-37). In an embodiment, the glucagon like peptide-1 (9- 37) has the sequence EGTFTSDVSSYLEGQAAKEFIAWLVKGRG.
  • the agent is directly administered to a retinal tissue for treating the retinopathy.
  • the agent comprises glucagon like peptide-1 (9-36) amide, the agent comprises a small organic molecule.
  • the agent is administered during or subsequent to a hyperglycemic event. In an embodiment, the agent is administered by self-administration. In an embodiment, the agent is administered continuously by a pump. [0055] Also provided is a method of treating diabetes or prediabetic hyperglycemia in a subject comprising administering to the subject an amount of an agent effective to the subject an amount of an agent effective to activate Aktl, to inhibit PP2A, to inhibit GSK- 3beta, to inhibit phosphorylation of VDAC threonine , or to increase association of VDAC with HK-II.
  • the subject is a human.
  • Also provided is a method of treating diabetes or prediabetic hyperglycemia in a subject comprising:
  • a method of treating diabetes or prediabetic hyperglycemia in a subject comprising:
  • the agent comprises a peptide.
  • the agent comprises glucagon like peptide- 1 (9-36) amide.
  • the agent comprises glucagon like peptide- 1 (9-37).
  • the agent comprises a small organic molecule.
  • the agent is administered during or subsequent to a hyperglycemic event.
  • the agent is administered by self-administration. In an embodiment, the agent is administered continuously by a pump. In an embodiment, the amount of agent administered decreases Akt inhibition in the vascular cells of the subject. In an embodiment, the amount of agent administered reduces the extent of VDAC phosphorylation associated with hyperglycemia in the subject. In an embodiment, the amount of agent administered reduces the extent of GSK-3P activation associated with hyperglycemia in the subject. In an embodiment, the amount of agent administered reduces the extent of PGI2 inactivation associated with hyperglycemia induced ROS production in the subject.
  • the subject is identified as undergoing a hyperglycemic spike by means of a blood glucose monitor.
  • the agent is administered within 5 minutes of a hyperglycemia threshold being crossed.
  • a hyperglycemia threshold is crossed when the glucose level in a subject's blood exceeds a predetermined threshold value, which is indicative of hyperglycemia.
  • the hyperglycemia threshold is l lmmol/L.
  • the hyperglycemia threshold is 12mmol/L.
  • the hyperglycemia threshold is 13mmol/L.
  • the hyperglycemia threshold is 14mmol/L.
  • the hyperglycemia threshold is 15mmol/L.
  • the hyperglycemia threshold is 16mmol/L.
  • the hyperglycemia threshold is 17mmol/L. In an embodiment, the hyperglycemia threshold is 18mmol/L. In an embodiment, the hyperglycemia threshold is 19mmol/L. In an embodiment, the hyperglycemia threshold is 20mmol/L. In an embodiment, the hyperglycemia threshold is 25mmol/L. In an embodiment, the agent is administered within 10 minutes of a hyperglycemia threshold being crossed. In an embodiment, the agent is administered within 15 minutes of a hyperglycemia threshold being crossed. In an embodiment, the agent is administered within 20 minutes of a hyperglycemia threshold being crossed. In an embodiment, the agent is administered within 25 minutes of a hyperglycemia threshold being crossed. In an embodiment, the agent is administered within 30 minutes of a hyperglycemia threshold being crossed.
  • the method is for treating diabetes. In an embodiment, the method is for treating prediabetic hyperglycemia.
  • Administration can be one of, or any combination of, auricular, buccal, conjunctival, cutaneous, subcutaneous, endocervical, endosinusial, endotracheal, enteral, epidural, via hemodialysis, interstitial, intrabdominal, intraamniotic, intra-arterial, intra-articular, intrabiliary, intrabronchial, intrabursal, intracardiac, intracartilaginous, intracaudal, intracavernous, intracavitary, intracerebral, intracisternal, intracorneal, intracoronary, intradermal, intradiscal, intraductal, intraepidermal, intraesophagus, intragastric, intravaginal, intragingival, intraileal, intraluminal, intralesional, intralymphatic, intramedullary, intrameningeal, intramuscular, intraocular, intraovarian, intrae
  • the amount of agent administered decreases Akt inhibition in the vascular cells of the subject. In an embodiment, the amount of agent administered reduces the extent of VDAC phosphorylation associated with hyperglycemia in the subject. In an embodiment, the amount of agent administered reduces the extent of GSK-3P activation associated with hyperglycemia in the subject. In an embodiment, the amount of agent administered reduces the extent of PGI2 inactivation associated with hyperglycemia induced ROS production in the subject. In an embodiment of the methods of treatment, the hyperglycemia is undetectable using HbAlc quantification. In an embodiment of the methods, the hyperglycemia does not increase HbAlc production in the subject.
  • the ROS production is persistent ROS production following brief spike(s) of hyperglycemia.
  • ROS reactive oxygen species
  • ROS reactive oxygen species
  • a method for reducing reactive oxygen species (ROS) production in a cell in a subject, wherein the ROS production is associated with a hyperglycemia in a subject comprising administering to the subject an amount of an agent effective to normalize Aktl activity, to normalize PP2A activity, to normalize GSK-3beta activity, to normalize phosphorylation of VDAC threonine, or to normalize association of VDAC with HK-II.
  • ROS reactive oxygen species
  • a method for treating a diabetes complication in a subject comprising administering to the subject an amount of an agent effective to activate Aktl, to inhibit PP2A, to inhibit GSK-3beta, to inhibit phosphorylation of VDAC threonine, or to increase association of VDAC with HK-II.
  • the complication is a retinopathy, an albuminuria or a microalbuminuria.
  • Also provided is a method for treating diabetes or prediabetic hyperglycemia in a subject comprising administering to the subject an amount of an agent effective to activate Aktl, to inhibit PP2A, to inhibit GSK-3beta, to inhibit phosphorylation of VDAC threonine, or to increase association of VDAC with HK-II.
  • the subject is a human.
  • ROS reactive oxygen species
  • the method further comprises physically recovering the candidate agent identified as reducing ROS production so as to obtain an agent which reduces hyperglycemia-induced ongoing ROS production.
  • the non-human animal is a diabetes-model animal.
  • the animal is a rat or a mouse.
  • it is vascular cell ROS production that is quantified.
  • treating a diabetes means that one or more symptoms of the disease, such as the diabetes itself, or a resultant symptom of the diabetes such as blindness, heart damage, lower limb ischemia or other parameters by which the disease is characterized, are reduced, ameliorated, inhibited, placed in a state of remission, or maintained in a state of remission.
  • the method inhibits further development of the diabetes.
  • the method inhibits further development of a pathology that results from the diabetes.
  • the diabetes is Type I diabetes.
  • the diabetes is Type II diabetes. This definition of treating a diabetes, mutatis mutandis, can apply to treating a diabetic complication, such as a retinopathy, albuminuria or microalbuminuria.
  • treating a prediabetes hyperglycemia means that one or more symptoms of the disease, such as ROS generation, or a damage resulting from hyperglycemia induced ROS generation or other parameters by which the disease is characterized, are reduced, ameliorated, inhibited, or maintained in a state of prediabetes.
  • the method inhibits further development of the prediabetes into diabetes.
  • the method inhibits further development of a pathology that results from the prediabetes hyperglycemia.
  • the subject treated has Hemoglobin Ale levels between 5.7% and 6.4% indicate increased risk of diabetes. In an embodiment, the subject treated has Hemoglobin Ale levels of 6.5% or higher.
  • a control amount is a value decided or obtained, usually beforehand, as a control.
  • the concept of a control is well-established in the field, and can be determined, in a non-limiting example, empirically from suitable systems, and may be normalized as desired (in non-limiting examples, for volume, mass, location, etc.) to negate the effect of one or more variables.
  • Hyperglycemia-induced overproduction of intracellular ROS initiates many of the complex series of molecular events that result in diabetic tissue damage, and transgenic expression of the antioxidant enzyme superoxide dismutase prevents each complication in experimental diabetes.
  • transient above-threshold spikes of hyperglycemia continue to generate excess ROS after they have ceased and thereby activate damaging mechanisms for days of subsequent normal glycemia. These brief spikes are too short to be reflected in the HbAlc values.
