WO2002097441A2 - Proteines de mammifere mediatrices du diabete - Google Patents

Proteines de mammifere mediatrices du diabete Download PDF

Info

Publication number
WO2002097441A2
WO2002097441A2 PCT/DK2002/000368 DK0200368W WO02097441A2 WO 2002097441 A2 WO2002097441 A2 WO 2002097441A2 DK 0200368 W DK0200368 W DK 0200368W WO 02097441 A2 WO02097441 A2 WO 02097441A2
Authority
WO
WIPO (PCT)
Prior art keywords
protein
proteins
diabetes
derivative
group
Prior art date
Application number
PCT/DK2002/000368
Other languages
English (en)
Other versions
WO2002097441A3 (fr
Inventor
Peter Mose Larsen
Stephen J. Fey
Allan E. Karlsen
Thomas Sparre
Jørn NERUP
Original Assignee
Syddansk Universitet
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 Syddansk Universitet filed Critical Syddansk Universitet
Priority to EP02742844A priority Critical patent/EP1421390A2/fr
Priority to AU2002344252A priority patent/AU2002344252A1/en
Publication of WO2002097441A2 publication Critical patent/WO2002097441A2/fr
Publication of WO2002097441A3 publication Critical patent/WO2002097441A3/fr
Priority to US10/960,652 priority patent/US7279274B2/en
Priority to US11/869,546 priority patent/US20080145868A1/en

Links

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
    • 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism

Definitions

  • Proteome analysis has allowed for the identification of proteins and their association to diabetes. These proteins, in themselves, either up-regulated or down-regulated, are indicators of diabetes in a patient. The pattern of regulation of a grouping of these proteins also serves as an indicator of diabetes. These proteins can be used as targets for the treatment of diabetes or for treatment itself. The proteins were identified by monitoring IL-l ⁇ induced protein changes in diabetes prone mammalian islets of Langerhans.
  • Type 1 Diabetes Mellitus is a multifactorial polygenetic autoimmune disease, where the insulin producing ⁇ -cells are selectively destroyed.
  • the initiating events and precise mechanisms leading to selective ⁇ -cell destruction remains unknown.
  • One current hypothesis [3] is that in genetically predisposed individuals the ⁇ -cells are influenced by factors from the internal or external environment which can damage the ⁇ -cells (e.g. cytokines, virus and chemicals) and then lead to release of ⁇ -cell specific proteins.
  • IL-l ⁇ is released by macrophages in the islets and the cytotoxic effects of IL-l ⁇ on the ⁇ -cells results in production of free radicals (e.g.
  • nitric oxide NO
  • super oxide (0 2 »)
  • hydroxyl OH ""
  • Free radicals and NO* are also produced in and secreted from macrophages in the islet infiltrate.
  • the effects of free radicals are attempted scavenged by ⁇ -cell protective proteins (e.g. haeme oxygenases and manganese superoxide dismutase (MnSOD)) [25, 26], A race between protective and destructive mechanisms is initiated, and when the destructive mechanisms exceed the protective mechanisms, the ⁇ -cells die [3].
  • ⁇ -cell protective proteins e.g. haeme oxygenases and manganese superoxide dismutase (MnSOD)
  • T1DM Autoimmune insulin-dependent diabetes mellitus
  • IL-l ⁇ interleukin-l ⁇
  • TNF- ⁇ tumor necrosis factor- ⁇
  • IFN- ⁇ Interferon- ⁇
  • Cytokines have been demonstrated to induce free radicals such as nitric oxide (NO «), catalyzed by inducible nitric oxide synthase (iNOS) and oxygen derived radical.
  • T1DM Type 1 Diabetes Mellitus
  • jnsulitis Langerhans
  • ⁇ -cells ⁇ -cells
  • Cytokines in particular interleukin-l ⁇ (IL-l ⁇ )
  • IL-l ⁇ interleukin-l ⁇
  • IL-l ⁇ influences many important cellular functions such as decreasing DNA synthesis, decreasing protein synthesis and intracellular energy production and induction of apoptosis. Many of these effects are mediated through induction of the inducible NO syntase (iNOS) and its product, the free radical nitric oxide (NO*) [5].
  • iNOS inducible NO syntase
  • NO* free radical nitric oxide
  • WF Wistar Furth
  • IL-l ⁇ induced significant changes in expression level of 105 proteins in WF islets where both protective proteins e.g. such as gaIectin-3 and HSP70 are up-regulated and deleterious protein changes e.g. mortalin and lamin A and Bl, were identified.
  • protective proteins e.g. such as gaIectin-3 and HSP70
  • deleterious protein changes e.g. mortalin and lamin A and Bl
  • proteins involved in mitochondrial energy production were suppressed e.g. adenylate kinase and mitochondrial ATP synthase regulatory subunit B [7].
  • the BioBreeding diabetes prone (BB-DP) rat spontaneously develops a diabetic syndrome with many characteristics common with human T1DM [8].
  • BB-DP rat strain has been breed from a WF rat colony [9]
  • Strain-dependent variations in ⁇ -cell sensitivity to IL-1B effects have been demonstrated in vitro and in vivo [10] [11].
  • Islets isolated from Brown Norway rats were less sensitive to IL-l ⁇ compared to Wistar Furth (WF), Lewis-Scripps (LS) and BB (both diabetes prone (DP) and diabetes resistant (DR)) rats.
  • WF Wistar Furth
  • LS Lewis-Scripps
  • BB both diabetes prone (DP) and diabetes resistant (DR) rats.
  • the BB-DP rat islets produce lower protective stress responses (HSP70) than the diabetes resistant BB rat which might promote enhanced vulnerability and ⁇ -cell destruction £2].
  • HSP70 protective stress responses
  • the present investigators have previously shown that there is no difference in nitric oxide (NO*) production and 24 hour accumulated insulin release in BB-DP and WF islets in response to exposure to 150pg/mi IL-l ⁇ for 24 hours [13].
  • NO* nitric oxide
  • the higher resistance to IL-1B induced inhibition of ⁇ -cell function in vitro and in vivo in BN rat islets was associated with lower expression of inducible nitric oxide synthase (iNOS) compared to Wistar Kyoto and LS rats [11].
  • iNOS inducible nitric oxide synthase
  • IL-l ⁇ induces reproducible and statistically significant changes in the expression level of 82 protein spots in BB-DP rat islets of Langerhans in vitro out of a total of 1.815 protein spots visualized by 2 dimensional gel electrophoresis. Twenty-two protein spots were up-regulated and 60 protein spots down-regulated [13].
  • the present investigators have identified proteins associated with diabetes by detecting the absolute or relative presence of the proteins of tables 1, 2 and 3 in a biological sample.
  • the biological sample is selected from the group consisting of urine, blood, CSF, saliva, lymphatic fluids, and tissue.
  • the tissue is pancreatic islets of Langerhans.
  • Proteome analysis applied to BB-DP rat islets exposed to IL-l ⁇ reveals insight into mechanisms responsible for ⁇ -cell destruction at the protein level as well as identifying proteins of relevance in the treatment of diabetes.
  • Furthermore comparison of protein changes identified by proteome analysis in WF and BB-DP rat islets exposed to IL-l ⁇ identify proteins and pathways involved in ⁇ -cell destruction specific for the diabetes prone BB rat.
  • the present invention relates in a first aspect to the novel proteins identified by proteome analysis and mass spectrometry.
  • a further aspect relates to the use of novel proteins and proteins of table 1 or table 2 or the tables of the mass spectra MWts for the previously unidentified proteins as markers or indicators for diabetes as well as to the use of known proteins whose presence, absence or prevalence has previously not been associated with diabetes.
  • the changes in protein expression and patterns of protein expression are considered to be important markers for diagnosis, prognosis and therapeutic applications and targets.
  • IL-l ⁇ induced protein changes in BB-DP islets from neonatal BB-DP rats islets were investigated by incubating BB-DP rats islets for 24 hours with or without recombinant human IL-l ⁇ . The medium was sampled for nitrite and insulin measurements [13]. Two dimensional gel analysis of the islets was performed and fluorographs of the islets were computer analysed. Protein spots that significantly changed expression level after exposure to IL-l ⁇ were cut out of preparatory gels and attempted identified by mass spectrometry.
  • Proteome analysis by 2-dimensional gel electrophoresis, mass spectrometry and bio- informatics, offered the opportunity to identify different pathways involved in IL-l ⁇ induced l ⁇ -cell destruction. Significantly changed proteins in BB-DP rat islets after IL-l ⁇ exposure was obtained.
  • the cytotoxic effect of cytokines on islets is inhibited by inhibitors of protein synthesis indicating that ⁇ -cell destruction is an active intracellular process requiring de novo synthesis of proteins.
  • the protein iNOS has previously been reported to change expression level after IL-l ⁇ exposure.
  • Heat shock protein 70, 90 and MnSOD others are has been referred to oxidative stress-proteins in connection to their protective response.
  • the proteins of the invention are involved in a variety of different pathways involved in ⁇ -cell destruction and may be classified according to the pathway.
  • the proteins of the invention may a) be involved in energy transduction and redox potentials and glycolytic enzymes (14 proteins); b) be involved in purine/pyrimidine synthesis, DNA/RNA synthesis, RNA processing, amino acid metabolism and protein synthesis (7 proteins); c) act as chaperones (5 proteins); d) be involved in differentiation, apoptosis, regulation and signal transduction; e) be involved in cellular defence; f) be involved in cellular structure; g) be involved in hormone/neurotransmitter metabolism; h) or have other functions.
  • proteins of the invention are involved in energy transduction and redox potentials and glycolytic enzymes (14 different proteins):
  • ATP synthase ATP synthase alpha chain
  • Vacuolar ATP synthase subunit B Alcohol dehydrogenase
  • Creatine kinase B chain was identified together with cytokeratine 8 polypeptide are each diabetes marker proteins of the present invention.
  • IL-l ⁇ induces NO production in the l ⁇ -cells which in turn nitrosylates the Fe-S complex in enzymes [31] and inactivates mitochondrial aconitase [32] whereby the oxidation of glucose in the Krebs Cycle is inhibited which results in decreased ATP production.
  • the malate dehydrogenase precursor has been found down-regulated in three spots, Aconitate hydratase, glyceralaldehyde-3-phosphate dehydrogenase, pyruvate kinase M2 isozyme and isocitrate dehydrogenase precursor are all down-regulated and involved in energy production in the Krebs Cycle.
  • Glucose-6-phosphate dehydrogenase the first step in the pentose phosphate pathway is up-regulated, which could be to compensate for the decreased energy production through the Krebs Cycle.
  • proteins involved in energy transduction and redox potentials are down-regulated in response to IL-l ⁇ exposure.
  • the proteins are H+ transporting ATP synthase, ATP synthase alpha chain, L-3-hydroxy-CoA dehydrogenase precursor, NADH dehydrogenase, carbonyl reductase, vacuolar ATP synthase subunit B and alcohol dehydrogenase.
  • Creatine kinase B chain was identified together with cytokeratine 8 polypeptide in an upregulated spot. Creatine kinase was found down-regulated in WF islets [7] indicating that creatine kinase could also be involved here.
  • proteins of the invention are involved in purine/pyrimidine synthesis, DNA/RNA synthesis, RNA processing, or amino acid metabolism and protein synthesis (8 different proteins:
  • Adenine phosphoribosyltransferase Heterogeneous nuclear ribonucleoprotein A2/1311; Heterogeneous nuclear ribonucleoprotein M (M4 protein deletion mutant); Aspartate aminotransferase (cytosolic); Aspartate aminotransferase (mitochondrial); and Ribosomal protein Lll are each further diabetes marker proteins of the present invention.
  • Heterogeneous nuclear ribonucleoprotein A2/1311 and M4 protein deletion mutant are both involved in processing of preRNA [33, 34] and were both down-regulated too.
  • Aspartate aminotransferase both the cytosolic and the mitochondrial form, involved in amino acid metabolism were down- regulated after exposure to IL-l ⁇ .
  • the down-regulation of all these proteins support that the ⁇ -cells focus their efforts in protecting themselves against the deleterious effects of IL-l ⁇ and down-regulate functions that are not involved in protection.
  • GRP 78 [39] 78kDa glucose regulated protein (GRP78);
  • Protein disulfide isomerase Protein disulfide isomerase
  • Calreticulin Calreticulin; Endoplasmic reticulum protein ERP 29; and
  • HSP70 are each further diabetes marker proteins of the present invention.
  • Endoplasmin one of two proteins in a down-regulated spot, belongs to the HSP 90 family which is involved in protein folding [38] and has been shown to have serine kinase activity which is enhanced by association with 78kDa glucose regulated protein (GRP78) [39].
  • GRP78 was found in three spots, one up-regulated, one down-regulated and together with endoplasmin in one down-regulated spot (demonstrating complex differential regulation of post-translational modifications). It is a member of the HSP 70 family involved in the folding and assembly of proteins in the endoplasmatic reticulum [40]. Endoplasmic reticulum protein ERP 29 is a member of the ER protein-processing machinery and is involved in protein secretory events. IL-l ⁇ exposure down-regulates this protein.
  • Protein disulfide isomerase involved in molecular chaperone of glycoprotein biosynthesis [41] is down-regulated by IL-l ⁇ together with probable disulfide isomerase P5. Both are involved in the formation and rearrangement of disulfide bonds in proteins. Interestingly, all these proteins are able to bind calcium like calreticulin [42] and may also be involved in signal transduction.
  • HSP70 expression has been demonstrated to diminish the inhibitory effect of IL-l ⁇ on islet insulin secretion and NO-induced mitochondrial impairment [43, 44].
  • insufficient HSP70 expression in islets has been correlated to sensitivity to NO * and oxygen radical toxicity [12].
  • Eukaryotic initiation factor 4A Eukaryotic initiation factor 4A
  • the Voltage dependent anion channel protein are each further diabetes marker proteins of the present invention.
  • IL-l ⁇ induces cell death through different pathways, among them gene controlled apoptosis [45-47].
  • Galectin-3 a protein involved in cell survival by inhibiting apoptosis [48 - 50] is up-regulated and present in 2 spots.
  • Eukaryotic initiation factor 4A like NUK 34, is involved in transcriptional/translational regulation [51] is up-regulated indicating specific activation of protein synthesis.
  • Toad 64 a protein up-regulated over the course of neurogenesis and playing a role in signal transduction processes involved in axon growing [52] is up-regulated after IL-l ⁇ exposure.
  • Calbindin D-28k Another calcium binding protein, secretagogin, specific for neuroendocrine cells and related to calbindin D-28k, is involved in cell growth and maturation [53] and is downregulated. Calbindin D-28k has been shown to be involved in protection from cytokine induced apoptosis in 13- ⁇ cells [54].
  • Voltage dependent anion channel is identified in a down-regulated spot together with L- 3-hydroxyacyl-CoA dehydrogenase precursor.
  • the Voltage dependent anion channel protein is able to form small pores in the outer mitochondrial membrane allowing movements of adenine nucleotides.
  • Bcl2 proteins binds to the channel in order to regulate the mitochondrial membrane potential and release of cytochrome c during apoptosis [57].
  • E. Cellular defence (2 proteins) Catalase; and Glutathione synthetase are each further diabetes marker proteins of the present invention.
  • Free radicals e.g. nitric oxide and hydrogen peroxide
  • Catalase which serves to protect cells from the toxic effects of hydrogen peroxide, is up-regulated In response to IL-l ⁇ exposure.
  • Glutathione synthetase is important for a variety of biologic functions including protection of cells from oxidative damage by free radicals and detoxifications [58, 60 and 61] and is here reduced to one eighth of the normal concentration (down-regulated) suggesting a reduced. potential for cellular defence against oxygen-derived free radicals.
  • Cellular structure (3 proteins) Tubulin beta-5 chain; Cytokeratin 8; and Keratin 2a are each further diabetes marker proteins of the present invention.
  • Tubulin beta-5 chain the major constituent of microtubules were present in 4 spots, possibly 4 different modifications, all down-regulated probably due to lower mitotic rate upon IL-l ⁇ exposure.
  • Cytokeratin 8 was present in the same up-regulated spot as creatine kinase-B. Cytokeratin 8 has been shown to play an essential role in regulation of growth and differentiation in the exocrine pancreas [62]. Furthermore keratin 2a is found together with tubulin beta-5 chain in a down-regulated spot.
  • Amyloid beta-peptide binding protein
  • Neuroendocrine convertase 2; and Beta-alanine oxoglutarate aminotransferase (GABA-transaminase) are each further diabetes marker proteins of the present invention.
  • Membrane associated progesterone component is a receptor for progesterone which is massively down-regulated after IL-l ⁇ exposure.
  • 25-Dx has 71 % sequence homology with the transmembrane domain of the precursor for the IL-6 receptor and a conserved consensus sequence found in the cytokine/growth factor prolactin receptor superfamily [63].
  • Amyloid beta-peptide binding protein is involved in androgen metabolism and has been postulated to be involved in apoptosis and amyloid toxicity [64]. Here, it is downregulated by lL-l ⁇ .
  • Beta-alanine oxoglutarate aminotransferase (GABA-transaminase) is responsible for the catabolism of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) and is down-regulated in IL- ⁇ exposed islets. It probably has a different function in the ⁇ -cells.
  • 5-aminolevulinate synthase precursor is a rate limiting nuclear-encoded mitochondrial enzyme in the heme biosy ⁇ thetic pathway which serve as part of several heme containing proteins such as hemoglobin and catalase.
  • IL-l ⁇ down-regulates the expression of 5-aminolevulinate synthase precursor and might then increase the ⁇ -cell susceptibility to IL-l ⁇ due to reduced levels of one of the substrates for catalase production.
  • cytomegalovirus protein immediate-early protein 1 has been identified in the BB-DP islets as down-regulated.
  • This protein is able to transactivate heterologous promoters [65] in rat cytomegalovirus.
  • the protein may come from a cytomegalovirus infection or may be a homologous rat protein.
  • proteins common for BB-DP and WF are marked with #. From Table 1 it is evident that proteins involved in chaperoning, protein folding and translocation and proteins involved in energy transduction and redox potentials are common pathways affected in the two strains. For both groups most of the proteins are down regulated suggesting that similar mechanisms are affected by IL-l ⁇ exposure in the two rat strains. Another pattern is seen for the glycolytic enzymes. Here the outcome of the changes in expression levels of proteins, a lower energy production, although there is only overlap in 2 out of 8 proteins in the BB-DP rat [7]. This suggests that IL-l ⁇ affects the glycolytic enzymes differently in the two rat strains with the same final result, a lower energy production.
  • catalase and glutathione synthetase are both changed in expression levels by IL-l ⁇ in BB-DP rats but not in WF rat islets.
  • the increased expression of catalase protects cells against the free radical hydrogen peroxide indicating that free radicals are present in the BB-DP islets after IL-l ⁇ exposure and that the islets are trying to protect themselves against the free radicals.
  • the previously described strain differences in rat islets regarding IL-l ⁇ sensitivity and NO* production after IL-l ⁇ exposure might be explained by different abilities to mount a free radical defense [10].
  • glutathione synthetase involved in energy requiring synthesis ATP is one of the substrates
  • glutathione detoxifying pathway is impaired by IL-l ⁇ in BB-DP rats.
  • IL-l ⁇ increased the NO* production over a 24 hour period, but iNOS was not identified as one of the up-regulated proteins. This could be explained by absence of IL- ⁇ in the 4 hours labeling period after the IL-l ⁇ incubation or that the difference in expression level does not fulfil our criteria for significant difference or is not present in the gels. The same was seen in WF islets incubated with IL-l ⁇ [7]. Increased mRNA levels for iNOS has been found in prediabetic NOD mice [67].
  • Some proteins are seen in more than one spot, e.g. calreticulin and GRP 78, which indicate that the protein is present in more than one form presumably due to post- translational modifications.
  • Post-translational modifications such as phosphorylations, methylations or glycosylations are important for the function or activation of the protein.
  • Proteins with known genes and chromosomal localisation close to known TI DM loci in the human genome might be of particular interest and might identify primary changes.
  • a first aspect of the invention relates to marker proteins.
  • the marker proteins are selected from the group consisting of: a) a protein of table 1, a protein of table 2, a protein of table 3, a protein of figure 1 (such as a protein marked with a number) and a protein having a spectrum as shown in any one of the figures 5-21; and b) a protein with at least 80% sequence homology with a protein in a).
  • the marker protein can be selected from the group consisting of: a) one or more proteins present in a significantly lower or significantly higher amount on a polyacrylamide gel of proteins from said biological sample in relation to a control; b) one or more proteins present on a polyacrylamide gel of proteins from said biological sample and absent on polyacrylamide gel of proteins of a control; and c) one or more proteins absent on a polyacrylamide gel of proteins from said biological sample and present on polyacrylamide gel of proteins of a control.
  • control is evident for a person skilled in the art of proteomics.
  • the control is an IOD% value of a spot (having the same position) from a gel of proteins from a sample originating from a human who is not predisposed for or having diabetes.
  • marker protein is meant to encompass the above mentioned proteins, as well as similar proteins from other species of mammals.
  • the human forms of the proteins are of particular interest.
  • the human forms of the rat proteins can be used as markers for diabetes in human beings.
  • the term “marker protein” encompasses the human form of the identified proteins in table 1 and the not yet identified proteins in table 2, as well as the proteins being up- or down-regulated in a mammalian tissue sample (e.g. human) as described elsewhere. Examples of the marker proteins of the invention are given below.
  • An other aspect of the invention relates a method for diagnosing or for determining the predisposition of diabetes in a mammal, e.g. in a human, the method comprising determining the presence, absence or level of expression of at least one marker protein in a biological sample from said mammal.
  • the biological sample can be selected from the group consisting of urine, blood, saliva, lymphatic fluids, and tissue, presently pancreatic tissue is preferred.
  • a presently preferred method comprises establishing the increased expression of at least one marker protein (an up-regulated marker protein) selected from the group consisting of: a) a protein which is defined in table 1 and/or 2, and marked as up-regulated; and b) a protein which is a modification or a derivative of a protein in a), so as to have at least 80% sequence homology with a protein in a), but of course the invention also relates to a method establishing the decreased expression of at least one down-regulated marker protein.
  • an up-regulated marker protein selected from the group consisting of: a) a protein which is defined in table 1 and/or 2, and marked as up-regulated; and b) a protein which is a modification or a derivative of a protein in a), so as to have at least 80% sequence homology with a protein in a), but of course the invention also relates to a method establishing the decreased expression of at least one down-regulated marker protein.
  • the invention in another embodiment, relates to a method for determining the predisposition for diabetes in a human, the method comprising: a) determining the increased expression of at least one marker protein in a biological sample originating from the human, said marker protein being selected from the group consisting of: i) an up-regulated protein defined in table 1, 2 and/or 3, and ii) a protein which is a modification or a derivative of a protein in i), so as to have at least 80% sequence homology with a protein in I); b) determining the decreased expression in a biological sample from the human of at least one marker protein, said marker protein being selected from the group consisting of: i) a down-regulated protein defined in table 1, 2 and/or 3 and ii) a protein which is a modification or a derivative of a protein in i), so as to have at least 80% sequence homology with a protein in i) ; or c) combinations of the determinations in a) and b).
  • the determination of whether a protein is up-regulated or down-regulated serves as useful indicators of diabetes susceptibility.
  • the pattern of up and down regulation may also serve as an indicator. That is to say that the level of expression of more than one protein is established and the pattern of expression of a grouping of proteins is used as an indicator.
  • At least one marker protein is selected from the group consisting of one or more proteins present in a significantly lower or significantly higher amount on a polyacrylamide gel of proteins from said biological sample in relation to a control, one or more proteins present on a polyacrylamide gel of proteins from said biological sample and absent on polyacrylamide gel of proteins of a control, one or more proteins absent on a polyacrylamide gel of proteins from said biological sample and present on polyacrylamide gel of proteins of a control.
  • the invention in another embodiment, relates to a method of treating diabetes, or preventing or delaying the onset or of diabetes, in a mammal, e.g. in a human, comprising altering the expressing of a least one marker protein.
  • the method preferably comprises administering a compound selected from the group consisting of: a) a protein which is defined in table 1, 2 and/or 3; b) a protein which is a modification or a derivative of a protein of table 1, 2 or 3, so as to have at least 80% sequence homology with a protein of table 1, 2 or 3; c) a protein having the same function as a protein in a) and/or b); d) a nucleotide sequence coding for a protein in a), b) or c); e) an antibody for a protein of a), b) or c); f) a nucleic acid fragment capable of binding to a protein of a) b) or c); and g) a compound capable of binding to a protein of a), b) or c) to said human. Also, the invention relates to the use of such a compound for the manufacture of a medicament for the treatment or prophylaxis of diabetes.
  • a single protein may be targeted for therapy or a grouping of proteins may be targeted.
  • the level of expression of these targeted proteins may be altered or the proteins themselves may be interfered with in order to alter their activity.
  • an interesting embodiment of a method of treating diabetes in a human comprises altering the expressing of a marker protein.
  • the invention in another embodiment, relates to a method of determining the likelihood of an agent having a therapeutic effect in the treatment of diabetes comprising determining the relative level of expression of one or more marker proteins before and after exposing a test model to said agent.
  • the invention relates to a method of determining the effect of a compound in the treatment of diabetes comprising determining the level of expression of proteins of one or more marker proteins.
  • the invention relates to a method of determining the level of effect of a compound used in the treatment of diabetes comprising determining the level of expression of one or more marker proteins before and after exposing a test model to said agent.
  • the invention relates to a method of determining the nature or cause of diabetes in a mammal, e.g. in a human, having or susceptible to said disease comprising establishing the level of expression of a at least one marker protein in relation to a model.
  • An other embodiment of the invention relates to a nucleic acid fragment comprising a nucleotide sequence which codes for a marker protein, or to a nucleic acid fragment which hybridizes with a such a nucleic acid fragment or a part thereof or with its complementary strand.
  • the invention relates to the use of the above mentioned nucleic acid fragments for detecting the presence of a marker protein.
  • the invention relates to an antibody able to bind to a marker protein.
  • an antibody can be a polyclonal antibody or a monoclonal antibody.
  • the invention also relates to the use of such an antibody for detecting the presence of a marker protein.
  • the invention relates to a test kit for diagnosing diabetes or a genetic predisposition for diabetes in a mammal, e.g. in a human, comprising: a) a binding means which specifically binds to at least one marker protein; or which binding means is an antibody for at least one said marker protein, a nucleic acid fragment capable of binding to at least one said marker protein, or a compound capable of binding to at least one said marker protein; b) a means for detecting binding, if any, or the level of binding, of the binding means to at least one of the marker proteins or to at least one of the nucleic acid fragments, and c) a means for correlating whether binding, if any, or the level of binding, to said binding means is indicative of the individual mammal having a significantly higher like- lihood of having diabetes or a genetic predisposition for having diabetes.
  • the invention relates to a method for determining the effect of a substance, the method comprising using a mammal, e.g. a human, which has been established to be an individual having a high likelihood of having diabetes or a genetic predisposition for having diabetes by use of a method of the invention, the method comprising administering the substance to the individual and determining the effect of the substance.
  • a mammal e.g. a human
  • the method comprising administering the substance to the individual and determining the effect of the substance.
  • the present investigators anticipate that a method of determining the nature or cause of diabetes in a human having or susceptible to said disease comprising establishing the level of expression of a protein of Table 1 in relation to a model serves for understanding the disease and potential therapies.
  • a very important embodiment of the invention relates to a pharmaceutical composition which comprises: a) a substance which is capable of regulating the expression of a nucleic acid fragment coding for at least part of a marker protein; b) a marker protein; c) a derivative, homologue or mimic of a marker protein; d) an antibody for a marker protein; e) a nucleic acid fragment capable of binding to a marker protein; and/or f) a compound capable of binding to a marker protein.
  • the pharmaceutical composition can be used as a medicament, such as for treatment or prophylaxis of diabetes.
  • a method for diagnosing or determining the predisposition of at least one disease related to diabetes comprising determining the presence, activity, concentration and/or level of expression of a combination of two markers would be preferred and three or more markers (e.g. at least 4, 5, 6 or 7 markers) would be strongly preferred.
  • treatment with more than one compound (e.g. at least 2, 3, 4, 5, 6 or 7 compounds) according to the invention e.g. more than one compound chosen from the group consisting of: a polypeptide, a nucleic acid fragment or an antibody according to the invention, said compounds combined being able to affect the level of more than one marker protein, would make the treatment of the disease even more efficient.
  • polypeptide in the present invention should have its usual meaning. That is an amino acid chain of any length, including a full-length protein, oligopeptides, short peptides and fragments thereof, wherein the amino acid residues are linked by covalent peptide bonds.
  • polypeptide amino acid chain of any length, including a full-length protein, oligopeptides, short peptides and fragments thereof, wherein the amino acid residues are linked by covalent peptide bonds.
  • polypeptide amino acid sequence
  • protein protein
  • the protein may be chemically or biochemically modified by being phosphorylated, methylated, sulphylated, glycosylated or by the addition of any form of lipid or fatty acid, ubiquitin or any other large side groups or by containing additional amino acids or any other forms of modification (of which there are over 200 known). These modifications occur at specific sites on the protein and a particular modification at one site can have different effects as the same modification at a different site on the same protein. They can be reversible in the cell where they are used for example to turn on and off enzymes and so the proteins can exist in a variety of forms - each with an associated activity level for each of the proteins functions. Furthermore the polypeptide may be cleaved e.g.
  • Each polypeptide may thus be characterized by specific amino acids and be encoded by specific nucleic acid sequences. It will be understood that such sequences include analogues and variants produced by recombinant or synthetic methods wherein such polypeptide sequences have been modified by substitution, insertion, addition or deletion of one or more amino acid residues in the recombinant polypeptide and still be immunogenic in any of the biological assays described herein. Substitutions are preferably "conservative". Conservative substitutions are known to a person skilled in the art.
  • amino acids belonging to the same grouping non-polar (G, A, P, I, L and V), polar-uncharged (C, S, T, M, N and Q), polar-charged (D, E, K and R) and aromatic (H, F, W and Y)).
  • amino acids belonging to the same grouping (non-polar (G, A, P, I, L and V), polar-uncharged (C, S, T, M, N and Q), polar-charged (D, E, K and R) and aromatic (H, F, W and Y)).
  • amino acids may be substituted for each other, but other substitutions are of course possible.
  • Each polypeptide is encoded by a specific nucleic acid sequence. It will be understood that such sequences include analogues and variants hereof wherein such nucleic acid sequences have been modified by substitution, insertion, addition or deletion of one or more nucleic acid residues (including the insertion of one or more introns (small or large)). Substitutions are preferably silent substitutions in the codon usage, which will not lead to any change in the amino acid sequence, but may be introduced to enhance the expression of the protein.
  • substantially pure polypeptide means a polypeptide preparation which contains at most 5% by weight of other polypeptide material with which it is natively associated (lower percentages of other polypeptide material are preferred, e.g. at most 4%, at most 3%, at most 2%, at most 1%, and at most 1 /2%). It is preferred that the substantially pure polypeptide is at least 96% pure, i.e. that the polypeptide constitutes at least 96% by weight of total polypeptide material present in the preparation, and higher percentages are preferred, such as at least 97%, at least 98%, at least 99%, at least 99,25%, at least 99,5%, and at least 99,75%.
  • the polypeptide fragment is in "essentially pure form", i.e. that the polypeptide fragment is essentially free of any other protein with which it is natively associated, i.e. free of any other protein from a mammal.
  • This can be accomplished by preparing the polypeptide of the invention by means of recombinant methods in a host cell as known to a person skilled in the art, or by synthesizing the polypeptide fragment by the well-known methods of solid or liquid phase peptide synthesis, e.g. by the method described by Merrifield (Merrifield, R. B. Fed. Proc. Am. Soc. Ex. Biol. 21: 412, 1962 and J. Am. Chem. Soc. 85: 2149, 1963) or variations thereof, or by means of recovery from electrophoretic gels.
  • protein also encompasses derivatives, analogues and mimetics of the above mentioned polypeptides.
  • a derivative, analogue and mimetic preferably have the same activity, e.g. the same kind of enzymatic activity, as the polypeptide which it is derived from.
  • the derivative, analogue or mimetic can have a lower level activity, the same level or preferably, a higher level of activity than the parent polypeptide.
  • a least one encompasses the integers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 etc. It should be understood that a single marker protein can be used, but it can be advantageous to use more than one marker protein in methods of the invention. That is to say that the level of expression of more than one protein is established and the pattern of expression of a grouping of proteins is used as an indicator. Obviously the reliability of identification increases as the number in the group increase.
  • a "peptide mimetic” is a molecule that mimics the biological activity of a peptide but is no longer peptidic in chemical nature.
  • a peptidomimetic is a molecule that no longer contains any peptide bonds (that is, amide bonds between amino acids).
  • the term peptide mimetic is sometimes used to describe molecules that are no longer completely peptidic in nature, such as pseudo-peptides, semi-peptides and pep- toids.
  • peptidomimetics according to this in- vention provide a spatial arrangement of reactive chemical moieties that closely resembles the three-dimensional arrangement of active groups in the peptide on which the peptidomimetic is based.
  • the peptidomimetic has effects on biological systems, which are similar to the biological activity of the peptide.
  • the present invention encompasses peptidomimetic compositions which are analogs that mimic the activity of biologically active peptides according to the invention, i.e. the peptidomimetics can be used for treatment of diabetes related diseases.
  • the peptidomimetic of this invention are preferably substantially similar in both three-dimensional shape and biological activity to the peptides or active sites of such as set forth above.
  • the mimetic can be an *antimimetic'.
  • Most current drugs are of this type.
  • Such antimimetlcs that are capable of interacting with the polypeptides of the invention are encompassed by the present invention.
  • peptides described above have utility in the development of such small chemical compounds with similar biological activities and therefore with similar therapeutic utilities.
  • the techniques of developing peptidomimetics are conventional.
  • peptide bonds can be replaced by non-peptide bonds that allow the peptidomimetic to adopt a similar structure, and therefore biological activity, to the original peptide.
  • Further modifications can also be made by replacing chemical groups of the amino acids with other chemical groups of similar structure.
  • the development of peptidomimetics can be aided by determining the tertiary structure of the original peptide by NMR spectroscopy, crystallography and/or computer-aided molecular modelling.
  • a potential peptidomimetic compound may be synthesized and assayed using the diagnostic assay described herein or an appropriate disease suppressor assay [see, Finlay et al. (1983), Cell, 57: 1083- 1093 and Fujiwara et al. (1993), Cancer Res., 53: 4129-4133, both incorporated herein by reference], to assess its activity.
  • the present invention provides com- pounds exhibiting enhanced therapeutic activity in comparison to the polypeptides described above.
  • the peptidomimetic compounds obtainable by the above methods, having the biological activity of the above named peptides and similar three dimensional structure, are encompassed by this invention. It will be readily apparent to one skilled in the art that a peptidomimetic can be generated from any of the modified peptides described previously or from a peptide bearing more than one of the modifications described previously. It will furthermore be apparent that the peptidomimetics of this invention can be further used for the development of even more potent non-peptidic compounds, in addition to their utility as therapeutic compounds.
  • nucleic acid fragment and “nucleic acid sequence” and the like are understood any nucleic acid molecule including DNA, RNA, LNA (locked nucleic acids), PNA, RNA, dsRNA and RNA-DNA-hybrids. Also included are nucleic acid molecules comprising non-naturally occurring nucleosides. The term includes nucleic acid molecules of any length, e.g. from 10 to 10000 nucleotides, depending on the use. When the nucleic acid molecule is for use as a pharmaceutical, e.g.
  • a molecule encoding at least a part of the polypeptide is preferably used, having a length from about 18 to about 1000 nucleotides, the molecule being optionally inserted into a vector.
  • a molecule having a length of 10-100 is preferably used.
  • molecule lengths can be used, for instance a molecule having at least 12, 15, 21, 24, 27, 30, 33, 36, 39, 42, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 or 1000 nucleotides (or nucleotide derivatives), or a molecule having at most 10000, 5000, 4000, 3000, 2000, 1000, 700, 500, 400, 300, 200, 100, 50, 40, 30 or 20 nucleotides (or nucleotide derivatives). It should be understood that these numbers can be freely combined to produce ranges.
  • the term "stringent" when used in conjunction with hybridization conditions is as defined in the art, i.e. the hybridization is performed at a temperature not more than 15-20°C under the melting point (Tm) of the nucleic acid fragment, cf. Sambrook et al Molecular Cloning; A laboratory manual, Cold Spring Harbor Laboratories, NY, 1989, pages 11.45- 11.49.
  • the conditions are "highly stringent", i.e. 5-10°C under the melting point (Tm).
  • the hybridisation conditions are preferably stringent.
  • sequence identity indicates a quantitative measure of the degree of homology between two amino acid sequences of equal length or between two nucleotide sequences of equal length. If the two sequences to be compared are not of equal length, they must be aligned to best possible fit possible with the insertion of gaps or alternatively truncation at the ends of the protein sequences.
  • sequence identity can be calculated as ⁇ Nnr ⁇ ar ' — # wherein N dif is the total number of non- identical residues in the two sequences when aligned and wherein N re r is the number of residues in one of the sequences.
  • Sequence identity can alternatively be calculated by the BLAST program e.g.
  • the degree of homology between two nucleic acid sequences is determined by using GAP version 8 from the GOG package with standard penalties for DNA: GAP weight 5.00, length weight 0.300, Matrix described in Gribskov and Burgess, Nucl. Acids Res. 14(16); 6745-6763 (1986), and the degree of homology between two amino acid sequences is determined by using GAP version 8 from the GCG package (Genetics Computer Group, 575 Science Drive, Madison, Wis. 53711, USA) with standard penalties for proteins: GAP weight 3.00, length weight 0.100, Matrix described in Gribskov and Burgess, Nucl. Acids Res. 14(16); 6745-6763 (1986).
  • a preferred minimum percentage of sequence homology is at least 70%, such as at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and at least 99.5%.
  • the invention also relates to the use of a polypeptide or nucleic acid of the invention for use as therapeutic vaccines as have been described in the literature exemplified by Lowry, D.B. et al 1999, Nature 400: 269-71.
  • a monoclonal or polyclonal antibody which is specifically reacting with a polypeptide of the invention in an immuno assay, or a specific binding fragment of said antibody, is also a part of the invention.
  • the antibodies can be produced by methods known to a person skilled in the art.
  • the polyclonal antibodies can be raised in a mammal, for example, by one or more injections of a polypeptide according to the present invention and, if desired, an adjuvant.
  • the monoclonal antibodies according to the present invention may, for example, be produced by the hybridoma method first described by Kohler and Milstein, Nature, 256:495 (1975), or may be produced by recombinant DNA methods such as described in U.S. Pat. No. 4,816,567.
  • the monoclonal antibodies may also be isolated from phage libraries generated using the techniques described by McCafferty et al, Nature, 348:552-554 (1990), for example. Methods for producing antibodies are described in the literature, e.g. in US 6,136,958.
  • an antibody, a nucleic acid fragment and/or a polypeptide of the invention can be used either alone, or as a constituent in a composition.
  • Such compositions are known in the art, and comprise compositions in which the antibody, the nucleic acid fragment or the polypeptide of the invention is coupled, preferably covalently, to at least one other molecule, e.g. a label (e.g. radioactive or fluorescent) or a carrier molecule.
  • the present invention is further directed to methods for using the compounds described above to therapeutically and/or prophylactically treat a patient for a diabetes related dis- ease.
  • the methods of the present invention include the steps of: a) incorporating one or more of the compounds of the present invention in a suitable pharmaceutical carrier; and b) administering either a therapeutically effective dosage or a prophylactically effective dosage of the compound or compounds incorporated in the carrier to a patient.
  • suitable pharmaceutical carrier refers to any carrier known in the pharmaceutical arts for administration of compounds to a patient. Any suitable pharmaceutical carrier can be used according to the present invention, so long as compatibility problems do not arise.
  • an effective dosage to a patient can be accomplished by parenteral injection, such as intravenously, intrathecally, intramuscularly or intra-arterially.
  • the compounds can also be administered orally or transdermally, or by any other means known to those skilled in the art, e.g. by means of an inhalator or a nasal spray. Oral administration is presently preferred.
  • therapeutically effective amount refers to that amount of one or more of the compounds of the present invention required to therapeutically treating a patient. Such treatment is appropriate for subjects having a diagnosed diabetes related disease.
  • prophylactically effective amount refers to that amount of one or more of the compounds of the present invention needed to prophylactically treat a patient.
  • Such treatment is appropriate for subjects who, for example, have not yet established any clinical symptoms of a diabetes related disease. It could be advantageous to start a prophylactic treatment as soon it is determined that the subject is in risk for developing a diabetes related disease, e.g. by means of a determination of a predisposition for diabetes by having an altered level of markers.
  • the dosage of compound given, the route of administration and the duration of therapy will be dependent not only the type of compound and its effectiveness in treating the disease but also upon the individual being treated, taking into consideration such factors as the body weight of the patient, other therapies being employed to treat the patient, and the condition, clinical response and tolerance of the patient. Dosage, administration, and duration of therapy can be determined by one skilled in the art upon evaluation of these and other relevant factors.
  • Preparatory 2D-gels were produced from a pool of approximately 200.000 neonatal WF islets isolated, prepared and separated on gels as described above. For localization of the spots, radioactively labelled tracer islets were mixed with the non-labelled islets.
  • Neonatal WF rat islets were used for preparatory gels because of the price and number of islets isolated per animal.
  • the pattern seen on 2D-gels of respectively BB-DP and WF rat islets are similar.
  • the cost of one WF rat is approximately one tenth of the price of a BB- DP rat (approximately 100 US$ each). From each rat is it possible to isolate in the range of 150-200 islets.
  • RPMI 1640 Hanks' balanced salt solution (HBSS) and Dulbecco's modified Eagle's medium (DMEM) (Gibco, Paisley, Scotland).
  • RPMI 1640 contained 11 mmol D-glucose and was supplemented with 20 mM HEPES buffer, 100,000 IU/1 penicillin and 100 mg/1 streptomycin.
  • Other reagents 2-mercaptoethanol, foetal calf serum (BSA), normal human serum (NHS), Tris HCI, Tris base, glycine, (Sigma, St.
  • RNA'se A DNA'se I (Worthington, Freehold, NJ, USA); [35S]-methionine (SJ 204, specific activity: >1.000 Ci/mmol, containing 0.1% 2-mercaptoethanol), Amplify® (Amersham International, Amersham, UK); urea (ultra pure) (Schwarz/Mann, Cambridge, MA, USA); acrylamide, bisacrylamide, 4N-tetra-methyl-ethylene-diamine (TEMED), ammonium persulphate (BioRad, Richmond, CA, USA); ampholytes: pH 5-7, pH 3.5-10, pH 7-9, pH 8-9.5 (Amasham Biode
  • Pregnant inbred WF rats were purchased from M&B, LI. Skensved, Denmark.
  • Four to five day-old BB/Wor/Mol-BB2 (BB-DP) rats were also purcased from M&B. The rats were picked up at M&B in the morning on the day of islet isolation, and transported in animal transport boxes. At M&B the BB-DP rats were housed separately in a specific pathogen- free environment.
  • Islets were isolated by collagenase digestion of the pancreata from 4-5 day old WF rats [Brunstedt, 1984][15]. After 4 days of preculture in RPMI 1640 + 10% fetal calf serum, islets were incubated for 24 h in 37°C humidified atmospheric air in 300 ⁇ l or 3000 ⁇ l RPMI 1640 + 0.5% normal human serum. Next islets were washed twice in HBSS and labelled for 4 h at 370C in 200 ⁇ l or 2000 ⁇ l home made methionine-free Dulbecco's modified Eagle's medium (DMEM) with 10% dialysed NHS, and 200 ⁇ Ci [35S]-methio- nine.
  • DMEM methionine-free Dulbecco's modified Eagle's medium
  • [35S]-methionine was freeze-dried for at least 4 h before labelling. After labelling, the islets were washed thrice in HBSS, the supernatant was removed and islets were immediately frozen at -80°C. Unlabelled islets for preparative gels were washed twice in HBSS and snapfrozen. For localization of the spots, radioactively labelled tracer islets were mixed with the non-labelled islets.
  • Sample preparation The frozen islets were re-suspended in 100 ⁇ l DNAsel/RNAse A solution and lysed by freeze-thawing twice. After the second thawing, the samples were left on ice for 30 min. for the digestion of nucleic acids and then freeze dried overnight. The samples were dissolved by shaking in 120 ⁇ l lysis buffer (8.5 M urea, 2% Nonidet P-40, 5% 2-mercaptoethanol and 2% ampholytes, pH range 7-9) for a minimum of 4 hours.
  • 120 ⁇ l lysis buffer 8.5 M urea, 2% Nonidet P-40, 5% 2-mercaptoethanol and 2% ampholytes, pH range 7-9
  • the amount of [35S]-methionine incorporation was quantified by adding 10 ⁇ l FCS (0.2 ⁇ g/ml H20) as a protein-carrier to 5 ⁇ l of a 1: 10 dilution of each sample in duplicate, followed by 0.5 ml of 10% TCA. This was left to precipitate for 30 min at 4°C before being filtered through 0.25 ⁇ m hydroxy appatit-WP (HAWP) filters. The filters were dried and placed into scintillation liquid for counting.
  • FCS 0.2 ⁇ g/ml H20
  • 2-DGE and preparative gels Preparative two dimensional gels (2-DG) were produced from a pool of approximately 200.000 neonatal WF rat islets isolated, cultured, labelled and separated on gels as described above. For localization of the spots, radioactively labelled tracer islets were mixed with the non-labelled islets. The procedure has been described earlier [O'Farrell, 1977 [14]; Fey, 1984 [18]; Fey, 1997 [17]. Briefly, first dimension gels contained 4% acrylamide, 0.25% bisacrylamlde, ampholytes and nonidet P-40. Equal amount of protein (175-200 ⁇ g for preparative gels) and counts per minute (106 cpm.) of each sample were applied to the gels.
  • Both isoelectric focusing (IEF; pH 3.5-7) and non-equilibrium pH-gradient electrophoresis (NEPHGE; pH 6.5-10.5) gels were made.
  • Second dimension gels contained 12,5% acrylamide and 0.063% bisacrylamlde and were run overnight. After electrophoresis, the gels were fixed and treated for fluorography with Amplify® before being dried. The gels were placed in contact with X-ray films and exposed at -70°C for 3 to 40 days. Each gel was exposed for at least 3 time periods to compensate for the lack of dynamic range of X-ray films.
  • Neonatal WF rat islets were used for preparative gels because of the price and higher number of islets isolated per animal.
  • the same protein spot pattern is seen on 2-DG of respectively BB-DP and WF rat islets and the BB-DP rat originates from the WF rat.
  • the cost of one WF rat is approximately one tenth of the price of a BB-DP rat (approximately 100 US$ each). From each rat was it possible to isolate approximately 150-200 islets.
  • Mr and pi for individual proteins on the gels were interpolated from landmark proteins.
  • Protein identification by matrix-assisted laser desorption/ionization (MALDI) MS Briefly, the 82 protein spots of interest were obtained by cutting them out of the dried gel using a scalpel. The proteins were enzymatically digested in the gel piece as described [Rosenfeld, 1992 [19]; Shevchenko, 1996 [20]] with minor modifications [Nawrocki, 1998][21], The excised gel pieces were washed in 50 mM NH4HC03/acetonitrile (60/40) and dried by vacuum centrifugation.
  • MALDI matrix-assisted laser desorption/ionization
  • Modified porcine trypsin (12 ng/ ⁇ L, Promega, sequencing grade) in digestion buffer 50 mM NH4HC03 was added to the dry gel pieces and incubated on ice for 1 h for reswelling. After removing the supernatant, 20-40 ⁇ L digestion buffer was added and the digestion was continued at 37o C for 4-18 hours.
  • the peptides were extracted as described [Shevchenko, 1996][20] and dried in a vacuum centrifuge. The residue was dissolved In 5 % formic acid and analysed by MALDI MS, Delayed extraction MALDI mass spectra of the peptide mixtures were acquired using a Bruker Reflex time-of-flight mass spectrometer (Bruker AG, Germany).
  • Protein information Information about the identified proteins known and putative biological functions were found at The ExPASy Molecular Biology Server (http://www.expasy.ch) and at The National Centre for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov).
  • Identified protein spots in neonatal BB-DP rats islets after exposure to IL-16 Changes in protein expression level are expressed as % IOD in neonatal BB-DP islets after IL-l ⁇ incubation in vitro compared to BB-DP islets without IL-l ⁇ .
  • Match numbers are arbitrary numbers given by the computer and corresponds to the number of the spot in the gel.
  • % IOD refers to the integrated optic density on the control gels.
  • % IOD ratio refers to the ratio of integrated optical density between gels compared. Numbers below 1 indicate proteins down-regulated and numbers above 1 are up-regulated in islets exposed to IL- 1B.
  • the proteins spots are ordered according to functional groups and % IOD ratio.
  • Protein name refers to the name found in the NCBI database (http://www.ncbi.nlm.nih.gov) through the database accession number. Given is the Mr and pi obtained directly by the gel analysis and the calculated theoretical Mr and pi calculated from the amino acid sequence. In some spots more than one protein was identified. These proteins are marked with *. Proteins mentioned more than once are found in more than one spot. Proteins previously identified as changes in expression in WF rat islets exposed to IL-l ⁇ as well [Mose Larsen, 2001][7] are marked with #. roteins that have not previously been described or supposed to be present in islets are marked with $.
  • Figures 5-21 Spectra of the proteins in table 3. Each spectrum is presented firstly as a total spectrum (e.g. figure 5A) and thereafter as enlarged regions so it is possible to read all the numbers and see the peak that they refer to (e.g. figure 5B, figure 5C, etc.). Thus, the spectra for the first protein in table 3 are called figure 5A, figure 5B, figure 5C and figure 5D.
  • Mandrup-Poulsen T The role of interleukin-1 in the pathogenesis of insulin-dependent diabetes mellitus. Diabetologia 39:1005-1029,1996
  • Bone AJ Animal models of type I diabetes, Current Opinion in Oncologic, Endocrine and Metabolic Investigational Drugs 2:192-200, 2000
  • Minturn JE Fryer HJ, Geschwind DH, Hockfield S: TOAD-64, a gene expressed early in neuronal differentiation in the rat, is related to unc-33, a C, elegans gene involved in axon outgrowth. Journal of neurosciencel5:6757-6766,1995

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Pathology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Cette invention concerne des protéines de mammifère médiatrices du diabète sécrétées et non sécrétées, y compris des protéines médiatrices du diabète protectrices et délétères, ainsi que des polynucléotides codants ces protéines, des méthodes de criblage de médicaments servant à identifier un composé de test pouvant modifier l'expression d'une protéine médiatrice du diabète, et des méthodes de prévention ou d'amélioration du diabète consistant à administrer un composé pouvant modifier l'expression d'une protéine médiatrice du diabète.
PCT/DK2002/000368 2001-05-29 2002-05-29 Proteines de mammifere mediatrices du diabete WO2002097441A2 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP02742844A EP1421390A2 (fr) 2001-05-29 2002-05-29 Proteines de mammifere mediatrices du diabete
AU2002344252A AU2002344252A1 (en) 2001-05-29 2002-05-29 Mammalian diabetes-mediating proteins
US10/960,652 US7279274B2 (en) 2001-05-29 2004-10-07 Method for diagnosing diabetes in a mammal
US11/869,546 US20080145868A1 (en) 2001-05-29 2007-10-09 Method for diagnosing diabetes in a mammal

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DKPA200100852 2001-05-29
DKPA200100852 2001-05-29
DKPA200200446 2002-03-22
DKPA200200446 2002-03-22

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US10479530 A-371-Of-International 2002-05-29
US10/960,652 Continuation US7279274B2 (en) 2001-05-29 2004-10-07 Method for diagnosing diabetes in a mammal

Publications (2)

Publication Number Publication Date
WO2002097441A2 true WO2002097441A2 (fr) 2002-12-05
WO2002097441A3 WO2002097441A3 (fr) 2004-03-25

Family

ID=26069028

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/DK2002/000368 WO2002097441A2 (fr) 2001-05-29 2002-05-29 Proteines de mammifere mediatrices du diabete

Country Status (3)

Country Link
EP (1) EP1421390A2 (fr)
AU (1) AU2002344252A1 (fr)
WO (1) WO2002097441A2 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003078456A2 (fr) * 2002-03-20 2003-09-25 Syddansk Universitet Proteines humaines mediatrices du diabete
WO2008074963A2 (fr) * 2006-12-19 2008-06-26 bioMérieux Procédé pour le diagnostic d'un diabète
EP2419536A1 (fr) * 2009-04-14 2012-02-22 Merck Sharp & Dohme Corp. Micro arn en tant que biomarqueur de la mobilisation de cellules béta des îlots pancréatiques

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998019271A1 (fr) * 1996-10-25 1998-05-07 Peter Mose Larsen Analyse de proteome pour caracteriser des proteines a regulation positive et negative dans des echantillons biologiques
WO2001001130A1 (fr) * 1999-06-25 2001-01-04 Proteome Sciences Plc PROCEDES ET COMPOSITIONS SE RAPPORTANT AU DYSFONCTIONS DES ILOTS PANCREATIQUES ET DES CELLULES $g(b)

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2375850A1 (fr) * 1999-06-01 2000-12-07 Proteome Sciences Plc Procede et compositions lies aux troubles de la resistance a l'insuline

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998019271A1 (fr) * 1996-10-25 1998-05-07 Peter Mose Larsen Analyse de proteome pour caracteriser des proteines a regulation positive et negative dans des echantillons biologiques
WO2001001130A1 (fr) * 1999-06-25 2001-01-04 Proteome Sciences Plc PROCEDES ET COMPOSITIONS SE RAPPORTANT AU DYSFONCTIONS DES ILOTS PANCREATIQUES ET DES CELLULES $g(b)

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
ANDERSEN H.U. ET AL.: "Interleukin-1beta induced changes in the protein expression of rat islets : a computerized database." ELECTROPHORESIS, vol. 18, 1997, pages 2091-2103, XP002902695 cited in the application *
CHRISTENSEN U.B. ET AL.: "Islet protein expression changes during diabetes development in islet syngrafts in BB-DP islet allografts." AUTOIMMUNITY, vol. 32, 2000, pages 1-15, XP002902697 cited in the application *
JOHN N.E. ET AL.: "Cytokine- or chemically derived nitric oxide alters the expression of proteins detected by two-dimensional gel electrophoresis in neonatal rat islets of Langerhans." DIABETES, vol. 49, November 2000 (2000-11), pages 1819-1829, XP002236131 *
MOSE LARSEN P. ET AL.: "Proteome analysis of interleukin-1beta-induced changes in protein expression in rat islets of Langerhans." DIABETES, vol. 50, May 2001 (2001-05), pages 1056-1063, XP002236130 *
See also references of EP1421390A2 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003078456A2 (fr) * 2002-03-20 2003-09-25 Syddansk Universitet Proteines humaines mediatrices du diabete
WO2003078456A3 (fr) * 2002-03-20 2004-01-15 Univ Syddansk Proteines humaines mediatrices du diabete
WO2008074963A2 (fr) * 2006-12-19 2008-06-26 bioMérieux Procédé pour le diagnostic d'un diabète
WO2008074963A3 (fr) * 2006-12-19 2009-01-15 Biomerieux Sa Procédé pour le diagnostic d'un diabète
EP2419536A1 (fr) * 2009-04-14 2012-02-22 Merck Sharp & Dohme Corp. Micro arn en tant que biomarqueur de la mobilisation de cellules béta des îlots pancréatiques
EP2419536A4 (fr) * 2009-04-14 2012-09-05 Merck Sharp & Dohme Micro arn en tant que biomarqueur de la mobilisation de cellules béta des îlots pancréatiques

Also Published As

Publication number Publication date
AU2002344252A1 (en) 2002-12-09
WO2002097441A3 (fr) 2004-03-25
EP1421390A2 (fr) 2004-05-26

Similar Documents

Publication Publication Date Title
Rocca et al. Progress in the emerging role of selenoproteins in cardiovascular disease: Focus on endoplasmic reticulum-resident selenoproteins
Lu et al. Protein phosphatase 2Cm is a critical regulator of branched-chain amino acid catabolism in mice and cultured cells
Piro et al. Chronic exposure to free fatty acids or high glucose induces apoptosis in rat pancreatic islets: possible role of oxidative stress
Hitomi et al. Inosine triphosphate protects against ribavirin-induced adenosine triphosphate loss by adenylosuccinate synthase function
Larsen et al. Proteome analysis of interleukin-1β–induced changes in protein expression in rat islets of Langerhans
Rinaldo et al. Fatty acid oxidation disorders
Sparre et al. IL-1β induced protein changes in diabetes prone BB rat islets of Langerhans identified by proteome analysis
de Baaij et al. Membrane topology and intracellular processing of cyclin M2 (CNNM2)
Chen et al. Quantitative organellar proteomics analysis of rough endoplasmic reticulum from normal and acute pancreatitis rat pancreas
Choi et al. Gender difference in proteome of brown adipose tissues between male and female rats exposed to a high fat diet
US20090155814A1 (en) Proteins in type 2 diabetes
Oonthonpan et al. Two human patient mitochondrial pyruvate carrier mutations reveal distinct molecular mechanisms of dysfunction
Dewey et al. Proteomic analysis of hearts from Akita mice suggests that increases in soluble epoxide hydrolase and antioxidative programming are key changes in early stages of diabetic cardiomyopathy
Kondo et al. Enhancement of acetaminophen-induced chronic hepatotoxicity in restricted fed rats: a nonclinical approach to acetaminophen-induced chronic hepatotoxicity in susceptible patients
Taurino et al. Mitochondrial proteome analysis reveals depression of the Ndufs3 subunit and activity of complex I in diabetic rat brain
Broer Diseases associated with general amino acid transporters of the solute carrier 6 family (SLC6)
Türkön et al. Increased levels of serum ischemia modified albumin in patients with ankylosing spondylitis
Rodriguez-Sosa et al. Proinflammatory cytokine MIF plays a role in the pathogenesis of type-2 diabetes mellitus, but does not affect hepatic mitochondrial function
US20050031605A1 (en) Compositions and methods of treating diabetes
US20080145868A1 (en) Method for diagnosing diabetes in a mammal
EP1421390A2 (fr) Proteines de mammifere mediatrices du diabete
EP1402257B1 (fr) Proteines utiles dans l'analyse proteomique du diabete
Xie et al. Proteomic analysis of mouse islets after multiple low-dose streptozotocin injection
EP2071338A2 (fr) Protéines liées au diabète humain
Hascilowicz et al. Regulation of ornithine decarboxylase by antizymes and antizyme inhibitor in zebrafish (Danio rerio)

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ CZ DE DE DK DK DM DZ EC EE EE ES FI FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SI SK SK SL TJ TM TN TR TT TZ UA UG US UZ VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 2002742844

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWP Wipo information: published in national office

Ref document number: 2002742844

Country of ref document: EP

NENP Non-entry into the national phase in:

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP