US20090253619A1 - Novel peptide involved in energy homeostasis - Google Patents

Novel peptide involved in energy homeostasis Download PDF

Info

Publication number
US20090253619A1
US20090253619A1 US12/012,627 US1262708A US2009253619A1 US 20090253619 A1 US20090253619 A1 US 20090253619A1 US 1262708 A US1262708 A US 1262708A US 2009253619 A1 US2009253619 A1 US 2009253619A1
Authority
US
United States
Prior art keywords
enho1
peptide
mice
seq
insulin
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US12/012,627
Other languages
English (en)
Inventor
Andrew A. Butler
James L. Trevaskis
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Louisiana State University and Agricultural and Mechanical College
Original Assignee
Louisiana State University and Agricultural and Mechanical College
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Louisiana State University and Agricultural and Mechanical College filed Critical Louisiana State University and Agricultural and Mechanical College
Priority to US12/012,627 priority Critical patent/US20090253619A1/en
Assigned to BOARD OF SUPERVISORS OF LOUISIANA STATE UNIVERSITY AGRICULTURAL AND MECHANICAL COLLEGE reassignment BOARD OF SUPERVISORS OF LOUISIANA STATE UNIVERSITY AGRICULTURAL AND MECHANICAL COLLEGE ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BUTLER, ANDREW A., TREVASKIS, JAMES
Publication of US20090253619A1 publication Critical patent/US20090253619A1/en
Priority to US12/848,308 priority patent/US8518892B2/en
Priority to US13/850,806 priority patent/US20130231280A1/en
Priority to US14/922,557 priority patent/US20160102126A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • 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
    • 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
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • 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
    • A61K38/2264—Obesity-gene products, e.g. leptin
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • This invention pertains to a novel gene and resulting protein, named “Energy Homeostasis Associated-1” (Enho1) which was found to be involved in the association of obesity with insulin resistance and lipidemia.
  • Obesity is an increasingly prevalent global disease and has reached epidemic proportions. Current estimates suggest that at least 50% of the Western population is either overweight or obese. Obesity, particularly abdominal obesity, combined with other conditions such as insulin resistance, dyslipidemia, hepatic steatosis, and hypertension is known as the Metabolic, or Insulin Resistance, Syndrome.
  • the central pathophysiological features of the dyslipidemia associated with insulin resistance and type 2 diabetes are increased plasma triglycerides (TG) in very low density lipoproteins (VLDL), and reduced high density lipoprotein (HDL) cholesterol.
  • TG plasma triglycerides
  • VLDL very low density lipoproteins
  • HDL high density lipoprotein
  • TGs are hydrolyzed into free fatty acids (FFA) and are taken up by peripheral tissues including the liver and can lead to hepatic steatosis, or non-alcoholic fatty liver.
  • FFA free fatty acids
  • Insulin resistance refers to reduced insulin-stimulated glucose uptake in skeletal muscle and fat, and an impaired suppression of liver glucose output (2).
  • Hyperglycemia and hyperlipidemia are both side effects of, and causative agents in, the pathophysiology of type 2 diabetes. Glucotoxicity and lipotoxicity further promote insulin resistance and type 2 diabetes due to suppression of insulin action and secretion from the ⁇ -cell.
  • Hyperinsulinemia is initially successful in suppressing liver glucose output, however the deleterious effects of increased insulin offset the gains associated with maintaining normal blood glucose levels (2). Hyperinsulinemia is thought to be a factor in a cluster of metabolic abnormalities, including hypertension, non-alcoholic fatty liver disease (NAFLD) and coronary heart disease (2).
  • NAFLD non-alcoholic fatty liver disease
  • NAFLD disease is commonly associated with insulin resistance, and requires two transcription factors: sterol regulatory element binding protein-1c (SREBP1c) and peroxisome proliferator receptor- ⁇ (PPAR ⁇ ) (3-6). Absence of SREBP1, or PPAR ⁇ signaling in liver inhibits the development of liver steatosis that occurs in obese insulin resistant mice (5-7).
  • SREBP1c sterol regulatory element binding protein-1c
  • PPAR ⁇ peroxisome proliferator receptor- ⁇
  • IRS1 and IRS2 are tyrosine phosphorylation of two adaptor proteins, IRS1 and IRS2, and are thought to function as part of a molecular scaffold that facilitates the formation of complexes of proteins with kinase, phosphatase or ubiquitin ligase function (12).
  • PI3K phosphoinositide 3′ kinase
  • a metabolic state conducive to the development of insulin resistance is thought to result from an imbalance of caloric intake with oxidative metabolism (13,14).
  • a well-characterized example of this is the adipocytokine leptin. Leptin acts in the hypothalamus and hindbrain to suppress appetite and through stimulation of the autonomic nervous system increases oxidative metabolism in skeletal muscle (16-20).
  • leptin can also improve hepatic insulin sensitivity independently of marked effects on food intake or body weight (17).
  • Adipocytokines such as leptin, adiponectin, and resistin regulate hepatic glucose production, glucose disposal in muscle, and the proliferation and storage of lipid in adipocytes (26).
  • Leptin regulates energy homeostasis through effects on neurons located in the hypothalamus and hindbrain, regulating ingestive behavior, autonomic nervous activity, and neuroendocrine system that govern metabolism (thyroid, adrenals) (16).
  • Leptin resistance or reduced serum adiponectin associated with obesity are factors that contribute to insulin resistance, through diminished insulin-sensitizing actions and by increasing risk for developing steatosis (intracellular fatty acid accumulation) (27,28).
  • Non-adipose tissues also secrete peptides that affect energy metabolism and insulin sensitivity, such as musclin from muscle (29) and angiopoietin-related growth factor from liver (30). These factors may also be targets for the treatment of the metabolic syndrome.
  • M4R neuronal melanocortin-4 receptor
  • FIGS. 1A-1E illustrate some of the known differences in wild-type mice (C57BL/6J) and the two knockout mice in terms of body mass as a function of either a low fat diet or a high fat diet.
  • FIGS. 2A-2E show the differences in hepatomegaly and steatosis in the two mouse strains, and also differences in expression of genes involved in lipid metabolism. (4,17,57)
  • Mc3rKO matched to Mc4rKO for fat mass (FM) exhibit a very modest impairment of glucose homeostasis.
  • a transgenic FVB/NJ strain of mouse was created which over expresses the Enho1 open reading frame, using Enho1 DNA (SEQ ID NO: 1) controlled by the human ⁇ -actin promoter which is expressed in all tissues.
  • Enho1 DNA SEQ ID NO: 1
  • Female FVB/NJ mice over expressing Enho1 had a significant reduction in fat mass, and a higher metabolic rate determined by measuring oxygen consumption (VO2) using indirect calorimetry (Oxymax, Columbus Instruments, Columbus, Ohio). The increase in metabolic rate observed in the transgenic mice had been predicted, based on the results from experiments using recombinant adenovirus expressing Enho1.
  • Enho1 Mice infected with recombinant adenovirus expressing Enho1 lost more weight during an overnight fast, suggesting an impaired ability to reduce metabolic rate to compensate during fasting.
  • FVB/NJ Enho1 transgenic mice exhibit the same exaggerated weight loss during a fast, associated with a higher metabolic rate.
  • a component of Enho1's anti-diabetic actions may therefore involve stimulation of pathways involved in oxidative metabolism. That is, Enho1 may improve the metabolic profile of obese, insulin resistant individuals partially through normalizing the balance of kJ consumption with kJ expended through effects on physical activity, basal metabolic rate, or a combination of both.
  • Full-length Enho1 peptide, or peptide derivatives, homologues, analogues, or mimetics thereof, delivered by oral intake, injection, subcutaneous patch, or intranasal routes, could be used as therapeutic or diagnostic agents for hypercholesterolemia, hypertriglyceridemia, insulin resistance, obesity, diabetes, and/or disorders of energy imbalance.
  • Antibodies (AB1 (SEQ ID NO:8) and AB2 (SEQ ID NO:9)) were raised against peptide fragments derived from the open reading frame predicted for BC021944, and shown in FIG. 5 . These antibodies were used to verify the presence of Enho1-immunoreactivity in human serum and in rat brain, strongly supporting the conclusion that the open reading frame predicted for BC021944 encodes a small secreted peptide. Enho1-immunoreactivity was detected in human plasma. (data not shown) In rat brain, neurons with Enho1 immunoreactivity have been identified in the arcuate nucleus of the hypothalamus.
  • the increased energy expenditure and physical activity of the FVB/NJ Enho1 transgenic mice may therefore involve action in the central nervous system, and more specifically through actions based on regulating activity of neurons in the arcuate nucleus of the hypothalamus.
  • FIG. 1A illustrates the differences in body mass in 6-month-old, female mice after 12 weeks feeding either a low-fat diet (LF) or a high-fat diet (HF) among three different strains, wild-type (WT), Mc3r ⁇ / ⁇ deficient mice (Mc3rKO), and Mc4r ⁇ / ⁇ deficient mice (Mc4rKO).
  • LF low-fat diet
  • HF high-fat diet
  • WT wild-type
  • Mc4r ⁇ / ⁇ deficient mice Mc4r ⁇ / ⁇ deficient mice
  • FIG. 1B illustrates the differences in body weight gain as a percent of starting weight in 6-month-old, female mice after 12 weeks feeding either a low-fat diet (LF) or a high-fat diet (HF) among three different strains, wild-type (WT), Mc3r ⁇ / ⁇ deficient mice (Mc3rKO), and Mc4r ⁇ / ⁇ deficient mice (Mc4rKO).
  • LF low-fat diet
  • HF high-fat diet
  • WT wild-type
  • Mc4r ⁇ / ⁇ deficient mice Mc4r ⁇ / ⁇ deficient mice
  • FIG. 1C illustrates the differences in percent body fat in 6-month-old, female mice after 12 weeks feeding either a low-fat diet (LF) or a high-fat diet (HF) among three different strains, wild-type (WT), Mc3r ⁇ / ⁇ deficient mice (Mc3rKO), and Mc4r ⁇ / ⁇ deficient mice (Mc4rKO).
  • LF low-fat diet
  • HF high-fat diet
  • WT wild-type
  • Mc4r ⁇ / ⁇ deficient mice Mc4r ⁇ / ⁇ deficient mice
  • FIG. 1D illustrates the differences in fat mass in 6-month-old, female mice after 12 weeks feeding either a low-fat diet (LF) or a high-fat diet (HF) among three different strains, wild-type (WT), Mc3r ⁇ / ⁇ deficient mice (Mc3rKO), and Mc4r ⁇ / ⁇ deficient mice (Mc4rKO).
  • LF low-fat diet
  • HF high-fat diet
  • WT wild-type
  • Mc4r ⁇ / ⁇ deficient mice Mc4r ⁇ / ⁇ deficient mice
  • FIG. 1E illustrates the differences in fat-free mass in 6-month-old, female mice after 12 weeks feeding either a low-fat diet (LF) or a high-fat diet (HF) among three different strains, wild-type (WT), Mc3r ⁇ / ⁇ deficient mice (Mc3rKO), and Mc4r ⁇ / ⁇ deficient mice (Mc4rKO).
  • LF low-fat diet
  • HF high-fat diet
  • WT wild-type
  • Mc4r ⁇ / ⁇ deficient mice Mc4r ⁇ / ⁇ deficient mice
  • FIG. 2A illustrates a comparison of liver histology, as shown in liver cross-sections stained with hemotoxylin and eosin, from female mice fed a high fat diet among three mice strains, wild-type (WT), Mc3r ⁇ / ⁇ deficient mice (Mc3rKO), and Mc4r ⁇ / ⁇ deficient mice (Mc4rKO).
  • WT wild-type
  • Mc4r ⁇ / ⁇ deficient mice Mc4r ⁇ / ⁇ deficient mice
  • FIG. 2B illustrates the differences in liver weight as a function of adiposity (percent body fat) in mice fed both low and high fat diets among three mice strains, wild-type (WT), Mc3r ⁇ / ⁇ deficient mice (Mc3rKO), and Mc4r ⁇ / ⁇ deficient mice (Mc4rKO).
  • FIG. 2D illustrates the differences in expression of stearoyl-CoA desaturase 1 (SCD1) in liver of mice fed a high fat diet among three mice strains, wild-type (WT), Mc3r ⁇ / ⁇ deficient mice (Mc3rKO), and Mc4r ⁇ / ⁇ deficient mice (Mc4rKO). Data is expressed as arbitrary units (a.u.). (“*” indicates p ⁇ 0.05 versus WT and Mc3rKO mice).
  • FIG. 2E illustrates the differences in expression of apolipoprotein A4 (ApoA4) in liver of mice fed a high fat diet in three mice strains, wild-type (WT), Mc3r ⁇ / ⁇ deficient mice (Mc3rKO), and Mc4r ⁇ / ⁇ deficient mice (Mc4rKO). (“*” indicates p ⁇ 0.05 versus WT and Mc3rKO mice; “#” indicates p ⁇ 0.05 versus WT mice, within gender).
  • WT wild-type
  • Mc3rKO Mc3r ⁇ / ⁇ deficient mice
  • Mc4rKO Mc4r ⁇ / ⁇ deficient mice
  • FIG. 3A illustrates the amount of hepatic AK009710 mRNA expression (given as a percent of WT expression) in wild-type (WT) mice and in obese Mc3r ⁇ / ⁇ deficient mice (Mc3rKO).
  • FIG. 3B illustrates the amount of hepatic AK009710 mRNA expression (given as a percent of WT expression) in wild-type mice (WT), Mc4r ⁇ / ⁇ deficient mice (Mc4rKO), and leptin-deficient Lep ob /Lep ob mice (two models of obesity).
  • FIG. 3C illustrates the amount of hepatic AK009710 mRNA expression (given as a percent of expression with saline) in leptin-deficient Lep ob /Lep ob mice injected 4 times over 2 days with either saline or leptin (0.5 mg/g).
  • FIG. 4 illustrates the nucleic acid sequence of the Enho1 gene (Mouse AK009710; SEQ ID NO:1), with the open reading frame encoding the Enho1 protein underlined, and its putative amino acid translation product Enho1 (SEQ ID NO:2).
  • FIG. 5 illustrates the alignment of the putative Enho1 protein sequences for mouse (SEQ ID NO:2), human (SEQ ID NO:14), rat (SEQ ID NO:12), dog (SEQ ID NO:15), pig (SEQ ID NO:16), cow (SEQ ID NO:17), sheep (SEQ ID NO:18), and chimpanzee (SEQ ID NO:13), showing the regions of the putative secreted polypeptide used to generate polyclonal antibodies (pAB1 ((SEQ ID NO:8) and pAB2 (SEQ ID NO:9)).
  • FIG. 6A illustrates the results of a Northern blot analysis using a radioactive-labeled portion of the AK009710 DNA sequence encoding Enho1 protein on human tissue samples (Lanes 1-8 represents RNA from heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas, respectively).
  • FIG. 6B illustrates the relative intensity of Enho1 mRNA bands from a Northern blot analysis using the full-length mouse AK009710 DNA probe on mouse tissue samples.
  • FIG. 6C illustrates the results of a Western blot analysis for FLAG immunoreactivity in media (M) and cell lysates (Pel) from HEK293 human-kidney derived cells transfected with pCMV-Enho1:Flag, pCMV-GFP, or in media from HEK293 cells infected with adenoviral vector expressing Enho1:Flag fusion protein [no transfected DNA (lanes 1, 2); transfected with a pCMV-Enho1:FLAG construct (lanes 3, 4); transfected with pCMV-Enho1 (lanes 5, 6); or transfected with pCMV-GFP (lanes 7, 8); infected with Ad5Enho1:FLAG (lane 9); infected with Ad5Enho1 (lane 10); or infected with Ad5GFP (lane 11)].
  • M media
  • Pel cell lysates
  • FIG. 6D illustrates the results of a Western blot analysis for ENHO1 protein in various tissues (liver, muscle, and brain) from Mc4r ⁇ / ⁇ mice injected with Ad5-ENHO1:FLAG 4 days prior to the assay, along with controls, both positive (HEK293 cells infected with Ad5-ENHO1:FLAG) and negative (Liver from non injected Mc4r ⁇ / ⁇ mice).
  • FIG. 7A illustrates the results of a Western blot analysis using lysate from HEK293 cells infected with recombinant Ad5Enho1 (native protein) or Ad5Enho1:FLAG (C-terminal FLAG-tagged fusion protein) after incubation with a polyclonal antibody (pAB1) against the N-terminus of Enho1 (as shown in FIG. 5 ).
  • Ad5Enho1 native protein
  • Ad5Enho1:FLAG C-terminal FLAG-tagged fusion protein
  • FIG. 7B illustrates the results of a Western blot analysis using lysate from HEK293 cells infected with recombinant Ad5Enho1 (native protein) or Ad5Enho1:FLAG (C-terminal FLAG-tagged fusion protein) after incubation with a polyclonal antibody (pAB2) against the C-terminus of Enho1 (as shown in FIG. 5 ).
  • Ad5Enho1 native protein
  • Ad5Enho1:FLAG C-terminal FLAG-tagged fusion protein
  • FIG. 7C illustrates the treatment protocol used to administer Ad5Enho1 or Ad5-GFP into the tail vein of various strains of mice to test the effects of Enho1 treatment on mice metabolism (results presented in Table 1).
  • AU arbitrary units
  • AU arbitrary units
  • AU arbitrary units
  • FIG. 9A illustrates the construction of the transgene (BAP-Enho1) used to generate transgenic mouse strains that over express Enho1.
  • FIG. 9B illustrates the amount of Enho1 expression in livers from transgenic mice pups (produced using the transgene of FIG. 9A ) from FVB/NJ founders at 5 weeks.
  • FIG. 9C illustrates the level of fasting triglycerides (TG) from serum of transgenic mice pups (produced using the transgene of FIG. 9A ) from FVB/NJ founders at 9 weeks.
  • FIG. 9D illustrates the body composition as measured by percent body fat (% body fat; left graph) and fat mass (showing both free fat mass (FFM) and fat mass (FM); right graph) in transgenic mice pups (produced using the transgene of FIG. 9A ) from FVB/NJ founders at 5 and 9 weeks as compared with control.
  • FIG. 9E illustrates change in fat mass (showing both free fat mass (FFM) and fat mass (FM)) of transgenic mice pups (produced using the transgene of FIG. 9A ) from FVB/NJ founders after 7 days on a 60% high fat diet.
  • FIG. 10 illustrates the results from a PCR screen for integration of BAP-Enho1 into the genome of C57BL/6J pups (from injection of BAP-Enho1 into C57BL/6J oocytes) showing six pups out of 23 with integration of the transgene into the genome.
  • FIG. 11A illustrates the increase in Erk phosphorylation in 3T3-L1 adipocytes 15 minutes after the application of the secreted portion of protein Enho1 (Enho1 34-76 ; SEQ ID NO:10).
  • FIG. 11B illustrates the increase in Erk phosphorylation in HepG2 hepatocytes 15 minutes after the application of the secreted protein Enho1 (Enho1 34-76 ; SEQ ID NO: 10).