  • HbAlc values In human vascular cells and in mice, several hours' exposure to high glucose activates a positive multicomponent feedback loop which maintains persistent overproduction of oxygen free radicals for days of subsequent exposure to normal glucose concentrations. Disruption of this loop, e.g. by selected peptides as shown herein, rapidly normalizes both the persistent free radical overproduction and its pathologic consequences that otherwise persist for days of subsequent exposure to normal glucose concentrations.
  • GLP-1 (9-36)amide e.g., EGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH 2 (SEQ ID NO: l)
  • GLP-1 (9-36)amide reversed the persistent left shift of the glucose concentration-ROS dose- response curve caused by transient hyperglycemia, and thereby reversed the persistent ROS dependent inactivation of PGI2.
  • GLP-1 (9-36)amide is a cleavage product of the incretin hormone GLP-1 (7- 36)amide.
  • GLP-1 (9-36)amide has been reported to have unique extrapancreatic insulin-like actions in the heart, vasculature, and liver, which appear not to be mediated through the GLP-1 receptor.
  • GLP-1 (9-36)amide activated cardioprotective signaling pathways while GLP-1 receptor agonists did not.

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Abstract

Methods for treating diabetes and prediabetes are provided. Methods for reducing transient hyperglycemia-induced ROS production are provided. Methods of obtaining agents for reducing transient hyperglycemia-induced ROS production are provided.

Description

REVERSAL OF PERSISTENT ROS GENERATION AFTER TRANSIENT
HYPERGLYCEMIA
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of U.S. Provisional Application No. 62/184,495, filed June 25, 2015, the contents of which are hereby incorporated by reference.
STATEMENT OF GOVERNMENT SUPPORT
[0002] This invention was made with government support under grant number DK033861 awarded by the National Institutes of Health. The government has certain rights in the invention.
BACKGROUND OF THE INVENTION
[0003] All publications, patents, patent application publications and books referred to herein are each hereby incorporated by reference in their entirety into the subject application to more fully describe the art to which the subject invention pertains.
[0004] Diabetic and pre-diabetic hyperglycemia continue to be a major cause of blindness, renal failure, heart attacks, and lower limb amputation. Hyperglycemia-induced overproduction of mitochondrial reactive oxygen species (ROS) initiates many of the complex series of molecular events that result in diabetic tissue damage. The assumption underlying current diabetes treatment is that lowering the level of time-averaged glucose concentrations, measured as HbAlc, prevents microvascular complications. However, recent analyses by the DCCT/EDIC study group showed that 89% of variation in risk of retinopathy, microalbuminuria or albuminuria is due to elements of glycemia not captured by mean HbAlc values. The assumption underlying current diabetes treatment that lowering the level of time-averaged glucose concentrations, measured as HbAlc, prevents microvascular complications is limited and improved methods are needed. Technologies targeting diabetic complications mostly target HbAlc values by providing improved insulin secretion or action. Developing technologies include iPSC differentiation, mechanical insulin or insulin-glucagon devices, and a variety of monomelic peptides engineered to be agonists for multiple peptide-hormone receptors. Small molecular weight compounds and peptides that increase tissue sensitivity to insulin action are also in development. None, to the inventor's knowledge, address the long-term effects of brief spikes of hyperglycemia.
[0005] Herein is disclosed a method for reversing persistent reactive oxygen species (ROS) production resulting from transient hyperglycemia in a subject and treating diabetes and prediabetes.
SUMMARY OF THE INVENTION
[0006] A method is provided for obtaining an agent which reduces hyperglycemia- induced ongoing or persistent or post-hyperglycemic spike reactive oxygen species (ROS) production in a cell in a subject, wherein the agent inhibits an ROS production feedback loop,
comprising contacting cells previously exposed to hyperglycemic glucose for over 4 hours followed by exposure to normoglycemic glucose for over 1 day with a candidate agent for a predetermined amount of time, and experimentally quantifying ROS production, VDAC phosphorylation, GSK-3beta activity or Akt activity, and comparing the experimentally quantified value against a control value of ROS production, VDAC phosphorylation, GSK- 3beta activity or Akt activity, respectively, wherein a quantified ROS production, VDAC phosphorylation or GSK-3beta activity below the control value or a quantified Akt activity above the control value, respectively, indicates the candidate agent reduces ongoing ROS production and wherein a quantified ROS production, VDAC phosphorylation or GSK- 3beta activity above the control value or an Akt activity below the control value, respectively, indicates the candidate agent does not reduce ROS production.
[0007] A method is provided for obtaining an agent which reduces hyperglycemia- induced ongoing reactive oxygen species (ROS) production in a cell in a subject, wherein the agent inhibits an ROS production feedback loop,
comprising contacting a system comprising VDAC, a GSK-3beta or an Akt with the candidate agent for a predetermined amount of time, and experimentally quantifying VDAC phosphorylation, GSK-3beta activity or Akt activity, and comparing the experimentally quantified value against a control value of VDAC phosphorylation, GSK-3beta activity or Akt activity, respectively, wherein a quantified VDAC phosphorylation or GSK-3beta activity below the control value or a quantified Akt activity above the control value, respectively, indicates the candidate agent reduces ROS production and wherein a quantified VDAC phosphorylation or GSK-3beta activity above the control value or an Akt activity below the control value, respectively, indicates the candidate agent does not reduce ROS production,
and physically recovering the candidate agent identified as reducing ROS production so as to obtain an agent which reduces hyperglycemia-induced ongoing reactive oxygen species (ROS) production in a cell in a subject.
[0008] Also provided is a method of obtaining an agent which reduces reactive oxygen species (ROS) production in a cell in a subject, wherein the ROS production is associated with a hyperglycemia in a subject, comprising contacting a VDAC, a GSK-3beta or an Akt with the candidate agent for a predetermined amount of time, and experimentally quantifying VDAC phosphorylation, GSK-3beta activity or Akt activity, and comparing the experimentally quantified value against a control value of VDAC phosphorylation, GSK- 3beta activity or an Akt activity, respectively, wherein a quantified VDAC phosphorylation or GSK-3beta activity below the control value or a quantified Akt activity above the control value, respectively, indicates the candidate agent reduces ROS production and wherein a quantified VDAC phosphorylation or GSK-3beta activity above the control value or an Akt activity below the control value, respectively, indicates the candidate agent does not reduce ROS production.
[0009] Also provided is a method of obtaining an agent which reduces reactive oxygen species (ROS) production in a cell in a subject, wherein the ROS production is associated with a hyperglycemia in a subject, comprising contacting system comprising a VDAC and an HKII with the candidate agent for a predetermined amount of time, and experimentally quantifying VDAC and HKII association, and comparing the experimentally quantified value against a control value of VDAC and HKII association, wherein a quantified VDAC and HKII association above the control value indicates the candidate agent reduces ROS production and wherein a quantified VDAC and HKII association below the control value indicates the candidate agent does not reduce ROS production. In an embodiment of the method, the method further comprises physically recovering the candidate agent identified as reducing ROS production so as to obtain an agent which reduces reactive oxygen species (ROS).
[0010] Also provided is a provided of method of obtaining an agent which reduces reactive oxygen species (ROS) production in a cell in a subject, wherein the ROS production is associated with a hyperglycemia in a subject, comprising contacting a human aortic endothelial cell (HAEC) with a candidate agent, wherein the HAEC has been exposed to an elevated glucose concentration for a predetermined amount of time, and experimentally quantifying ROS production by the HAEC, and comparing the quantified ROS production against a control wherein an ROS production below the control value indicates the candidate agent reduces ROS production and wherein an ROS production in excess of the control value indicates the candidate agent does not reduces ROS production, and physically recovering the candidate agent identified as reducing ROS production so as to obtain an agent which reduces ROS production in a cell.
[0011] Also provided is a method of reducing reactive oxygen species (ROS) production in a cell in a subject, wherein the ROS production is associated with a hyperglycemia in a subject, comprising administering to the subject an amount of an agent effective to inhibit reactive oxygen species production by a cell of a subject.
[0012] Also provided is a method of treating diabetes complication in a subject comprising administering to the subject an amount of an agent effective to inhibit reactive oxygen species production by a cell of a subject.
[0013] A method is provided for reducing reactive oxygen species (ROS) production in a cell in a subject, wherein the ROS production is associated with a hyperglycemia in a subject, comprising administering to the subject an amount of an agent effective to activate Aktl, to inhibit PP2A, to inhibit GSK-3beta, to inhibit phosphorylation of VDAC threonine, or to increase association of VDAC with HK-II.
[0014] Also provided is a method for treating diabetes complication in a subject comprising administering to the subject an amount of an agent effective to activate Aktl, to inhibit PP2A, to inhibit GSK-3beta, to inhibit phosphorylation of VDAC threonine, or to increase association of VDAC with HK-II.
[0015] Also provided is a method for treating diabetes or prediabetic hyperglycemia in a subject comprising administering to the subject an amount of an agent effective to the subject an amount of an agent effective to activate Aktl, to inhibit PP2A, to inhibit GSK- 3beta, to inhibit phosphorylation of VDAC threonine, or to increase association of VDAC with HK-II.
BRIEF DESCRIPTION OF THE DRAWINGS [0016] Figure 1A-1D. Transient exposure to high glucose induces persistent mitochondrial ROS production by shifting the glucose concentration-ROS curve to the left. (1A) ROS levels in HAEC exposed to 25mM glucose for 6hrs, (green bar), and 25 mM glucose for 6 hrs followed by 2 days of subsequent incubation in 5mM glucose (red bars). In the indicated groups, cells were infected with MnSOD or control adenoviral vectors before transient exposure to 25mM glucose. (IB) ROS levels in HAEC exposed to 25mM glucose for varying times. (1C) ROS levels after 2 days incubation in 5mM glucose following transient exposure to 25mM glucose for varying times. (ID) Glucose concentration-ROS dose-response curves from HAEC transiently exposed to 25mM glucose (6 hrs) and then to 5mM glucose for 2 days (red line), and from HAEC exposed to 5mM glucose for 6 hrs, continued for 2 days (blue line). ROS levels were measured by CMH2DCFDA. Data are the mean +/- S.E.M from 3 independent experiments with at least 8 technical replicates. * p<0.05.
[0017] Figure 2A-2H. Persistent mitochondrial ROS production after transient exposure to high glucose is maintained by activation of a multi-component feedback loop. (2A) Inner mitochondrial transmembrane potential and ROS level in HAEC after transient exposure to 25mM glucose or dissociation of HK-II from VDAC with the cell-permeable HK-II N- terminal peptide HK-TAT in 5mM glucose. Upper panel: Representative photomicrographs of increased JC-1 red fluorescence indicating increased mitochondrial membrane potential. Lower panel: ROS production measured by CM-H2DCFDA. (2B-G) HAEC were exposed to 25 mM glucose for the indicated times with (red bars) or without (green bars) subsequent incubation in 5 mM glucose for 2 days. Cells exposed to 5mM glucose were used as controls (blue bars). (2B) HK-II/VDAC association after transient exposure of HAEC to high glucose, after 2 days of 5mM glucose following 6 hrs of 25mM glucose, and after 6 hrs exposure to 25mM in the presence of a peptide inhibitor of GSK-3 . Upper panel: IP:WB of VDAC VDAC and VDAC:HK-II. Lower panel: Quantitation of IP-WB data from upper panel. (2C) VDAC phosphorylation after transient exposure of HAEC to high glucose, after 2 days of 5mM glucose following 6 hrs of 25mM glucose, and after 6 hrs exposure to 25mM in the presence of a peptide inhibitor of GSK-3 . Upper panel: IP:WB of VDACVDAC and VDAC:P-Thr. Lower panel: Quantitation of IP-WB data from upper panel. (2D)GSK-3 activity after transient exposure of HAEC to high glucose, after 2 days of 5mM glucose following 6 hrs of 25mM glucose, and after 6 hrs exposure to 25mM in the presence of Adenoviral constitutively active Aktl. Activity was measured by 32P incorporation into a GSK-3 -specific substrate. (2E) Aktl activity after transient exposure of HAEC to high glucose, after 2 days of 5mM glucose following 6 hrs of 25mM glucose, and after 6 hrs exposure to 25mM in the presence of PP2A inhibitors. (2F) PP2A activity after transient exposure of HAEC to high glucose, after 2 days of 5mM glucose following 6 hrs of 25mM glucose, and after 6 hrs exposure to 25mM in the presence of either Ad- MnSOD, Ad-catalase, or deferoxamine. (2G) Free iron concentration after transient exposure of HAEC to high glucose, after 2 days of 5mM glucose following 6 hrs of 25mM glucose, and after 6 hrs exposure to 25mM in the presence of Ad-MnSOD. Free iron in HAEC was measured by Electron Paramagnetic Spectroscopy (EPR). (2H) Effect of Akt- DN or GSK-3 -CA on inner mitochondrial transmembrane potential and ROS level in HAEC in 5mM glucose. HAEC were exposed to 5mM glucose for 6 hrs after infection with adenoviral vectors expressing either Akt-DN, GSK-3 -CA, or vector alone. Upper panel: Representative photomicrographs of increased JC-1 red fluorescence indicating increased mitochondrial membrane potential. Lower panel: ROS production measured by CM- H2DCFDA. ROS data are the mean +/- S.E.M from 3 independent experiments with at least 8 technical replicates. All data shown are the mean +/- S.D. from 5 independent experiments. * p<0.05.
[0018] Figure 3. Schematic representation of the multi-component feedback loop which maintains persistently increased ROS production after transient exposure to high glucose. Transient exposure to high glucose induces a transient increase in ETC flux increasing ΔΨ and thereby increasing mitochondrial superoxide production. In the mitochondria, this superoxide causes release of Fe2+ from ferritin and iron sulfur cluster-containing proteins. This released free iron reacts with diffused superoxide-derived hydrogen peroxide to form hydroxyl radicals, which increase PP2A activity. Activated PP2A then dephosphorylates Aktl, decreasing its activity. Decreased Aktl activity increases GSK-3 activity, which then increases VDAC threonine phosphorylation. Increased VDAC Thr phosphorylation decreases HK-II association with VDAC, which increases the ΔΨ at physiologic levels of glucose. Operation of this loop causes a stable left-shift of the glucose concentration-ROS dose-response curve, thereby maintaining increased ROS levels at normal glucose levels for days after transient exposure of cells to high glucose. [0019] Figure 4A-4F. GLP-1 (9-36)amide reverses the persistent left shift of the glucose concentration-ROS dose-response curve caused by transient hyperglycemia. (4A) Glucose concentration-ROS dose-response curves. Glucose concentration-ROS dose response curve from HAEC exposed to 12mM for 6 hrs followed by 2 days of 5mM glucose, and then transiently treated with a peptide GSK-3 inhibitor (gold curve). This curve is shown in comparison to the glucose concentration-ROS curves from cells exposed to 5mM glucose for 2 days (blue curve), and the curve from cells exposed to 6 hrs 12mM glucose followed by 5mM glucose for 2 days (red curve) from Figure ID. ROS levels were measured by CM- H2DCFDA. ROS data are the mean +/- S.E.M from 3 independent experiments with at least 8 technical replicates. * p<0.05. (4B-4E) Effect of GLP-1 (9-36)amide on feedback loop components. HAEC were exposed to 12 mM glucose for 6 hrs with or without 100 pM GLP-1 (9-36)amide. Cells exposed to 5mM glucose were used as controls (blue bars). (4B) HK-II/VDAC association. A peptide inhibitor of GSK-3 was used as control (4C) VDAC phosphorylation: Quantitation of IPWB data. (4D) GSK-3 phosphorylation. Upper panel: IP:WB of GSK-3 : GSK-3 and GSK-3 :p-GSK-3 (S9) . Lower panel: Quantitation of IPWB data from upper panel. (4E) Aktl activity. All data shown are the mean +/- S.D. from 5 independent experiments. * pO.01. (F) Effect of GLP-1 (9-36)amide on glucose concentration-ROS dose-response curves. Glucose concentration-ROS dose-response curve from HAEC exposed to 25mM for 6 hrs followed by 2 days of 5mM glucose, and then transiently treated with GLP-1 (9-36)amide (green curve). This curve has been superimposed on the glucose concentration-ROS curves from Figure 4A. ROS levels were measured by CM-H2DCFDA. ROS data are the mean +/- S.E.M from 3 independent experiments with at least 8 technical replicates. * p<0.05. The data show prevention of the original high glucose-generated ROS.
[0020] Figure 5. GLP-1 (9-36)amide reverses the persistent reduction of prostacyclin synthase caused by transient hyperglycemia in vivo. (Upper panel: Experiment schematic. Lower panel: C57B16 mice (n=5 /group) received 4 sequential injections of glucose (3 g/kg IP) (red bars) or an equivalent volume of 0.9% saline (blue bar) at 2 hr intervals. Two days after transient hyperglycemia a micro-osmotic pump with GLP-1 (9-36)amide (300μg/ml) was inserted in the mice for 24 hours and then removed. Aortas were removed and prostacyclin synthase activity measured in all groups 6 days after initial glucose or saline injections. Data are shown as mean +/- S.D. * pO.01. DETAILED DESCRIPTION OF THE INVENTION
[0021] Herein it is disclosed that treatment with a peptide (non-hypoglycemic), or small molecule drug, that does not affect glycemia can disrupt and reverse the damaging ROS- generating loop. In patients, this can significantly reduce the duration of exposure to the damaging consequences of hyperglycemia, and thereby reduce the rate at which diabetes- associated tissue damage develops. It is disclosed herein that ROS overproduction during hyperglycemia involves increased glucose metabolism causing increased mitochondrial electron leak, which can also activate other ROS -generating proteins such as NADPH oxidases and monomeric ENOS. However, when glucose levels are normal, there is no increased glucose metabolism in target cells of complications. That increased ROS production can persist during normal glycemia after transient spikes of hyperglycemia was previously unknown, as was the feed-forward loop which causes this. Treatment of this continuous ROS overproduction existing during normoglycemia, but subsequent to post- hyperglyciemic spikes, is a novel intervention.
[0022] A method is provided for obtaining an agent which reduces hyperglycemia- induced ongoing reactive oxygen species (ROS) production in a cell in a subject, wherein the agent inhibits an ROS production feedback loop,
comprising contacting cells previously exposed to hyperglycemic glucose for over 4 hours followed by exposure to normoglycemic glucose for over 1 day with a candidate agent for a predetermined amount of time, and experimentally quantifying ROS production, VDAC phosphorylation, GSK-3beta activity or Akt activity, and comparing the experimentally quantified value against a control value of ROS production, VDAC phosphorylation, GSK- 3beta activity or Akt activity, respectively, wherein a quantified ROS production, VDAC phosphorylation or GSK-3beta activity below the control value or a quantified Akt activity above the control value, respectively, indicates the candidate agent reduces ongoing ROS production and wherein a quantified ROS production, VDAC phosphorylation or GSK- 3beta activity above the control value or an Akt activity below the control value, respectively, indicates the candidate agent does not reduce ROS production.
[0023] A method is provided for obtaining an agent which reduces hyperglycemia- induced ongoing reactive oxygen species (ROS) production in a cell in a subject, wherein the agent inhibits an ROS production feedback loop, comprising contacting cells previously exposed to hyperglycemic glucose for over 4 hours followed by exposure to normoglycemic glucose for over 1 day with the candidate agent for a predetermined amount of time, and experimentally quantifying ROS, VDAC phosphorylation, GSK-3beta activity or Akt activity, and comparing the experimentally quantified value against a control value of ROS,VDAC phosphorylation, GSK-3beta activity or Akt activity, respectively, wherein a quantified ROS, VDAC phosphorylation or GSK-3beta activity below the control value or a quantified Akt activity above the control value, respectively, indicates the candidate agent reduces ongoing ROS production and wherein a quantified ROS, VDAC phosphorylation or GSK-3beta activity above the control value or an Akt activity below the control value, respectively, indicates the candidate agent does not reduce ROS production,
and physically recovering the candidate agent identified as reducing ongoing ROS production so as to obtain an agent which reduces hyperglycemia-induced ongoing reactive oxygen species (ROS) production in a cell in a subject. In an embodiment, the cell is a vascular cell.
[0024] In an embodiment, candidate agents are selected from a peptide library. In an embodiment, candidate agents are selected from a peptide library wherein the peptiodes are from 5 to 40 amino acids in length.
[0025] Hyperglycemic glucose and normoglycemic glucose are easily determined by the species of mammal the cell being tested. In an embodiment, the cells are human cells, and the hyperglycemic glucose and normoglycemic glucose are hyperglycemic glucose for a human and normoglycemic glucose for a human. In an embodiment, the cells are previously exposed to hyperglycemic glucose for 5.75 - 6.25 hours. In an embodiment, the cells are previously exposed to hyperglycemic glucose for 6 hours. In an embodiment, the cells are exposed to normoglycemic glucose for over 1.5 days with the candidate agent. In an embodiment, the cells are exposed to normoglycemic glucose for 2.0 days with the candidate agent.
[0026] A method is provided for obtaining an agent which reduces hyperglycemia- induced ongoing reactive oxygen species (ROS) production in a cell in a subject, wherein the agent inhibits an ROS production feedback loop,
comprising contacting a syetem comprising VDAC, a GSK-3beta or an Akt with the candidate agent for a predetermined amount of time, and experimentally quantifying VDAC phosphorylation, GSK-3beta activity or Akt activity, and comparing the experimentally quantified value against a control value of VDAC phosphorylation, GSK-3beta activity or Akt activity, respectively, wherein a quantified VDAC phosphorylation or GSK-3beta activity below the control value or a quantified Akt activity above the control value, respectively, indicates the candidate agent reduces ROS production and wherein a quantified VDAC phosphorylation or GSK-3beta activity above the control value or an Akt activity below the control value, respectively, indicates the candidate agent does not reduce ROS production,
and physically recovering the candidate agent identified as reducing ROS production so as to obtain an agent which reduces hyperglycemia-induced ongoing reactive oxygen species (ROS) production in a cell in a subject. In an embodiment, the cell is a vascular cell.
[0027] In an embodiment the VDAC, a GSK-3beta or an Akt is contacted with the candidate agent.
[0028] Also provided is a method of obtaining an agent which reduces reactive oxygen species (ROS) production in a cell in a subject, wherein the ROS production is associated with a hyperglycemia in a subject, comprising contacting a VDAC, a GSK-3beta or an Akt with the candidate agent for a predetermined amount of time, and experimentally quantifying VDAC phosphorylation, GSK-3beta activity or Akt activity, and comparing the experimentally quantified value against a control value of VDAC phosphorylation, GSK- 3beta activity or an Akt activity, respectively, wherein a quantified VDAC phosphorylation or GSK-3beta activity below the control value or a quantified Akt activity above the control value, respectively, indicates the candidate agent reduces ROS production and wherein a quantified VDAC phosphorylation or GSK-3beta activity above the control value or an Akt activity below the control value, respectively, indicates the candidate agent does not reduce ROS production. In an embodiment, the cell is a vascular cell.
[0029] In an embodiment of the methods, the method further comprises physically recovering the candidate agent identified as reducing ROS production so as to obtain an agent which reduces hyperglycemia-induced ongoing ROS production in a vascular cell or an agent which reduces ROS production in a vascular cell, respectively.
[0030] In an embodiment of the methods, VDAC phosphorylation, GSK-3beta activity or Akt activity are experimentally quantified using Western blot and/or immunoprecipitation. [0031] In an embodiment of the methods, the candidate agent is a peptide. In an embodiment of the methods, the candidate agent is a peptide of less than 20 amino acid residues. In an embodiment of the methods, the candidate agent is a peptide of less than 10 amino acid residues.
[0032] In an embodiment of the methods, the candidate agent is a small organic molecule of 1,500 daltons or less.
[0033] In an embodiment of the methods, the hyperglycemia of the hyperglycemia- induced ongoing ROS production or the hyperglycemia associated with the ROS production is transient. In an embodiment of the methods, the hyperglycemia is from about 4 to 6 hours. In an embodiment of the methods, the hyperglycemia is undetectable using HbAlc quantification. In an embodiment of the methods, the hyperglycemia does not increase HbAlc production in the subject.
[0034] In an embodiment of the methods, the candidate the agent does not affect glycemia in the subject. In an embodiment of the methods, the candidate the agent does not affect insulin production or affect insulin activity in a subject.
[0035] Also provided is a method of obtaining an agent which reduces reactive oxygen species (ROS) production in a cell in a subject, wherein the ROS production is associated with a hyperglycemia in a subject, comprising contacting system comprising a VDAC and an HKII with the candidate agent for a predetermined amount of time, and experimentally quantifying VDAC and HKII association, and comparing the experimentally quantified value against a control value of VDAC and HKII association, wherein a quantified VDAC and HKII association above the control value indicates the candidate agent reduces ROS production and wherein a quantified VDAC and HKII association below the control value indicates the candidate agent does not reduce ROS production. In an embodiment of the method, the method further comprises physically recovering the candidate agent identified as reducing ROS production so as to obtain an agent which reduces reactive oxygen species (ROS). In an embodiment, the cell is a vascular cell.
[0036] Also provided is a provided of method of obtaining an agent which reduces reactive oxygen species (ROS) production in a cell in a subject, wherein the ROS production is associated with a hyperglycemia in a subject, comprising contacting a human aortic endothelial cell (HAEC) with a candidate agent, wherein the HAEC has been exposed to an elevated glucose concentration for a predetermined amount of time, and experimentally quantifying ROS production by the HAEC, and comparing the quantified ROS production against a control wherein an ROS production below the control value indicates the candidate agent reduces ROS production and wherein an ROS production in excess of the control value indicates the candidate agent does not reduces ROS production, and physically recovering the candidate agent identified as reducing ROS production so as to obtain an agent which reduces ROS production in a cell. In an embodiment, the cell is a vascular cell.
[0037] In an embodiment, the HAEC has been obtained from a diabetic human. In an embodiment, the HAEC has been obtained from a non-diabetic human.
[0038] In an embodiment, the elevated glucose concentration is equivalent to over 190 mg/dL blood. In an embodiment, the elevated glucose concentration is equivalent to over 210 mg/dL blood. In an embodiment, the elevated glucose concentration is equivalent over 20 mM. In an embodiment, the elevated glucose concentration is 23 - 27 mM. In an embodiment, the elevated glucose concentration is 25 mM.
[0039] In an embodiment, the candidate agent is a peptide. In an embodiment, the candidate agent is a peptide of less than 20 amino acid residues. In an embodiment, the candidate agent is a peptide of less than 10 amino acid residues. In an embodiment, the candidate agent is a small organic molecule of 1,500 daltons or less. In an embodiment, the candidate agent is a small organic molecule of 1,000 daltons or less. In an embodiment, the candidate agent is a small organic molecule of 500 daltons or less.
[0040] In an embodiment, ROS levels are quantified using CM-H2DCFDA. In an embodiment, ROS levels are quantified using fluorimetry.
[0041] Also provided is a method of reducing reactive oxygen species (ROS) production in a cell in a subject, wherein the ROS production is associated with a hyperglycemia in a subject, comprising administering to the subject an amount of an agent effective to inhibit reactive oxygen species production by a cell of a subject. In an embodiment, the cell is a vascular cell.
[0042] Also provided is a method of reducing reactive oxygen species (ROS) production in a cell in a subject, wherein the ROS production is fatty acid-induced ROS production, comprising administering to the subject an amount of an agent effective to inhibit reactive oxygen species production by a cell of a subject. In an embodiment, the cell is a vascular cell. [0043] Also provided is a method of treating diabetes complication in a subject comprising administering to the subject an amount of an agent effective to inhibit reactive oxygen species production by a cell of a subject.
[0044] Normal fasting plasma glucose (FPG) is generally understood to be < 100 mg/dL (5.6 mmol/L). Two-hour glucose during oral glucose tolerance test (OGTT) has blood glucose <140 mg/dL (7.8 mmol/L). In an embodiment of the methods, the subject has an increased risk for diabetes. Subjects with increased risk for diabetes include those with impaired fasting glcose (IFG) - which is generally understood to be a fasting plasma glucose of between 100 and 125 mg/dL (5.6 to 6.9 mmol/L). Subjects with increased risk for diabetes also include those with impaired glucose tolerance (IGT) - a two-hour plasma glucose value during a 75 g oral glucose tolerance test between 140 and 199 mg/dL (7.8 to 11.0 mmol/L). In an embodiment of the methods, the subject has diabetes type 2 diabetes or diabtes mellitus. Subjects with diabetes mellitus have an FPG at or above 126 mg/dL (7.0 mmol/L), a two-hour value in an OGTT (2-h PG) at or above 200 mg/dL (11.1 mmol/L), or a random (or "casual") plasma glucose concentration >200 mg/dL (11.1 mmol/L) in the presence of symptoms. In an embodiment, the subject has near normal FPG. In an embodiment, the subject has diabetes. "Near normal" FPG in someone who already has diabetes would be 100-145 or 150 mg/dL, while 200-250 would be quite high but not unusual in many ambulatory diabetic patients.
[0045] In an embodiment, the cell is a vascular cell. In an embodiment, the complication is a retinopathy, an albuminuria or a microalbuminuria. In an embodiment, the complication is a peripheral neuropathy, a non-healing diabetic foot ulcer, a cognitive decline, a diabetic cardiomyopathy, an atherosclerosis, or a post-myocardial infarction arrhythmia.
[0046] A method of reducing reactive oxygen species (ROS) production in a cell in a subject, wherein the ROS production is fatty acid-induced ROS, comprising administering to the subject an amount of an agent effective to activate Aktl, to inhibit PP2A, to inhibit GSK-3beta, to inhibit phosphorylation of VDAC threonine, or to increase association of VDAC with HK-II.
[0047] A method of treating diabetes or prediabetic hyperglycemia in a subject comprising: a) receiving an identification of the subject as having a persistent or an ongoing fatty acid- induced ROS;
b) administering to the subject so identified an amount of an agent effective to inhibit reactive oxygen species production by a cell of a subject and thereby treat the diabetes or prediabetic hyperglycemia.
[0048] Also provided is a method of treating diabetes or prediabetic hyperglycemia in a subject comprising administering to the subject an amount of an agent effective to inhibit reactive oxygen species production by a cell of a subject. In an embodiment, the cell is a vascular cell.
[0049] Also provided is a method of preventing or reducing development of diabetes, or preventing or reducing development of prediabetic hyperglycemia, in a subject comprising administering to the subject an amount of an agent effective to inhibit reactive oxygen species production by a cell of a subject. In an embodiment, the cell is a vascular cell.
[0050] In an embodiment, the method is for treating diabetes. In an embodiment, the method is for treating prediabetic hyperglycemia. In an embodiment, the agent comprises a peptide.
[0051] In an embodiment of the methods, the peptide is a non-hypoglycemic peptide. In an embodiment, the agent comprises glucagon like peptide- 1 (9-36) amide. In an embodiment, the agent does not comprise glucagon like peptide-1 (9-36) amide. In an embodiment, the glucagon like peptide-1 (9-36) amide has the sequence EGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH2 (SEQ ID NO: l). In an embodiment, the agent comprises glucagon like peptide-1 (9-37). In an embodiment, the agent does not comprise glucagon like peptide-1 (9-37). In an embodiment, the glucagon like peptide-1 (9- 37) has the sequence EGTFTSDVSSYLEGQAAKEFIAWLVKGRG.
[0052] In an embodiment, the agent is directly administered to a retinal tissue for treating the retinopathy.
[0053] In an embodiment, the agent comprises glucagon like peptide-1 (9-36) amide, the agent comprises a small organic molecule.
[0054] In an embodiment, the agent is administered during or subsequent to a hyperglycemic event. In an embodiment, the agent is administered by self-administration. In an embodiment, the agent is administered continuously by a pump. [0055] Also provided is a method of treating diabetes or prediabetic hyperglycemia in a subject comprising administering to the subject an amount of an agent effective to the subject an amount of an agent effective to activate Aktl, to inhibit PP2A, to inhibit GSK- 3beta, to inhibit phosphorylation of VDAC threonine , or to increase association of VDAC with HK-II.
[0056] In an embodiment, the subject is a human.
[0057] Also provided is a method of treating diabetes or prediabetic hyperglycemia in a subject comprising:
a) receiving an identification of the subject as having a persistent or an ongoing ROS production under normoglycemic conditions after a hyperglycemia spike;
b) administering to the subject so identified an amount of an agent effective to inhibit reactive oxygen species production by a cell of a subject and thereby treat the diabetes or prediabetic hyperglycemia.
[0058] A method of treating diabetes or prediabetic hyperglycemia in a subject comprising:
a) receiving an identification of the subject as having a hyperglycemic spike in their blood glucose;
b) administering to the subject so identified an amount of an agent effective to inhibit persistent or ongoing ROS production under normoglycemic conditions after a
hyperglycemia spike and thereby treat the diabetes or prediabetic hyperglycemia.
[0059] In an embodiment of the methods, the agent comprises a peptide. In an embodiment, the agent comprises glucagon like peptide- 1 (9-36) amide. In an embodiment, the agent comprises glucagon like peptide- 1 (9-37). In an embodiment, the agent comprises a small organic molecule. In an embodiment, the agent is administered during or subsequent to a hyperglycemic event.
[0060] In an embodiment, the agent is administered by self-administration. In an embodiment, the agent is administered continuously by a pump. In an embodiment, the amount of agent administered decreases Akt inhibition in the vascular cells of the subject. In an embodiment, the amount of agent administered reduces the extent of VDAC phosphorylation associated with hyperglycemia in the subject. In an embodiment, the amount of agent administered reduces the extent of GSK-3P activation associated with hyperglycemia in the subject. In an embodiment, the amount of agent administered reduces the extent of PGI2 inactivation associated with hyperglycemia induced ROS production in the subject.
[0061] In an embodiment, the subject is identified as undergoing a hyperglycemic spike by means of a blood glucose monitor.
[0062] In an embodiment, the agent is administered within 5 minutes of a hyperglycemia threshold being crossed. A hyperglycemia threshold is crossed when the glucose level in a subject's blood exceeds a predetermined threshold value, which is indicative of hyperglycemia. In an embodiment, the hyperglycemia threshold is l lmmol/L. In an embodiment, the hyperglycemia threshold is 12mmol/L. In an embodiment, the hyperglycemia threshold is 13mmol/L. In an embodiment, the hyperglycemia threshold is 14mmol/L. In an embodiment, the hyperglycemia threshold is 15mmol/L. In an embodiment, the hyperglycemia threshold is 16mmol/L. In an embodiment, the hyperglycemia threshold is 17mmol/L. In an embodiment, the hyperglycemia threshold is 18mmol/L. In an embodiment, the hyperglycemia threshold is 19mmol/L. In an embodiment, the hyperglycemia threshold is 20mmol/L. In an embodiment, the hyperglycemia threshold is 25mmol/L. In an embodiment, the agent is administered within 10 minutes of a hyperglycemia threshold being crossed. In an embodiment, the agent is administered within 15 minutes of a hyperglycemia threshold being crossed. In an embodiment, the agent is administered within 20 minutes of a hyperglycemia threshold being crossed. In an embodiment, the agent is administered within 25 minutes of a hyperglycemia threshold being crossed. In an embodiment, the agent is administered within 30 minutes of a hyperglycemia threshold being crossed.
[0063] In an embodiment, wherein the subject is a human.
[0064] In an embodiment, the method is for treating diabetes. In an embodiment, the method is for treating prediabetic hyperglycemia.
[0065] Administration, as in the methods described herein, unless otherwise specified can be one of, or any combination of, auricular, buccal, conjunctival, cutaneous, subcutaneous, endocervical, endosinusial, endotracheal, enteral, epidural, via hemodialysis, interstitial, intrabdominal, intraamniotic, intra-arterial, intra-articular, intrabiliary, intrabronchial, intrabursal, intracardiac, intracartilaginous, intracaudal, intracavernous, intracavitary, intracerebral, intracisternal, intracorneal, intracoronary, intradermal, intradiscal, intraductal, intraepidermal, intraesophagus, intragastric, intravaginal, intragingival, intraileal, intraluminal, intralesional, intralymphatic, intramedullary, intrameningeal, intramuscular, intraocular, intraovarian, intraepicardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrasinal, intraspinal, intrasynovial, intratendinous, intratesticular, intrathecal, intrathoracic, intratubular, intratumor, intratympanic, intrauterine, intravascular, intravenous, intraventricular, intravesical, intravitreal, laryngeal, nasal, nasogastric, ophthalmic, oral, oropharyngeal, parenteral, percutaneous, periarticular, peridural, rectal, inhalationally, retrobulbar, subarachnoid, subconjuctival, sublingual, submucosal, topically, transdermal, transmucosal, transplacental, transtracheal, ureteral, uretheral, and vaginal.
[0066] In an embodiment, the amount of agent administered decreases Akt inhibition in the vascular cells of the subject. In an embodiment, the amount of agent administered reduces the extent of VDAC phosphorylation associated with hyperglycemia in the subject. In an embodiment, the amount of agent administered reduces the extent of GSK-3P activation associated with hyperglycemia in the subject. In an embodiment, the amount of agent administered reduces the extent of PGI2 inactivation associated with hyperglycemia induced ROS production in the subject. In an embodiment of the methods of treatment, the hyperglycemia is undetectable using HbAlc quantification. In an embodiment of the methods, the hyperglycemia does not increase HbAlc production in the subject.
[0067] In an embodiment of the methods disclosed herein, the ROS production is persistent ROS production following brief spike(s) of hyperglycemia.
[0068] A method is provided for reducing reactive oxygen species (ROS) production in a cell in a subject, wherein the ROS production is associated with a hyperglycemia in a subject, comprising administering to the subject an amount of an agent effective to activate Aktl, to inhibit PP2A, to inhibit GSK-3beta, to inhibit phosphorylation of VDAC threonine, or to increase association of VDAC with HK-II. Also provided is a method is provided for reducing reactive oxygen species (ROS) production in a cell in a subject, wherein the ROS production is associated with a hyperglycemia in a subject, comprising administering to the subject an amount of an agent effective to normalize Aktl activity, to normalize PP2A activity, to normalize GSK-3beta activity, to normalize phosphorylation of VDAC threonine, or to normalize association of VDAC with HK-II. To normalize as used herein means to bring the activity or association, as relevant, within the range of that seen in a normal, non-diabetic individual. [0069] Also provided is a method for treating a diabetes complication in a subject comprising administering to the subject an amount of an agent effective to activate Aktl, to inhibit PP2A, to inhibit GSK-3beta, to inhibit phosphorylation of VDAC threonine, or to increase association of VDAC with HK-II. In an embodiment, the complication is a retinopathy, an albuminuria or a microalbuminuria.
[0070] Also provided is a method for treating diabetes or prediabetic hyperglycemia in a subject comprising administering to the subject an amount of an agent effective to activate Aktl, to inhibit PP2A, to inhibit GSK-3beta, to inhibit phosphorylation of VDAC threonine, or to increase association of VDAC with HK-II.
[0071] In an embodiment of the methods, the subject is a human.
[0072] Also provided is a method of obtaining an agent which reduces reactive oxygen species (ROS) production in a cell in a subject, wherein the ROS production is persistent ROS production subsequent to a hyperglycemic spike in a subject, comprising
administering the agent to a non-human animal with the agent and inducing a hypercglycemic spike in the animal,
quantifying the ROS production in the animal during the normoglycemia immediately subsequent to the subsequent to the hyperglycemic spike,
and comparing the experimentally quantified value against a control value of ROS production during the normoglycemia subsequent to the hyperglycemic spike,
wherein a quantified ROS production during the normoglycemia below the control value indicates the candidate agent reduces ROS production and wherein a quantified ROS production during the normoglycemia above the control value indicates the candidate agent does not reduce ROS production. In an embodiment, the method further comprises physically recovering the candidate agent identified as reducing ROS production so as to obtain an agent which reduces hyperglycemia-induced ongoing ROS production. In an embodiment, the non-human animal is a diabetes-model animal. In an embodiment, the animal is a rat or a mouse. In an embodiment, it is vascular cell ROS production that is quantified.
[0073] As used herein, "treating" a diabetes means that one or more symptoms of the disease, such as the diabetes itself, or a resultant symptom of the diabetes such as blindness, heart damage, lower limb ischemia or other parameters by which the disease is characterized, are reduced, ameliorated, inhibited, placed in a state of remission, or maintained in a state of remission. In an embodiment, the method inhibits further development of the diabetes. In an embodiment, the method inhibits further development of a pathology that results from the diabetes. In an embodiment of the methods, the diabetes is Type I diabetes. In an embodiment of the methods, the diabetes is Type II diabetes. This definition of treating a diabetes, mutatis mutandis, can apply to treating a diabetic complication, such as a retinopathy, albuminuria or microalbuminuria.
[0074] As used herein, "treating" a prediabetes hyperglycemia means that one or more symptoms of the disease, such as ROS generation, or a damage resulting from hyperglycemia induced ROS generation or other parameters by which the disease is characterized, are reduced, ameliorated, inhibited, or maintained in a state of prediabetes. In an embodiment, the method inhibits further development of the prediabetes into diabetes. In an embodiment, the method inhibits further development of a pathology that results from the prediabetes hyperglycemia.
[0075] In an embodiment, the subject treated has Hemoglobin Ale levels between 5.7% and 6.4% indicate increased risk of diabetes. In an embodiment, the subject treated has Hemoglobin Ale levels of 6.5% or higher.
[0076] As used herein, a control amount is a value decided or obtained, usually beforehand, as a control. The concept of a control is well-established in the field, and can be determined, in a non-limiting example, empirically from suitable systems, and may be normalized as desired (in non-limiting examples, for volume, mass, location, etc.) to negate the effect of one or more variables.
[0077] This invention will be better understood from the examples follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims that follow thereafter.
Example 1
[0078] Hyperglycemia-induced overproduction of intracellular ROS initiates many of the complex series of molecular events that result in diabetic tissue damage, and transgenic expression of the antioxidant enzyme superoxide dismutase prevents each complication in experimental diabetes. [0079] Herein it is shown that transient above-threshold spikes of hyperglycemia continue to generate excess ROS after they have ceased and thereby activate damaging mechanisms for days of subsequent normal glycemia. These brief spikes are too short to be reflected in the HbAlc values. In human vascular cells and in mice, several hours' exposure to high glucose activates a positive multicomponent feedback loop which maintains persistent overproduction of oxygen free radicals for days of subsequent exposure to normal glucose concentrations. Disruption of this loop, e.g. by selected peptides as shown herein, rapidly normalizes both the persistent free radical overproduction and its pathologic consequences that otherwise persist for days of subsequent exposure to normal glucose concentrations.
[0080] Since hyperglycemia-induced overproduction of mitochondrial ROS also initiates many of the other complex series of molecular events that result in diabetic tissue damage, these data suggest that hyperglycemic spikes high enough to activate persistent ROS production during subsequent periods of normal glycemia but too brief to affect the hemoglobin Ale value are a major determinant of the 89% of diabetic complications risk not captured by hemoglobin Ale.
[0081] In the present study, GLP-1 (9-36)amide (e.g., EGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH2 (SEQ ID NO: l)) was shown to prevent the excess VDAC phosphorylation induced by transient hyperglycemia, the excess activation of GSK-3 responsible for this, the decreased association of HKII with VDAC, and to prevent the inhibition of Akt activity caused by transient hyperglycemia. As a result, GLP-1 (9-36)amide reversed the persistent left shift of the glucose concentration-ROS dose- response curve caused by transient hyperglycemia, and thereby reversed the persistent ROS dependent inactivation of PGI2.
[0082] GLP-1 (9-36)amide is a cleavage product of the incretin hormone GLP-1 (7- 36)amide. GLP-1 (9-36)amide has been reported to have unique extrapancreatic insulin-like actions in the heart, vasculature, and liver, which appear not to be mediated through the GLP-1 receptor. In human endothelial cells, GLP-1 (9-36)amide activated cardioprotective signaling pathways while GLP-1 receptor agonists did not.
[0083] The data presented here in Figs. 1-5 suggest that the 89% of variation in risk of diabetic microalbuminuria or albuminuria that is not captured by mean HbAlc values in the DCCT cohort may reflect transient above-threshold spikes of hyperglycemia which continue to activate damaging mechanisms for days of subsequent near-normal glycemia. The phenomenon and mechanism described in this study provide a basis for novel therapeutic agents, including GLP-1 (9-36)amide, for the prevention and treatment of diabetic and prediabetic complications.

Claims

What is claimed is:
1. A method for obtaining an agent which reduces hyperglycemia-induced ongoing reactive oxygen species (ROS) production in a cell in a subject, wherein the agent inhibits an ROS production feedback loop,
comprising contacting cells previously exposed to hyperglycemic glucose for over 4 hours followed by exposure to normoglycemic glucose for over 1 day with a candidate agent for a predetermined amount of time, and experimentally quantifying ROS production, VDAC phosphorylation, GSK-3beta activity or Akt activity, and comparing the experimentally quantified value against a control value of ROS production, VDAC phosphorylation, GSK- 3beta activity or Akt activity, respectively, wherein a quantified ROS production, VDAC phosphorylation or GSK-3beta activity below the control value or a quantified Akt activity above the control value, respectively, indicates the candidate agent reduces ongoing ROS production and wherein a quantified ROS production, VDAC phosphorylation or GSK- 3beta activity above the control value or an Akt activity below the control value, respectively, indicates the candidate agent does not reduce ROS production.
2. The method of Claim 1, further comprising physically recovering the candidate agent identified as reducing ongoing ROS production so as to obtain an agent which reduces hyperglycemia-induced ongoing reactive oxygen species (ROS) production in a cell in a subject.
3. A method of obtaining an agent which reduces hyperglycemia-induced ongoing reactive oxygen species (ROS) production in a cell in a subject, wherein the agent inhibits an ROS production feedback loop,
comprising contacting a sytem comprising VDAC, a GSK-3beta or an Akt with the candidate agent for a predetermined amount of time, and experimentally quantifying VDAC phosphorylation, GSK-3beta activity or Akt activity, and comparing the experimentally quantified value against a control value of VDAC phosphorylation, GSK-3beta activity or Akt activity, respectively, wherein a quantified VDAC phosphorylation or GSK-3beta activity below the control value or a quantified Akt activity above the control value, respectively, indicates the candidate agent reduces ROS production and wherein a quantified VDAC phosphorylation or GSK-3beta activity above the control value or an Akt activity below the control value, respectively, indicates the candidate agent does not reduce ROS production,
and physically recovering the candidate agent identified as reducing ROS production so as to obtain an agent which reduces hyperglycemia-induced ongoing reactive oxygen species (ROS) production in a cell in a subject.
4. A method of obtaining an agent which reduces reactive oxygen species (ROS) production in a cell in a subject, wherein the ROS production is associated with a hyperglycemia in a subject, comprising contacting a VDAC, a GSK-3beta or an Akt with the candidate agent for a predetermined amount of time, and experimentally quantifying VDAC phosphorylation, GSK-3beta activity or Akt activity, and comparing the experimentally quantified value against a control value of VDAC phosphorylation, GSK- 3beta activity or an Akt activity, respectively, wherein a quantified VDAC phosphorylation or GSK-3beta activity below the control value or a quantified Akt activity above the control value, respectively, indicates the candidate agent reduces ROS production and wherein a quantified VDAC phosphorylation or GSK-3beta activity above the control value or an Akt activity below the control value, respectively, indicates the candidate agent does not reduce ROS production.
5. The method of Claim 4, further comprising physically recovering the candidate agent identified as reducing ROS production so as to obtain an agent which reduces hyperglycemia-induced ongoing ROS production in a vascular cell or an agent which reduces ROS production in a vascular cell, respectively.
6. The method of any of Claims 1-5, wherein VDAC phosphorylation, GSK-3beta activity or Akt activity are experimentally quantified using Western blot and/or immunoprecipitation.
7. The method of any of Claims 1-5, wherein the candidate agent is a peptide.
8. The method of any of Claims 1-6, wherein the candidate agent is a peptide of less than 20 amino acid residues.
9. The method of any of Claims 1-8, wherein the candidate agent is a peptide of less than 10 amino acid residues.
10. The method of any of Claims 1-5, wherein the candidate agent is a small organic molecule of 1,500 daltons or less.
11. The method of any of Claims 1-10, wherein the hyperglycemia of the hyperglycemia-induced ongoing ROS production or the hyperglycemia associated with the ROS production is transient.
12. The method of Claim 11, wherein the hyperglycemia is from about 4 to 6 hours.
13. The method of any of Claims 1-12, wherein the hyperglycemia is undetectable using HbAlc quantification.
14. The method of any of Claims 1-13, wherein the hyperglycemia does not increase HbAlc production in the subject.
15. The method of any of Claims 1-14, wherein the candidate the agent does not affect glycemia in the subject.
16. The method of any of Claims 1-15, wherein the candidate the agent does not affect insulin production or affect insulin activity in a subject.
17. A method of obtaining an agent which reduces reactive oxygen species (ROS) production in a cell in a subject, wherein the ROS production is associated with a hyperglycemia in a subject, comprising contacting a human aortic endothelial cell (HAEC) with a candidate agent, wherein the HAEC has been exposed to an elevated glucose concentration for a predetermined amount of time, and experimentally quantifying ROS production by the HAEC, and comparing the quantified ROS production against a control wherein an ROS production below the control value indicates the candidate agent reduces ROS production and wherein an ROS production in excess of the control value indicates the candidate agent does not reduces ROS production, and physically recovering the candidate agent identified as reducing ROS production so as to obtain an agent which reduces ROS production in a cell.
18. The method of Claim 17, wherein the HAEC has been obtained from a diabetic human.
19. The method of Claim 17, wherein the HAEC has been obtained from a non-diabetic human.
20. The method of any of Claims 17 - 19, wherein the elevated glucose concentration is equivalent to over 190 mg/dL blood.
21. The method of any of Claims 17 - 20, wherein the elevated glucose concentration is equivalent over 20 mM.
22. The method of any of Claims 17 - 21, wherein the elevated glucose concentration is 23 -27 mM.
23. The method of any of Claims 17 - 22, wherein the elevated glucose concentration is 25 mM.
24. The method of any of Claims 17 - 23, wherein the candidate agent is a peptide.
25. The method of any of Claims 17 - 24, wherein the candidate agent is a peptide of less than 20 amino acid residues.
26. The method of any of Claims 17 - 25, wherein the candidate agent is a peptide of less than 10 amino acid residues.
27. The method of any of Claims 17 - 23, wherein the candidate agent is a small organic molecule of 1,500 daltons or less.
28. The method of any of Claims 17 - 27, wherein ROS levels are quantified using CM- H2DCFDA.
29. The method of any of Claims 17 - 28, wherein ROS levels are quantified using fluorimetry.
30. A method of reducing reactive oxygen species (ROS) production in a cell in a subject, wherein the ROS production is associated with a hyperglycemia in a subject, comprising administering to the subject an amount of an agent effective to inhibit reactive oxygen species production by a cell of a subject.
31. A method of treating diabetes complication in a subject comprising administering to the subject an amount of an agent effective to inhibit reactive oxygen species production by a cell of a subject.
32. The method of Claim 31, wherein the complication is a retinopathy, an albuminuria or a microalbuminuria.
33. A method of treating diabetes or prediabetic hyperglycemia in a subject comprising administering to the subject an amount of an agent effective to inhibit reactive oxygen species production by a cell of a subject.
34. The method of Claim 33, wherein the method is for treating diabetes.
35. The method of Claim 33, wherein the method is for treating prediabetic
hyperglycemia.
36. The method of any of Claims 30-35, wherein the agent comprises a peptide.
37. The method of Claim 31 or 32, wherein the agent comprises glucagon like peptide-1 (9-36) amide.
38. The method of Claim 30 - 36, wherein the agent comprises glucagon like peptide-1 (9-37).
38. The method of any of Claims 30- 35, wherein the agent comprises a small organic molecule.
39. The method of any of Claims 30 - 38, wherein the agent is administered during or subsequent to a hyperglycemic event.
40. The method of any of Claims 30 - 39, wherein the agent is administered by self- administration.
41. The method of any of Claims 30 - 39, wherein the agent is administered
continuously by a pump.
42. The method of any of Claims 30 - 41, wherein the amount of agent administered decreases Akt inhibition in the vascular cells of the subject.
43. The method of any of Claims 30 - 42, wherein the amount of agent administered reduces the extent of VDAC phosphorylation associated with hyperglycemia in the subject.
44. The method of any of Claims 30 - 43, wherein the amount of agent administered reduces the extent of GSK-3P activation associated with hyperglycemia in the subject.
45. The method of any of Claims 30 - 44, wherein the amount of agent administered reduces the extent of PGI2 inactivation associated with hyperglycemia induced ROS production in the subject.
The method of any of Claims 1-45, wherein the cell is a vascular cell.
47. The method of any of Claims 1-46, wherein the ROS production is post- hyperglycemic spike persistent ROS production.
48. A method of reducing reactive oxygen species (ROS) production in a cell in a subject, wherein the ROS production is associated with a hyperglycemia in a subject, comprising administering to the subject an amount of an agent effective to activate Aktl, to inhibit PP2A, to inhibit GSK-3beta, to inhibit phosphorylation of VDAC threonine, or to increase association of VDAC with HK-II.
49. A method of treating diabetes complication in a subject comprising administering to the subject an amount of an agent effective to activate Aktl, to inhibit PP2A, to inhibit GSK-3beta, to inhibit phosphorylation of VDAC threonine, or to increase association of VDAC with HK-II.
50. The method of Claim 49, wherein the complication is a retinopathy, an albuminuria or a microalbuminuria.
51. A method of treating diabetes or prediabetic hyperglycemia in a subj ect comprising administering to the subject an amount of an agent effective to the subject an amount of an agent effective to activate Aktl, to inhibit PP2A, to inhibit GSK-3beta, to inhibit phosphorylation of VDAC threonine , or to increase association of VDAC with HK-II.
52. The method of any of Claims 1-51, wherein the subject is a human.
53. A method of treating diabetes or prediabetic hyperglycemia in a subject comprising: a) receiving an identification of the subject as having a hyperglycemic spike in their blood glucose;
b) administering to the subject so identified an amount of an agent effective to inhibit persistent or ongoing ROS production under normoglycemic conditions after a
hyperglycemia spike and thereby treat the diabetes or prediabetic hyperglycemia.
54. A method of treating diabetes or prediabetic hyperglycemia in a subject comprising: a) receiving an identification of the subject as having a persistent or an ongoing ROS production under normoglycemic conditions after a hyperglycemia spike;
b) administering to the subject so identified an amount of an agent effective to inhibit reactive oxygen species production by a cell of a subject and thereby treat the diabetes or prediabetic hyperglycemia.
55. The method of Claim 53, 54 or 55, wherein the agent comprises a peptide.
56. The method of Claims 53-55, wherein the agent comprises glucagon like peptide-1 (9-36) amide.
57. The method of Claims 53-56, wherein the agent comprises glucagon like peptide-1 (9-37).
58. The method of Claims 53-54, wherein the agent comprises a small organic molecule.
59. The method of any of Claims 53-58, wherein the agent is administered during or subsequent to a hyperglycemic spike in the subject.
60. The method of any of Claims 53-59, wherein the agent is administered by self- administration.
61. The method of any of Claims 53-60, wherein the agent is administered continuously by a pump.
62. The method of any of Claims 53-61, wherein the amount of agent administered decreases Akt inhibition in the vascular cells of the subject.
63. The method of any of Claims 53-61, wherein the amount of agent administered reduces the extent of VDAC phosphorylation associated with hyperglycemia in the subject.
64. The method of any of Claims 53-61, wherein the amount of agent administered reduces the extent of GSK-3P activation associated with hyperglycemia in the subject.
65. The method of any of Claims 53-61, wherein the amount of agent administered reduces the extent of PGI2 inactivation associated with hyperglycemia induced ROS production in the subject.
66. The method of any of Claims 53-65, wherein the method is for treating diabetes.
67. The method of any of Claims 53-65, wherein the method is for treating prediabetic hyperglycemia.
68. The method of any of Claims 53-67, wherein the subject is identified as undergoing a hyperglycemic spike by means of a blood glucose monitor.
69. The method of any of Claims 53-68, wherein the agent is administered within 5 minutes of a hyperglycemia threshold being crossed.
70. The method of any of Claims 53-68, wherein the agent is administered within 30 minutes of a hyperglycemia threshold being crossed.
71. The method of any of Claims 53-67, wherein the subject is a human.
72. A method of obtaining an agent which reduces reactive oxygen species (ROS) production in a cell in a subject, wherein the ROS production is persistent ROS production subsequent to a hyperglycemic spike in a subject, comprising
administering the agent to a non-human animal with the agent and inducing a hypercglycemic spike in the animal,
quantifying the ROS production in the animal during the normoglycemia immediately subsequent to the subsequent to the hyperglycemic spike,
and comparing the experimentally quantified value against a control value of ROS production during the normoglycemia subsequent to the hyperglycemic spike, wherein a quantified ROS production during the normoglycemia below the control value indicates the candidate agent reduces ROS production and wherein a quantified ROS production during the normoglycemia above the control value indicates the candidate agent does not reduce ROS production.
73. The method of Claim 72, further comprising physically recovering the candidate agent identified as reducing ROS production so as to obtain an agent which reduces hyperglycemia-induced ongoing ROS production.
74. The method of Claim 72, wherein the non-human animal is a diabetes-model animal.
75. The method of Claim 72, 73 or 74, wherein the animal is a rat or a mouse.
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