  • FIG. 13A illustrates the physical activity (measured in beam breaks/10 min) in wild-type (WT) and BAP-Enho1 transgenic mice (Tg) over 3 days.
  • FIG. 13B illustrates the physical activity (measured in average number beam breaks/10 min for the period) in dark and light periods in wild-type (WT) and BAP-Enho1 transgenic mice (Tg) over a 24 hr.
  • FIG. 13C illustrates whole body fat oxidation (RER) in dark and light periods in wild-type (WT) and BAP-Enho1 transgenic mice (Tg) over a 24 hr.
  • FIG. 13D illustrates the metabolic rate (measured as VO2 (ml/h ⁇ 10 3 )) in dark and light periods in wild-type (WT) and BAP-Enho1 transgenic mice (Tg) over a 24 hr.
  • FIG. 13E illustrates the metabolic rate as a function of free fat mass (measured as VO2 (ml/h/gFFM ⁇ 10 3 )) in dark and light periods in wild-type (WT) and BAP-Enho1 transgenic mice (Tg) over a 24 hr.
  • FIG. 13F illustrates the metabolic rate as a function of body weight (measured as VO2 (ml/h/gBW ⁇ 10 3 )) in dark and light periods in wild-type (WT) and BAP-Enho1 transgenic mice (Tg) over a 24 hr.
  • the present invention discloses a novel secreted protein (Enho1) and identifies some of its functions.
  • the sequence of this protein was found to be highly conserved across several mammalian species, and the sequences are shown in SEQ ID NOS:2 and 12-18.
  • the nucleic acids that encode this protein was used to make mice that over expressed the Enho1 protein, either by infection with a recombinant adenovirus expressing Enho1 or by making a transgenic strain using Enho1 DNA controlled by an actin promoter.
  • the Enho1 protein, fragment, derivative or analogs are used therapeutically to prevent a pathophysiology associated with increased body mass, e.g., obesity, hyperglycemia, hyperinsulinemia, insulin resistance, hyperlipidemia, and non-insulin dependent (type 2) diabetes mellitus.
  • a pathophysiology associated with increased body mass e.g., obesity, hyperglycemia, hyperinsulinemia, insulin resistance, hyperlipidemia, and non-insulin dependent (type 2) diabetes mellitus.
  • the purified Enho1 protein, fragment, derivative or analog is isolated from various mammals, or made synthetically, or made using cell cultures that express the protein.
  • antibodies are made to the Enho1 protein, its fragments, derivatives or analogs. These antibodies can be used in a kit to identify the Enho1 protein from various samples, including body fluids.
  • transgenic animals are made that over express the Enho1 protein by linking the Enho1 sequence to an active promoter, e.g., an actin promoter.
  • an active promoter e.g., an actin promoter.
  • a transformation vector comprising at least the open reading frame of SEQ ID NO: 1 (the portion underlined in FIG. 4 ) are made.
  • Microarrays analyzing hepatic gene expression in lean and obese Mc3rKO were performed using arrays printed from libraries of 16,463-18,400 70-mer oligonucleotides (Mouse Array-Ready Oligo Set Version 2.0, Qiagen Operon, Alameda, Calif.) (34-36).
  • the microarray data indicated increased expression of genes involved in lipid metabolism (apoliproteins) and oxidative stress secondary to obesity.
  • the expression of only three genes was reduced in liver of Mc3rKO irrespective of age, gender, and adiposity.
  • neuraminidase 3 (neu3), encoding an enzyme that cleaves sialic acid from glycoproteins and glycolipids, and solute carrier family 21 (slc21a1) which encodes an organic anion transporter.
  • slc21a1 solute carrier family 21 which encodes an organic anion transporter.
  • the third gene (AK009710) was novel and had no assigned function in the databases.
  • Quantitative RealTime PCR confirmed the microarray data showing a tendency for a modest reduction in the expression of AK009710 in liver of Mc3rKO ( FIG. 3A ). Intriguingly, a more dramatic reduction in the expression of AK009710 was observed in liver of severely insulin resistant Mc4rKO and leptin-deficient (Lep ob /Lep ob ) mice ( FIG. 3B ). In addition, short term treatment with leptin (4 injections of 0.5 mg/g leptin over 2 days) significantly increased Enho1 in the liver of Lep ob /Lep ob mice. ( FIG. 3C ).
  • Oligonucleotide primers targeted to AK009710 mRNA were utilized to measure AK009710 gene expression in liver cDNA from various mouse models of obesity and insulin resistance. Sequences of primers were: sense 5-cctgagggtgctgtctgtcatg-3′ (SEQ ID NO:3), antisense 5′-cagtagcagcaagaagcctacg-3′ (SEQ ID NO:4), probe 5′-6FAM-ctctcatcgccatcgtctgca-BHQ-3′ (SEQ ID NO:5).
  • AK009710 mRNA was down regulated in the liver of Mc3r ⁇ / ⁇ mice compared to WT mice (by 55%, p ⁇ 0.05; FIG. 3A ).
  • Mc4r ⁇ / ⁇ mice and leptin-deficient Lep ob /Lep ob mice.
  • AK009710 mRNA was expressed at approximately 10-fold lower levels in both of these models compared to wild-type mice (WT) ( FIG. 3B ). When all animals were grouped together negative relationships between AK009710 mRNA expression and glucose and insulin values were observed (Data not shown).
  • AK009710 gene expression was not altered in the liver of young and aged WT mice fed ad libitum, fasted for 16 h, or fasted for 24 h and re-fed for 4 h. (data not shown) AK009710 is not thought to be altered by nutritional status.
  • AK009710 is a mouse tongue cDNA clone 1247 bp in length and belongs to the Unigene cluster Mm.34074, 2310040A07Rik: RIKEN cDNA 2310040A07 gene. It hypothetically could encode a 534 amino acid protein immediately from the 5′ end of the sequence. Given that this putative protein did not start with a methionine residue, it was listed as a truncated product.
  • BLAST analysis of AK009710 for homologous EST or cDNA sequences on the NCBI database revealed a significant match with NM198573, a human transcript discovered by a large-scale Secreted Protein Discovery Initiative encoding a hypothetical protein of 87 aa (UNQ470/GAAI470) (37).
  • Translation of AK009710 in six frames revealed an open reading frame encoding a 76 amino acid protein (SEQ ID NO:2).
  • pAB1 SEQ ID NO: 8
  • pAB2 SEQ ID NO:9 refer to the regions of the putative secreted polypeptide used to generate polyclonal antibodies. The predicted signal sequence is underlined.
  • peptides were synthesized corresponding to amino acids 34 through 76 (Enho1 34-76 ; SEQ ID NO:10), and to amino acids 39 through 76 (Enho1 39-76 ; SEQ ID NO:11) of SEQ ID NO:2, the mouse protein.
  • the human sequence contains 1 possible serine site, and 1 possible threonine phosphorylation sites, all 3′ to the predicted cleavage point, as well as 6 possible glycosylation sites, also all 3′ to the cleavage site.
  • a Northern blot was performed using the 210 nt sequence containing the 76 amino acid protein ENHO1 and a human Multiple Tissue Northern blot (BD Biosciences, Palo Alto, Calif.).
  • a single band of ⁇ 1.35 kb was detected in humans in brain and liver samples only, with highest levels detected in the liver.
  • Enho1 was found in mice in the liver, brain, and muscle.
  • Bioinformatics analysis predicted the presence of a putative signal sequence suggesting that AK009710 is a secreted peptide.
  • the coding sequence of the 76 amino acid protein was amplified from mouse liver cDNA by PCR using primers with NotI (5′-ggggcggccgcaccatgggggcagccatctcccaa-3′ (SEQ ID NO:6)) and Xho1 (5′-gggctcgagggccagagcccttcagggctgcag-3′ (SEQ ID NO:7)) restriction enzyme sites attached.
  • the product was ligated into a pCMV-Tag1 vector with a FLAG epitope at the C-terminal end (pCMV-Enho1:FLAG), then transiently transfected into HEK human kidney-derived cells.
  • This product was also used to create two adenoviral constructs—one with Enho1 attached to a FLAG epitope (Ad-Enho1:FLAG), and another without the FLAG epitope (Ad-Enho1).
  • HEK293 cells were transfected with pCMV-Enho1, or pCMVEnho1:FLAG. Media was collected after 16 h, immunoprecipitated, and then run on a 20% polyacrylamide gel to be visualized by an anti-FLAG antibody. These experiments were repeated using recombinant adenovirus (Ad5) expressing native or FLAG-tagged Enho1.
  • Lanes 10 and 11 show no FLAG immunoreactivity in media of cells infected with adenovirus containing Enho1 without the FLAG epitope, or with GFP, respectively.
  • the presence of FLAG immunoreactivity in cultured media of HEK-293 cells transfected with an expression vector expressing an epitope-tagged fusion protein (pCMV-Enho1FLAG) confirmed that an undefined portion of the 76 aa protein is secreted.
  • adenoviral vector-mediated expression has been used to investigate regulation of liver metabolism and insulin sensitivity (39-41).
  • Three recombinant adenovirus vectors were constructed expressing the 76 aa protein (Ad5-Enho1), a C-terminal FLAG-tagged fusion protein (Ad5-Enho1:FLAG), or green fluorescent protein for the negative control (Ad5-GFP). Expression of protein following tail vein injection was confirmed using anti-FLAG antibody ( FIG. 6D and data not shown).
  • Synthesis of Enho1 was confirmed in HEK293 cells transfected with Ad5-Enho1 using polyclonal antibodies (pAB1 & pAB2 in FIG. 5 ) against N- and C-terminal regions of the putative secreted protein (Sigma Genosys, The Woodlands, Tex.) ( FIG. 7A , B).
  • An intraperitoneal glucose tolerance test (IPGTT) was performed one week prior to injection of Ad5-ENHO1 or Ad5-GFP, and all mice were observed to be glucose intolerant.
  • IPGTT were performed on mice after an overnight fast, with a single intraperitoneal injection of 1 g/kg glucose, and blood glucose measured with a blood glucose meter and test strips (Glucometer Elite, Bayer Corp., Elkhart, Ind.) from the tail blood of the animals at several intervals, as described by W. Fan et al., “The Central Melanocortin System Can Directly Regulate Serum Insulin Levels,” Endocrinology, vol. 141, pp. 9 3072-3079 (2000).
  • mice were injected with 5 ⁇ 10 8 pfu of Ad5-ENHO1 or Ad5-GFP in 100 ⁇ l of diluent (DMEM) into the tail vein. Animals were observed and weighed daily throughout the 4-day experiment. On day 4 animals were given another IPGTT (0.4 mg glucose/g body weight).
  • DMEM diluent
  • mice Four days after injection of Ad5-ENHO1, the mice demonstrated improved glucose tolerance as measured using IPGTT (data not shown). There was no change in body weight, blood glucose or cholesterol levels throughout the experiment. There was a trend for a decrease in insulin and serum triglyceride levels (Table 1).
  • ITT insulin tolerance test
  • adenoviral constructs were further purified to remove any contaminating viral particles and cellular debris.
  • IPGTT revealed that glucose tolerance was not significantly improved by Ad5-ENHO1 treatment compared to Ad5-GFPAd5-GFP treatment.
  • Liver weight of Ad5-ENHO1 mice tended to be lower than liver weight of Ad5-GFP controls (Table 2).
  • Body weight, and serum glucose and insulin levels were not significantly different between Ad5-ENHO1 and Ad5-GFP controls (Table 2).
  • ENHO1 mRNA was elevated approximately 4-fold in the liver of KK-A y Ad5-ENHO1-treated mice compared to Ad5-GFP-treated mice (data not shown).
  • the injected mice were not given an IPGTT, but were monitored for body weight changes, and then sacrificed 7 days post injection after an overnight fast.
  • Ad5-GFP-treated mice gained approximately 1 g of body weight over the 7-day period.
  • Ad5-ENHO1 treatment blocked weight gain in these obese Lep ob /Lep ob mice (data not shown).
  • Mean body weight was not statistically different between groups, and food intake was not measured.
  • Similar phenotypic changes to that observed in KK-A y mice were also observed.
  • Ad5-GFP Ad5-GFP
  • a trend for a reduction in liver weight was observed that was not significant when expressed as a percentage of body weight (p 0.12).
  • Trends for reduced fasting blood glucose and insulin levels in the Ad5-ENHO1 group did not reach statistical significance.
  • Ad5-Enho1 or Ad5-GFP was administered into the tail vein of KKAy, B6 Ay/a, or OBOB mice purchased from the Jackson Laboratory (Bar Harbor, Me.).
  • the treatment protocol, shown in FIG. 7C was based on the pilot experiments demonstrating peak expression of Ad5-Enho1:FLAG during this period (data not shown). Animal and food weight were recorded daily. Ad5-Enho1 infection was well tolerated in mouse strains used for these experiments. There were no marked differences in the body weight (Table 2) of mice infected with Ad5-Enho1 compared to controls infected with Ad5-GFP over the treatment period. A 4-5 fold increase in Enho1 mRNA was observed in mice infected with Ad5-Enho1, compared to Ad5-GFP treated controls (data not shown).
  • Fasn mRNA and protein levels were measured using quantitative RealTime PCR and western blot, as described in D. C. Albarado et al., “Impaired Coordination of Nutrient Intake and Substrate Oxidation in Melanocortin-4 Receptor Knockout Mice,” Endocrinology, vol. 145, pp. 243-252 (2004).
  • FIG. 8C illustrates the expression level of acetyl CoA carboxylase (Acc) mRNA in liver from obese leptin-deficient (Lep ob /Lep ob ) mice eight days after injection with either Ad5-ENHO1 or Ad5-GFP.
  • Acc acetyl CoA carboxylase
  • FIG. 8D illustrates the expression level of a gene involved in insulin resistance (suppressor of cytokine signaling 3 or Socs3) in liver from obese leptin-deficient (Lep ob /Lep ob ) mice eight days after injection with either Ad5-ENHO1 or Ad5-GFP.
  • the presence of Enho1 again decreased the gene express of Socs3.
  • FIG. 9A Transgenic strains over expressing Enho1 were generated using a construct (BAP-Enho1) containing the human ⁇ -actin promoter, a synthetic exon 1 and intron, and an open reading frame encoding the 76 aa Enho1 protein ( FIG. 9A ; see FIG. 4 , the open reading frame is underlined).
  • Eight founders were obtained (2 FVB/NJ, 6 C57BL/6J), with one of the FVB strains [FVB/NJ.Tg-(BAP-Enho1)AAB20], hereafter referred to as FVB.Tg, exhibiting an increase in hepatic Enho1 expression at 5-6 wk of age ( FIG. 9B ).
  • FIG. 10 illustrates the results of PCR screening the pups in the first round of injection of BAP-Enho1 into C57BL/6J oocytes, showing six pups (6, 8, 14, 19, 21, and 22) with integration of the transgene into the genome.
  • VO2 and RER were measured using indirect calorimetry (15,46,50).
  • the Pennington Biomedical Research Center has a 16 chamber comprehensive laboratory animal monitoring system (CLAMS) housed in a temperature controlled incubator.
  • CLAMS simultaneously measured oxygen consumption (VO2), respiratory exchange ratio (RER, an indicator of whole animal substrate oxidation), physical activity in the X and Z axis, and food intake. Mice were placed in the CLAMS system, and the parameters indicated recorded for 72 h. Mice were fed ad libitum for 48 h, with a fast for final 24 h.
  • FVB.Tg The results for FVB.Tg are shown in FIGS. 13A-13F .
  • Increased weight loss was observed after an overnight fast in obese KKAy, in obese B6 Ay/a mice expressing Ad5Enho1 (% weight loss after overnight fast for Ad5-Enho1 vs Ad5-GFP: 5.5 ⁇ 0.4% vs. 3.5 ⁇ 0.4%, P ⁇ 0.01), and in 9 wk old lean FVB.Tg (13.6 ⁇ 1.1% vs. 10.0 ⁇ 0.7%, P ⁇ 0.05).
  • Enho1 34-76 SEQ ID NO:10
  • ERK1/2 and p38 ⁇ are important in the regulation of lipolysis and thermogenesis in adipocytes.
  • Enho1 34-76 was associated with a robust increase in the phosphorylation of Erk1 in fully differentiated 3T3-L1 adipocytes ( FIG. 11A ) and in HepG2 cells ( FIG. 11B ). These results indicate that functional receptors for Enho1 34-76 are active in adipocytes and hepatocytes, indicating that the synthetic peptide is biologically active.
  • a shorter, second peptide was synthesized using amino acids 39 through 76 of SEQ ID NO:2, and called “Enho1 39-76 ” (SEQ ID NO:11). Similar results using adipocytes and hepatocytes were observed with this peptide. The loss of four amino acids did not appear to affect Enho1 function.
  • Hypothalamic Enho1 may be involved in the regulation of energy homeostasis.
  • Preliminary analysis using pAB1 demonstrated the presence of Enho1-immunoreactivity in neurons located in the arcuate nucleus of the hypothalamus (data not shown). It was predicted that a negative correlation exists between hypothalamic Enho1 expression and obesity and insulin resistance. In other words, reduced synthesis of Enho1 in liver and hypothalamus in situations of obesity may be a factor contributing to obesity and insulin resistance.
  • Enho1 mRNA expression was measured by quantitative RT-PCR in mediobasal hypothalamic blocks from control (low fat diet) and diet-induced obese C57BL/6J, Mc3rKO and Mc4rKO mice ( FIGS. 12A-12B ). As predicted, Enho1 mRNA abundance in the hypothalamus was reduced in the obese state ( FIG. 12A ). Enho1 expression was also low in mice with elevated HOMA-IR values, an indicator of insulin resistance ( FIG. 12B ).
  • Socs3 mRNA expression was elevated in situations of obesity and insulin resistance ( FIG. 11C ). This data indicates that reduced synthesis of Enho1 in situations of obesity is not be limited to the liver. Reduced Enho1 activity in the hypothalamus would also contribute to the development of obesity and insulin resistance.
  • an expression vector was constructed using a partial cDNA sequence encoding the predicted 76 residues of ENHO1 (between nucleotides 207 and 437 of BC021944), with an epitope-tag inserted onto the carboxyterminal end to allow visualization of the protein on western blot using a commercially available antibody. Using this construct, it was verified that the reported sequences encoded a secreted protein.
  • This expression vector and an expression vector encoding the native protein without an epitope label were then used to construct a recombinant adenovirus (Ad5-ENHO1) for use in mouse studies.
  • ENHO1 peptide SEQ.ID.NO. 2
  • peptide derivatives, homologues, analogues, or mimetics thereof delivered by oral intake, injection, subcutaneous patch, or intranasal routes, could be used as therapeutic or diagnostic agents for hypercholesterolemia, hypertriglyceridemia, insulin resistance, obesity, diabetes, and/or energy imbalance.
  • substitutions within the native coding sequence can be made to make derivatives of ENHO1 with increased stability and/or biological potency.
  • the ENHO1 peptide can be used to identify its cell receptor which can then be used as as-yet-unidentified receptor(s) for ENHO1 is (are) a potential drug target(s) for the development of therapies aimed at reducing total cholesterol, triglycerides, insulin resistance, obesity, diabetes and/or energy imbalance.
  • Enho1 A novel secreted protein, Enho1, that is an important factor in the etiology of insulin resistance and hepatic steatosis in the obese state has been identified by Microarray analysis of gene expression in mouse models of moderate- to severe-diabesity. Enho1 significantly reduces HOMA-IR, an index of fasting insulin and glucose, and serum lipids in mouse models of type 2 diabetes, and significantly reduces hepatic lipids indicating a reversal of hepatic steatosis. Transgenic over expression of Enho1 is associated with a lean phenotype. Enho1 may act in a manner similar to leptin and adiponectin, improving metabolic profile in the obese insulin state by stimulating energy expenditure and increasing oxidative metabolism.
  • Preliminary data from Ad5-Enho1 and BAP-Enho1 transgenics indicate improved insulin action and lipid metabolism.
  • FVB.Tg mice increased oxidative metabolism is a likely factor explaining these effects. It may not be possible to dissect the anti-diabetic actions of Enho1 from those secondary to reductions of FM.
  • Preliminary data using recombinant adenovirus indicate that Enho1 reduces hyperinsulinemia and hyperlipidemia independently of effects on obesity. Further experiments using recombinant adenovirus and synthetic peptide may therefore provide important information regarding ‘direct’ effects of Enho1 on liver metabolism and insulin sensitivity.
  • RIA/ELISA assays
  • Mice shall be weighed on the day of adenovirus injection, and pre- and post-fasting. If possible, body composition shall be determined using nuclear magnetic resonance (NMR) (15,32,46). Mice shall be acclimated to housing in wire-mesh caging that allows for measurement of food intake and spillage, as previously described (15).
  • NMR nuclear magnetic resonance
  • the Ad5 experiments will be modified to investigate the response of males and females. Most of the experiments investigating the response of obese insulin resistant mice to Ad5-Enho1 treatment used males; it may be that females will exhibit a different response, perhaps exhibiting weight loss in addition to improved insulin sensitivity. This would not be unprecedented, for example sexual dimorphism has been observed in the response of male and female rats to the anorectic actions of insulin and leptin (47,48).
  • Ad5-ENHO1 will increase insulin-stimulated activity of the insulin receptor tyrosine kinase cascade in liver.
  • Ad5-ENHO1 may also improve insulin signaling in muscle and/or adipose tissue.
  • VO2 and RER of obese Ay/a and lean and diet-induced obese C57BL/6J mice infected with Ad5-Enho1 or Ad5-GFP shall be measured using indirect calorimetry (15,46,50).
  • the Pennington Biomedical Research Center has a 16 chamber comprehensive laboratory animal monitoring system (CLAMS) housed in a temperature controlled incubator.
  • CLAMS simultaneously measures oxygen consumption (VO2), respiratory exchange ratio (RER, an indicator of whole animal substrate oxidation), physical activity in the X and Z axis, and food intake.
  • mice shall be placed in the CLAMS system 96 h after adenovirus injection, and the parameters indicated recorded for 72 h. Mice shall be allowed to feed ad libitum for 48 h, with a fast for final 24 h.
  • transcription factors i.e., SREBP1c, PPAR ⁇
  • mice infected with Ad5-Enho1 indicate increased basal metabolic rate, a finding corroborated by fasting weight loss and indirect calorimetry data from BAP-Enho1 transgenics.
  • FIGS. 13A-F It is predicted that infection with Ad5-Enho1 will increase VO2, although this may only be evident during the fasting phase.
  • An increase in mitochondrial oxidative enzyme activity in liver only would be consistent with Enho1 acting as an autocrine/paracrine factor.
  • Increased mitochondrial oxidative enzyme activity in skeletal muscle and brown adipose tissue would suggest an endocrine function, either acting through the autonomic nervous system or through receptors expressed in muscle and/or brown adipose tissue.
  • Ad5-Enho1 is markedly increasing energy expenditure (as observed in FVB.Tg) but is not affecting body weight, then a compensatory increase in food intake would be predicted. A reduction in the expression of genes involved in lipogenesis, as observed in OBOB mice treated with Ad5-Enho1, is also predicted. ( FIG. 8A-8D ).
  • Transgenics A sequence encoding the 76 aa protein has been ligated into a synthetic transgene controlled by the human ⁇ -actin promoter (BAP) ( FIG. 9A ).
  • Enho1 is a secreted polypeptide ( FIG. 4 ), and thus tissue-selectivity is not important for these transgenic studies. Promoters specific for tissues have not been used where the endogenous gene is abundantly expressed, because suppression of the endogenous gene may limit efficacy of over expression (53,54).
  • a comprehensive analysis of mRNA expression shall be completed using quantitative RT-PCR (qRT-PCR) as previously described (31,32,46) and Northern blot analysis.
  • Protein levels shall be measured by Western blot using the two polyclonal antibodies against the N- and C-terminus of Enho 34-76 (pAB1, pAB2) ( FIG. 5 ). This may require using immunoprecitipitation to detect protein. Alternatively, if antibodies in hand or in development are useful for developing sensitive and quantitative assays, then these shall be used. Transgene copy number shall be determined by Southern blot analysis. A sub-aim of this experiment is therefore a more comprehensive analysis of Enho1 mRNA expression in mouse tissues (liver, hypothalamus, forebrain, hindbrain, skeletal and cardiac muscle, retroperitoneal and inguinal adipose depots, stomach, intestine, pancreas, kidney). Major organs (heart, kidney, gut, liver) shall be weighed and inspected histologically for major morphological changes.
  • Enho1 is one of a small group of secreted polypeptides (leptin, adiponectin) that, when over expressed, improves metabolic profile (i.e. increased insulin sensitivity, reduced hepatic lipogenesis) in mouse models of obesity and insulin resistance.
  • the over expression of Enho1 has leptin-like effects on energy metabolism, protecting against diet-induced obesity and insulin resistance.
  • Administering Enho1 can reverse insulin resistance and dyslipidemia associated with diet- and genetically-induced obesity, and can prevent or delay onset of diabesity.
  • Examples 16 to 25 were carried out using human Enho1, a 76 amino acid peptide having the sequence:
  • the effect of Enho-1 upon weight loss was tested over the course of 3 days using KKAy mice.
  • the mice were fed Breeder Chow (Purina 5015) and were approximately 12-14 weeks of age at the start of the experiment.
  • Six control mice received vehicle only while six test mice each received doses of either 900 nmol/kg/d or 9000 nmol/kg/d.
  • the injections were given as 3 intraperitoneal (ip) injections at 0600, 1400 and 2000 h on days 1 and 2; over the 3 day period, the mice received a total of 7 injections. All animals were euthanized 5 hours after the last injection given the morning of the third day. Food was removed after the final injection.
  • the six control mice had a mean pre-treatment weight of 35.3 g (SE ⁇ 1.2 g) and a mean post-treatment weight of 33.6 g (SE ⁇ 1.0).
  • the difference of ⁇ 1.7 g (SE ⁇ 0.3) represents a ⁇ 4.8% (SE ⁇ 0.6) reduction in weight.
  • the six test mice which received 900 nmol/kg/d of Enho 1 had a pre-treatment weight of 35.3 g (SE ⁇ 1.1) and post-treatment weight of 33.0 g (SE ⁇ 1.0).
  • the difference of ⁇ 2.3 g (SE ⁇ 0.2) represents a ⁇ 6.5% (SE ⁇ 0.6) reduction in weight.
  • the six test mice which received 9000 nmol/kg/d of Enho 1 had a pre-treatment weight of 35.6 g (SE ⁇ 0.6) and post-treatment weight of 33.1 g (SE ⁇ 0.4).
  • the difference of ⁇ 2.6 g (SE ⁇ 0.3) represents a ⁇ 7.1% (SE ⁇ 0.8) reduction in weight.
  • the effect of Enho-1 upon insulin and glucose was tested over the course of 3 days using KKAy mice.
  • the mice were fed Breeder Chow (Purina 5015) and were approximately 12-14 weeks of age at the start of the experiment.
  • Six control mice received vehicle only while five or six test mice each received doses of either 900 nmol/kg/d or 9000 nmol/kg/d, respectively.
  • the injections were given as 3 ip injections at 0600, 1400 and 2000 h on days 1 and 2; over the 3 day period, the mice received a total of 7 injections. All animals were euthanized 5 hours after the last injection given the morning of the third day. Food was removed after the final injection.
  • the HOMA-IR for each treatment was calculated using the following formula: ((glucose mg/dL ⁇ 18) ⁇ (insulin ng/ml ⁇ 25.05)) ⁇ 22.5
  • the six control mice demonstrated a post-test mean blood glucose level of 524 mg/dL (SE ⁇ 48) and an insulin level of 7.4 ng/ml (SE ⁇ 0.6); the HOMA-IR value for the control group was determined to be 234 (SE ⁇ 16).
  • the five test mice which received 900 nmol/kg/d of Enho 1 demonstrated a posttest mean blood glucose level of 474 mg/dL (SE ⁇ 29) and an insulin level of 5.7 ng/ml (SE ⁇ 0.5); the HOMA-IR value for this group was determined to be 167 (SE ⁇ 18), representing a decrease of 29% as compared to the control group.
  • the blood glucose levels decrease by approximately 10% while the insulin levels were reduced by about 22%.
  • the six test mice which received 9000 nmol/kg/d of Enho 1 demonstrated a posttest mean blood glucose level of 480 mg/dL (SE ⁇ 38) and an insulin level of 7.0 ng/ml (SE ⁇ 0.8); the HOMA-IR value for this group was determined to be 213 (SE ⁇ 37), representing a decrease of 9% as compared to the control group.
  • mice were fed Breeder Chow (Purina 5015) and were approximately 12-14 weeks of age at the start of the experiment.
  • Six control mice received vehicle only while five or six test mice each received doses of either 900 nmol/kg/d or 9000 nmol/kg/d, respectively.
  • the injections were given as 3 ip injections at 0600, 1400 and 2000 h on days 1 and 2; over the 3 day period, the mice received a total of 7 injections. All animals were euthanized 5 hours after the last injection given the morning of the third day. Food was removed after the final injection.
  • the six control mice exhibited a mean liver weight of 1.6 g (SE ⁇ 0.1), representing 4.6% (SE ⁇ 0.2) of total body weight.
  • the mean liver lipid level for the control group was 94 mg/g (SE ⁇ 6), with total lipids measuring 147 mg/g (SE ⁇ 14).
  • the five test mice which received 900 nmol/kg/d of Enho 1 demonstrated a posttest mean liver weight of 1.6 g (SE ⁇ 0.1), representing 5.0% (SE ⁇ 0.2) of body weight.
  • the mean liver lipid level for this group was determined to be 82 mg/g (SE ⁇ 9), with total lipids measuring 137 mg/g (SE ⁇ 18).
  • the six test mice which received 9000 nmol/kg/d of Enho 1 demonstrated a posttest mean liver weight of 1.6 g (SE ⁇ 0.1), representing 4.9% (SE ⁇ 0.3) of body weight.
  • the mean liver lipid level for this group was determined to be 87 mg/g (SE ⁇ 6), with total lipids measuring 143 mg/g (SE ⁇ 17).
  • Enho 1 is capable of inducing a dose-dependent reversal of hepatic steatosis.
  • mice receiving the 900 nmol/kg/d and the 9000 nmol/kg/d dosage the livers, the TG was reduced by 32% and 46%, respectively, as compared to the control group.
  • Enho-1 upon weight loss was tested over the course of 3 days using diet induced obese (DIO) C57BL/6J mice (Jackson Laboratories, Bar Harbor, Me.). The mice were fed Research Diets 12492 (60% kJ/fat) for 12 weeks, resulting in obesity and moderate hyperglycemia in the animals. The mice were approximately 20-22 weeks of age and 30-50 g in weight at the start of the experiment. Fasting blood glucose was determined to be approximately 170-220 mg/dL. Six control mice received vehicle only while six test mice each received doses of either 90 nmol/kg/d, 900 nmol/kg/d or 9000 nmol/kg/d.
  • mice exhibited mean pre- and post-treatment weights of 42.7 g (SE ⁇ 2.4 g) and 41.7 g (SE ⁇ 2.3), respectively, representing a 2.4% (SE ⁇ 0.5) reduction in total body weight.
  • the six test mice receiving 90 nmol/kg/d Enho 1 exhibited mean pre- and post-treatment weights of 42.9 g (SE ⁇ 1.2) and 41.7 g (SD ⁇ 0.9), respectively, representing a 2.9% (SE ⁇ 0.6) reduction in total body weight.
  • the six test mice receiving 900 nmol/kg/d Enho 1 exhibited mean pre- and post-treatment weights of 42.8 g (SE ⁇ 2.9) and 41.3 g (SE ⁇ 2.7), respectively, representing a 3.5% (SE ⁇ 0.7) reduction in total body weight.
  • the six test mice receiving 9000 nmol/kg/d Enho 1 exhibited mean pre- and post-treatment weights of 42.7 g (SE ⁇ 1.6) and 40.9 g (SE ⁇ 1.6), respectively, representing a 4.1% (SE ⁇ 0.6) reduction in total body weight.
  • the HOMA-IR for each treatment was calculated using the following formula: ((glucose mg/dL ⁇ 18) ⁇ (insulin ng/ml ⁇ 25.05)) ⁇ 22.5
  • the six test mice which received 90 nmol/kg/d of Enho1 demonstrated a post-test mean blood glucose level of 204 mg/dL (SE ⁇ 7) and an insulin level of 3.8 ng/ml (SE ⁇ 0.4); the HOMA-IR value for this group was determined to be 50 (SE ⁇ 5), representing a decrease of 15% as compared to the control group.
  • the six test mice which received 900 nmol/kg/d of Enho 1 demonstrated a posttest mean blood glucose level of 166 mg/dL (SE ⁇ 13; P>0.05) and an insulin level of 4.2 ng/ml (SE ⁇ 0.5); the HOMA-IR value for this group was determined to be 42 (SE ⁇ 3), representing a decrease of 29% (p ⁇ 0.02) as compared to the control group.
  • the blood glucose levels decreased by approximately 15% while the insulin levels were reduced by about 14%.
  • the six test mice which received 9000 nmol/kg/d of Enho 1 demonstrated a posttest mean blood glucose level of 198 mg/dL (SE ⁇ 9) and an insulin level of 4.2 ng/ml (SE ⁇ 0.3); the HOMA-IR value for this group was determined to be 52 (SE ⁇ 4), representing a decrease of 12% as compared to the control group.
  • the six control mice exhibited a mean liver weight of 1.6 g (SE ⁇ 0.2), representing 3.7% (SE ⁇ 0.2) of total body weight.
  • the mean liver lipid level for the control group was 155 mg/g (SE ⁇ 39), with total lipids measuring 272 mg/g (SE ⁇ 89).
  • Liver TG for the control group was determined to be 58 mg/g (SE ⁇ 11) while the total liver TG was determined to be 87 mg/g (SE ⁇ 15).
  • the six test mice which received 90 nmol/kg/d of Enho 1 demonstrated a post-test mean liver weight of 1.5 g (SE ⁇ 0.0), representing 3.6% (SE ⁇ 0.1) of body weight.
  • the mean liver lipid level for this group was determined to be 126 mg/g (SE ⁇ 9), with total lipids measuring 188 mg/g (SE ⁇ 13).
  • Liver TG for this group was determined to be 57 mg/g (SE ⁇ 8) while the total liver TG was determined to be 86 mg/g (SE ⁇ 14).
  • the six test mice which received 900 nmol/kg/d of Enho 1 demonstrated a posttest mean liver weight of 1.5 g (SE ⁇ 0.2), representing 3.5% (SE ⁇ 0.2) of body weight.
  • the mean liver lipid level for this group was determined to be 137 mg/g (SE ⁇ 34), with total lipids measuring 226 mg/g (SE ⁇ 77).
  • Liver TG for this group was determined to be 48 mg/g (SE ⁇ 12) while the total liver TG was determined to be 66 mg/g (SE ⁇ 14).
  • the six test mice which received 9000 nmol/kg/d of Enho 1 demonstrated a posttest mean liver weight of 1.4 g (SE ⁇ 0.2), representing 3.4% (SE ⁇ 0.3) of body weight.
  • the mean liver lipid level for this group was determined to be 139 mg/g (SE ⁇ 42), with total lipids measuring 230 mg/g (SE ⁇ 96).
  • Liver TG for this group was determined to be 47 mg/g (SE ⁇ 9) while the total liver TG was determined to be 63 mg/g (SE ⁇ 10).
  • Enho1 is capable of inducing a dose-dependent reversal of hepatic steatosis.
  • mice receiving the 9000 nmol/kg/d dosage the livers, the liver TG was reduced by 18-19% while serum TG was reduced by 20% (p ⁇ 0.05; 1-tailed t-test) as compared to the control group.
  • mice The effect of Enho-1 upon liver serum lipid content was tested over the course of 8 days using obese (ob/ob) mice.
  • the mice were fed Research Diets 12450 and were approximately 9-10 weeks of age at the start of the experiment.
  • Control mice received vehicle only while test mice each received doses of either 90 nmol/kg/d, 900 nmol/kg/d or 9000 nmol/kg/d.
  • the injections were given as 3 ip injections at 0800, 1200 and 1600 h for 7 days. On day 8, the mice were given the 0800 h injection and were sacrificed 1-3 h later, at which time blood samples were taken.
  • Blood glucose levels were measured using an Accu-Chek glucometer. Insulin levels were measured by ELISA (Mercodia Mouse Insulin ELISA, ALPCO) while triglycerides were measured using a Triglyceride L-Type TG H kit (Wako Diagnostics).
  • ELISA Mercodia Mouse Insulin ELISA, ALPCO
  • triglycerides were measured using a Triglyceride L-Type TG H kit (Wako Diagnostics).
  • mice which received 9000 nmol/kg/d of Enho1 demonstrated a post-test blood TG level of 22.0 mg/dL (SE ⁇ 2.2), a decrease of 26% from the control.
  • Enho-1 upon weight loss was tested over the course of 7 days using DIO C57BL/6J mice (Charles River Laboratories).
  • the mice were fed a high fat diet (Research Diets 12492) for 10 weeks prior to the start of the experiment, which was begun when the mice were 15-16 weeks of age.
  • Control mice received vehicle only while test mice each received doses of either 90 nmol/kg/d, 900 nmol/kg/d or 9000 nmol/kg/d.
  • the injections were given as 3 ip injections at 0800, 1200 and 1600 h for 7 days. On day 8, the mice were given the 0800 h injection and were sacrificed 1-3 h later.
  • mice exhibited mean pre- and post-treatment weights of 34.2 g (SE ⁇ 1.5 g) and 32.6 g (SE ⁇ 1.0), respectively, representing a 4.5% (SE ⁇ 1.5) reduction in total body weight.
  • test mice receiving 9000 nmol/kg/d Enho1 exhibited mean pre- and post-treatment weights of 33.8 g (SE ⁇ 10.8) and 31.6 g (SE ⁇ 0.7), respectively, representing a 6.7% (SE ⁇ 1.3) reduction in total body weight.
  • mice were fed a high fat diet (Research Diets 12492) for 10 weeks prior to the start of the experiment, which was begun when the mice were 15-16 weeks of age.
  • Control mice received vehicle only while test mice each received doses of either 90 nmol/kg/d, 900 nmol/kg/d or 9000 nmol/kg/d.
  • the injections were given as 3 ip injections at 0800, 1200 and 1600 h for 7 days. On day 8, the mice were given the 0800 h injection and were sacrificed 1-3 h later at which time blood samples were collected and levels of insulin and glucose determined.
  • the HOMA-IR for each treatment was calculated using the following formula: ((glucose mg/dL ⁇ 18) ⁇ (insulin ng/ml ⁇ 25.05)) ⁇ 22.5
  • mice demonstrated a post-test mean blood glucose level of 191 mg/dL (SE ⁇ 10) and an insulin level of 1.9 ng/ml (SE ⁇ 0.3); the HOMA-IR value for the control group was determined to be 22 (SE ⁇ 4).
  • the eight test mice which received 90 nmol/kg/d of Enho 1 demonstrated a posttest mean blood glucose level of 182 mg/dL (SE ⁇ 17) and an insulin level of 1.3 ng/ml (SE ⁇ 0.1); the HOMA-IR value for this group was determined to be 15 (SE ⁇ 2), representing a decrease of 15% as compared to the control group.
  • the seven test mice which received 900 nmol/kg/d of Enho1 demonstrated a post-test mean blood glucose level of 212 mg/dL (SE ⁇ 11) and an insulin level of 2.2 ng/ml (SE ⁇ 0.3); the HOMA-IR value for this group was determined to be 29 (SE ⁇ 4), representing a decrease of 29% (p ⁇ 0.02) as compared to the control group.
  • the seven test mice which received 9000 nmol/kg/d of Enho 1 demonstrated a post-test mean blood glucose level of 190 mg/dL (SE ⁇ 16) and an insulin level of 1.3 ng/ml (SE ⁇ 0.2); the HOMA-IR value for this group was determined to be 16 (SE ⁇ 4), representing a decrease of 27 as compared to the control group.
  • mice which received 9000 nmol/kg/d of Enho 1 demonstrated a post-test blood TG level of 18.5 mg/dL (SE ⁇ 1.7), a decrease of 19% from the control.
  • an “effective amount” of a Enho1 protein or peptide is an amount that decreases the level of insulin resistance or of dyslipidemia, or that prevents, delays or reduces the incidence of the onset of type 2 diabetes in obese insulin resistant patients by a statistically significant degree. “Statistical significance” is determined as the P ⁇ 0.05 level, or by such other measure of statistical significance as is commonly used in the art for a particular type of experimental determination.
  • Enho1 used herein and in the claims refers to the protein Enho1 (SEQ.ID.NO. 2), its functional peptides (e.g., Enho1 34-76 ), derivatives and analogs.
  • the terms “derivatives” and “analogs” are understood to be proteins that are similar in structure to Enho1 and that exhibit a qualitatively similar effect to the unmodified Enho1.
  • the term “functional peptide” refers to a piece of the Enho1 protein that still binds to the Enho1 receptor or is able to activate changes inside body cells, e.g., adipocytes or hepatocytes.
  • Enho1 its functional peptides, its analogs and derivatives in accordance with the present invention may be used to reverse insulin resistance and dyslipidemia, to delay onset to type 2 diabetes in obese insulin resistant subjects, and to prevent or delay onset of obesity.
  • These compounds can also be used as therapeutic or diagnostic agents for hypercholesterolemia, hypertriglyceridemia, insulin resistance, obesity, and diabetes.
  • terapéuticaally effective amount refers to an amount of Enho1 protein, a fragment, or a derivative or analog sufficient either increase body energy expenditure, decrease serum triglyceride, decrease serum cholesterol, decrease hyperlipidemia, or decrease insulin resistance to a statistically significant degree (p ⁇ 0.05).
  • the dosage ranges for the administration of Enho1 protein are those that produce the desired effect. Generally, the dosage will vary with the age, weight, condition, sex of the patient. A person of ordinary skill in the art, given the teachings of the present specification, may readily determine suitable dosage ranges. The dosage can be adjusted by the individual physician in the event of any contraindications.
  • the effectiveness of treatment can be determined by monitoring either the body metabolism, body weight, or the serum glucose, triglyceride, cholesterol levels by methods well known to those in the field.
  • Enho1 can be applied in pharmaceutically acceptable carriers known in the art.
  • This method of treatment may be used in vertebrates generally, including human and non-human mammals.
  • Peptides in accordance with the present invention may be administered to a patient by any suitable means, including oral, intravenous, parenteral, subcutaneous, intrapulmonary, and intranasal administration.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, or intraperitoneal administration.
  • the compound may also be administered transdermally, for example in the form of a slow-release subcutaneous implant, or orally in the form of capsules, powders, or granules. It may also be administered by inhalation.
  • compositions for parenteral administration include sterile, aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • the active therapeutic ingredient may be mixed with excipients that are pharmaceutically acceptable and are compatible with the active ingredient.
  • Suitable excipients include water, saline, dextrose, glycerol and ethanol, or combinations thereof.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like.
  • Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, inert gases, and the like.
  • compositions for injection may be provided in the form of an ampule, each containing a unit dose amount, or in the form of a container containing multiple doses.
  • the compound may be formulated into therapeutic compositions as pharmaceutically acceptable salts.
  • These salts include acid addition salts formed with inorganic acids, for example hydrochloric or phosphoric acid, or organic acids such as acetic, oxalic, or tartaric acid, and the like. Salts also include those formed from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and organic bases such as isopropylamine, trimethylamine, histidine, procaine and the like.
  • the compositions may be administered intravenously, subcutaneously, intramuscularly.
  • Controlled delivery may be achieved by admixing the active ingredient with appropriate macromolecules, for example, polyesters, polyamino acids, polyvinyl pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, prolamine sulfate, or lactide/glycolide copolymers.
  • suitable macromolecules for example, polyesters, polyamino acids, polyvinyl pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, prolamine sulfate, or lactide/glycolide copolymers.
  • the rate of release of the active compound may be controlled by altering the concentration of the macromolecule.
  • Another method for controlling the duration of action comprises incorporating the active compound into particles of a polymeric substance such as a polyester, peptide, hydrogel, polylactide/glycolide copolymer, or ethylenevinylacetate copolymers.
  • an active compound may be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, by the use of hydroxymethylcellulose or gelatin-microcapsules or poly(methylmethacrylate) microcapsules, respectively, or in a colloid drug delivery system.
  • Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • nucleic acid sequence (SEQ ID NO: 1) (e.g., the open reading frame underlined in FIG. 4 ) can be used to make plasmids or vectors to incorporate the Enho1 gene into organisms to increase the production of the Enho1 protein.
  • nucleic acid sequence of SEQ ID NO: 1 is not the only sequence that can be used to produce the Enho1 protein.
  • the genetic code may be found in numerous references concerning genetics or biology, including, for example, FIG. 9.1 on page 214 of B. Lewin, Genes VI (Oxford University Press, New York, 1997).
  • FIG. 9.3 on page 216 of Lewin directly illustrates the degeneracy of the genetic code.
  • the codon for asparagine may be AAT or AAC.
  • the invention also encompasses nucleotide sequences encoding Enho1 proteins having one or more silent amino acid changes in portions of the molecule not involved with receptor binding or protein secretion.
  • alterations in the nucleotide sequence that result in the production of a chemically equivalent amino acid at a given site are contemplated; thus, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another hydrophobic residue, such as glycine, or may be substituted with a more hydrophobic residue such as valine, leucine, or isoleucine.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Obesity (AREA)
  • Hematology (AREA)
  • Diabetes (AREA)
  • Endocrinology (AREA)
  • Biophysics (AREA)
  • Toxicology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Marine Sciences & Fisheries (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
US12/012,627 2005-08-05 2008-02-05 Novel peptide involved in energy homeostasis Abandoned US20090253619A1 (en)

Priority Applications (4)

Application Number Priority Date Filing Date Title
US12/012,627 US20090253619A1 (en) 2005-08-05 2008-02-05 Novel peptide involved in energy homeostasis
US12/848,308 US8518892B2 (en) 2005-08-05 2010-08-02 Methods to treat symptoms of pathophysiology related to body mass
US13/850,806 US20130231280A1 (en) 2005-08-05 2013-03-26 Novel Peptide Involved in Energy Homeostasis
US14/922,557 US20160102126A1 (en) 2005-08-05 2015-10-26 Novel Peptides Involved in Energy Homeostasis

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US70594005P 2005-08-05 2005-08-05
PCT/US2006/030686 WO2007019426A2 (fr) 2005-08-05 2006-08-07 Nouveau peptide intervenant dans l'homeostasie energetique
US12/012,627 US20090253619A1 (en) 2005-08-05 2008-02-05 Novel peptide involved in energy homeostasis

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2006/030686 Continuation-In-Part WO2007019426A2 (fr) 2005-08-05 2006-08-07 Nouveau peptide intervenant dans l'homeostasie energetique

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US12/848,308 Continuation US8518892B2 (en) 2005-08-05 2010-08-02 Methods to treat symptoms of pathophysiology related to body mass

Publications (1)

Publication Number Publication Date
US20090253619A1 true US20090253619A1 (en) 2009-10-08

Family

ID=37727967

Family Applications (4)

Application Number Title Priority Date Filing Date
US12/012,627 Abandoned US20090253619A1 (en) 2005-08-05 2008-02-05 Novel peptide involved in energy homeostasis
US12/848,308 Expired - Fee Related US8518892B2 (en) 2005-08-05 2010-08-02 Methods to treat symptoms of pathophysiology related to body mass
US13/850,806 Abandoned US20130231280A1 (en) 2005-08-05 2013-03-26 Novel Peptide Involved in Energy Homeostasis
US14/922,557 Abandoned US20160102126A1 (en) 2005-08-05 2015-10-26 Novel Peptides Involved in Energy Homeostasis

Family Applications After (3)

Application Number Title Priority Date Filing Date
US12/848,308 Expired - Fee Related US8518892B2 (en) 2005-08-05 2010-08-02 Methods to treat symptoms of pathophysiology related to body mass
US13/850,806 Abandoned US20130231280A1 (en) 2005-08-05 2013-03-26 Novel Peptide Involved in Energy Homeostasis
US14/922,557 Abandoned US20160102126A1 (en) 2005-08-05 2015-10-26 Novel Peptides Involved in Energy Homeostasis

Country Status (10)

Country Link
US (4) US20090253619A1 (fr)
EP (1) EP1928487A4 (fr)
JP (1) JP2009502208A (fr)
KR (1) KR101004662B1 (fr)
CN (1) CN101505786A (fr)
AU (2) AU2006278406B2 (fr)
CA (1) CA2618047C (fr)
IL (2) IL189065A (fr)
RU (2) RU2409590C2 (fr)
WO (1) WO2007019426A2 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8450350B2 (en) 2010-05-05 2013-05-28 Infinity Pharmaceuticals, Inc. Triazoles as inhibitors of fatty acid synthase
US8546432B2 (en) 2010-05-05 2013-10-01 Infinity Pharmaceuticals, Inc. Tetrazolones as inhibitors of fatty acid synthase
WO2020247012A1 (fr) * 2018-06-08 2020-12-10 Saint Louis University Procédé et compositions pour traiter une capacité cognitive réduite
EP3675893A4 (fr) * 2017-08-30 2021-05-26 Hadasit Medical Research Services&Development Ltd. Méthode de traitement et de diagnostic d'un cancer du foie métastatique

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110028570A (zh) * 2018-10-31 2019-07-19 华中科技大学 一种肌肉素的表达方法及其在代谢疾病中的应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030096951A1 (en) * 1998-08-14 2003-05-22 Kenneth Jacobs Secreted proteins and polynucleotides encoding them

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2339047A1 (fr) * 1998-08-14 2000-02-24 Genetics Institute, Inc. Proteines secretees et polynucleotides les codant
US6783969B1 (en) * 2001-03-05 2004-08-31 Nuvelo, Inc. Cathepsin V-like polypeptides

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030096951A1 (en) * 1998-08-14 2003-05-22 Kenneth Jacobs Secreted proteins and polynucleotides encoding them
US7344861B2 (en) * 1998-08-14 2008-03-18 Ono Pharmaceutical Co., Ltd. Secreted proteins and polynucleotides encoding them

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8450350B2 (en) 2010-05-05 2013-05-28 Infinity Pharmaceuticals, Inc. Triazoles as inhibitors of fatty acid synthase
US8546432B2 (en) 2010-05-05 2013-10-01 Infinity Pharmaceuticals, Inc. Tetrazolones as inhibitors of fatty acid synthase
US9346769B2 (en) 2010-05-05 2016-05-24 Infinity Pharmaceuticals, Inc. Tetrazolones as inhibitors of fatty acid synthase
EP3675893A4 (fr) * 2017-08-30 2021-05-26 Hadasit Medical Research Services&Development Ltd. Méthode de traitement et de diagnostic d'un cancer du foie métastatique
US11246912B2 (en) * 2017-08-30 2022-02-15 Hadasit Medical Research Services & Development Ltd. Methods for treating and diagnosing metastatic liver cancer
WO2020247012A1 (fr) * 2018-06-08 2020-12-10 Saint Louis University Procédé et compositions pour traiter une capacité cognitive réduite

Also Published As

Publication number Publication date
US20100299769A1 (en) 2010-11-25
US20130231280A1 (en) 2013-09-05
IL189065A0 (en) 2008-08-07
IL189065A (en) 2012-10-31
AU2006278406B2 (en) 2010-03-25
AU2010202600A1 (en) 2010-07-15
CA2618047C (fr) 2015-04-14
US8518892B2 (en) 2013-08-27
KR101004662B1 (ko) 2011-01-03
KR20080042866A (ko) 2008-05-15
JP2009502208A (ja) 2009-01-29
CA2618047A1 (fr) 2007-02-15
IL207764A0 (en) 2010-12-30
EP1928487A4 (fr) 2009-11-04
RU2409590C2 (ru) 2011-01-20
CN101505786A (zh) 2009-08-12
AU2006278406A1 (en) 2007-02-15
WO2007019426A2 (fr) 2007-02-15
RU2010119559A (ru) 2011-11-20
AU2010202600B2 (en) 2012-07-19
EP1928487A2 (fr) 2008-06-11
WO2007019426A3 (fr) 2009-04-23
US20160102126A1 (en) 2016-04-14
RU2008108509A (ru) 2009-09-10

Similar Documents

Publication Publication Date Title
US20160102126A1 (en) Novel Peptides Involved in Energy Homeostasis
US10172918B2 (en) SorCS1 for the treatment of obesity
EP1180114B1 (fr) Utilisation d'anticorps spécifiques contre la resistine dans le traitement du diabète
Lee et al. SENP2 suppresses browning of white adipose tissues by de-conjugating SUMO from C/EBPβ
CA2422068A1 (fr) Procede de traitement de la resistance a l'insuline dans l'obesite et le diabete
US20090104203A1 (en) Compositions and Methods for Regulating Osteoclast Differentiation, Activation and Bone Resorption
WO2023010049A1 (fr) Utilisations thérapeutiques de la protéine isthmine
AU2020346948A1 (en) Application of non-IGF1R-binding substance in prevention and/or treatment of inflammatory diseases
WO2007002830A2 (fr) Diagnostic d'un hyperinsulinisme et d'un diabete de type ii et protection contre ceux-ci a partir de proteines exprimees de façon differentielle dans la peau et identifiees par proteomique
Ono et al. sererer denser kkk kkkk
Rajala Functional characterization of resistin, a novel adipokine
Patel A role for CEACAM2 in insulin homeostasis and action
Pajvani Functional consequences of adiponectin oligomerization state: The role of adiponectin in maintaining systemic insulin sensitivity
US20120079613A1 (en) Homeobox Transcription Factor BSX and Uses Thereof for Treating Diseases, in Particular Obesity

Legal Events

Date Code Title Description
AS Assignment

Owner name: BOARD OF SUPERVISORS OF LOUISIANA STATE UNIVERSITY

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:BUTLER, ANDREW A.;TREVASKIS, JAMES;REEL/FRAME:023238/0555

Effective date: 20090825

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION