WO2005023231A1

WO2005023231A1 - Polypyrimidine tract binding protein promotes insulin secretory granule biogenesis

Info

Publication number: WO2005023231A1
Application number: PCT/EP2004/010167
Authority: WO
Inventors: Michele Solimena; Klaus-Peter Knoch
Original assignee: Technische Universitaet Dresden; Max Planck Gesellschaft zur Foerderung der Wissenschaften
Current assignee: Technische Universitaet Dresden; Max Planck Gesellschaft zur Foerderung der Wissenschaften
Priority date: 2003-09-10
Filing date: 2004-09-10
Publication date: 2005-03-17
Anticipated expiration: 2006-03-10

Abstract

The present invention relates to a method for stimulating production of secretory granules in peptide hormone-secreting endocrine cells or neurons comprising the step of promoting the presence of polypyrimidine tract binding protein (PTB) or a biologically active fragment or derivative thereof in the cytoplasm of said cells or neurons. Preferably, the method alternatively or further comprises promoting the activity of PTB or said biologically active fragment or derivative thereof in the cytoplasm of said cells or neurons. It is also preferred that said promotion comprises the promotion of the nucleocytoplasmic transport of PTB. In another aspect, the invention relates to a method of screening for an agent capable of stimulating production of secretory granules in peptide hormone-secreting endocrine cells or neurons comprising the steps of (a) contacting a cell capable of forming secretory granules and expressing polypyrimidine tract binding protein (PTB) or a biologically active fragment or derivative thereof with one or more compounds; and (b) assessing whether said one or more compounds promote the presence or activity of said polypyrimidine tract binding protein (PTB) or said biologically active fragment or derivative thereof in the cytoplasm of said cell. Comprised by the invention are further methods of screening for an agent useful as a cure for diabetes, sleeping disorders or depression as well as various medical uses of an agent capable of the promotion/reduction of the presence or activity of polypyrimidine tract binding protein (PTB) or of a biologically active fragment or derivative thereof. In alternative embodiments, the reduction or downregulation of PTB or said biologically active fragment or derivative thereof is considered.

Description

Polypyrimidine tract binding protein promotes insulin secretory granule biogenesis

The present invention relates to a method for stimulating production of secretory granules in peptide hormone-secreting endocrine cells or neurons comprising the step of promoting the presence of polypyrimidine tract binding protein (PTB) or a biologically active fragment or derivative thereof in the cytoplasm of said cells or neurons. Preferably, the method alternatively or further comprises promoting the activity of PTB or said biologically active fragment or derivative thereof in the cytoplasm of said cells. It is also preferred that said promotion comprises the promotion of the nucleocytoplasmic transport of PTB. In another aspect, the invention relates to a method of screening for an agent capable of stimulating production of secretory granules in peptide hormone-secreting endocrine cells or neurons comprising the steps of (a) contacting a cell capable of forming secretory granules and expressing polypyrimidine tract binding protein (PTB) or a biologically active fragment or derivative thereof with one or more compounds; and (b) assessing whether said one or more compounds promote the presence or activity of said polypyrimidine tract binding protein (PTB) or said biologically active fragment or derivative thereof in the cytoplasm of said cell. Comprised by the invention are further methods of screening for an agent useful as a cure for diabetes, sleeping disorders or depression as well as various medical uses of an agent capable of the promotion/reduction of the presence or activity of polypyrimidine tract binding protein (PTB) or of a biologically active fragment or derivative thereof. In alternative embodiments, the reduction or downregulation of PTB or said biologically active fragment or derivative thereof is considered.

In the specification, a number of documents is cited. The disclosure content of these documents, including manufacturers' manuals, is herewith incorporated by reference in its entirety. Secretory granules (SGs) are the organelles devoted to the storage of peptide hormones in peptide-secreting endocrine cells. Chemical stimuli induce the fusion of SGs with the plasma membrane and the release of their content into the extracellular space. Like most secretory proteins, peptide hormones are co-translationally translocated into the lumen of the rough endoplasmic reticulum, then they are carried to the Golgi complex and finally they are sorted into nascent SGs. Along this route peptide hormones can undergo multiple post-translational modifications, including proteolytic cleavage and glycosylation. Thus, the generation of SGs is a slow process, which requires more than 30 minutes. As sustained stimulation leads to a progressive depletion of SGs, cells must quickly activate transcriptional and post- transcriptional mechanisms to renew their pool of these organelles.

β-cells of pancreatic islets are the endocrine cells that produce insulin, the most important hormone for the control of glucose homeostasis in vertebrates. Glucose stimulates both the Ca²⁺-dependent exocytosis of insulin SGs as well as the biosynthesis of insulin and other SG components [1 ,2], including chromogranin A [1], and the prohormone convertases 1/3 (PC1/3) [3,4] and 2 (PC2) [4]. Glucose enhances both the transcription [5-8] and the translation [9] of the insulin gene. Increased translation, in particular, accounts entirely for up-regulating insulin biosynthesis during the first 2 hours following stimulation [10,11]. Such increase results from the stimulation of initiation, elongation and signal-recognition-particle- mediated translocation of the nascent preproinsulin polypeptide into the lumen of the endoplasmic reticulum [2,11] as well as from a reduced degradation of insulin mRNA [8]. Recently it has been shown that glucose stimulation increases the stability of insulin mRNA by inducing the binding of polypyrimidine-tract binding protein (PTB) to its 3'-UTR [12,13]. Specifically, Tillmar at al. [12] describe experiments with isolated pancreatic islets in the determination of the effect of glucose on insulin mRNA abundance. In addition, effects on insulin mRNA stability were assessed. The authors found that insulin is abundantly expressed in the islet cells and also that induction of insulin mRNA is only affected by long-term glucose induction but not by short-term glucose induction. Further, they determined that a 55-60 kD protein bound to a pyrimidine-rich region in the 3'-UTR of the insulin mRNA. Binding of this protein that could be identified as PTB by analysis with monoclonal antibodies, resulted in a stabilization of insulin mRNA. The binding was found to be increased in response to glucose as well as to reducing agents such as DTT.

PTB, also known as heterogeneous nuclear ribonucleoprotein I, is a pre-mRNA splicing repressor [14,15], which has also been implicated in cap-independent translation [16,17], cytoplasmic RNA transport [18], poly (A) site cleavage [19], and mRNA stability [12]. The PTB gene encodes a protein of 59 kD with four RNA recognition motif domains [20].

In a recent contribution, Xie et al. (PNAS 100 (2003), 8776-8781) disclose that the nucleocytoplasmic transport of PTB is regulated by the 3',5'-cAMP-dependant protein kinase K (PKA). PKA directly phosphorylates PTB on conserved Ser-16 and PKA activation in PC12 cells and Xenopus oocytes induces Ser-16 phosphorylation. A mutation from Ser-16 to AIa-16 abolished nucleocytoplasmic transport. The authors admit that the effect of PKA stimulation on PTB is not yet clear.

Thus, whereas the biochemistry of peptide hormones and neuropeptides and in particular insulin from transcription including post-translational modifications until secretion from the granules of cells secreting such hormones or neuropeptides is well known in the art, until today, however, no factor that regulates SG biogenesis has been identified. The technical problem underlying the present invention was therefore to provide means and methods that allow the controlled regulation of SG biogenesis. Advantageously, this includes the identification of such factors and, furthermore, furnishing such factors.

Accordingly, the present invention relates to a method for stimulating production of secretory granules in peptide hormone-secreting endocrine cells or neurons comprising the step of promoting the presence of polypyrimidine tract binding protein (PTB) or a biologically active fragment or derivative thereof in the cytoplasm of said cells (i.e. said endocrine cells or neurons, in this and all pertinent following embodiments).

The term "stimulating production of secretory granules (SGs) in peptide hormone- secreting endocrine cells or neurons" refers to the induction or enhancement of a process in peptide hormone-secreting endocrine cells or neurons that leads to increase of production of such granules of at least 20%, preferably at least 30%, more preferred at least 50%, even more preferred at least 75% and most preferred at least 100% as compared to a physiological, normal and optionally non-pathogenic status of corresponding endocrine cells or neurons. Advantageously, the increase may be at least 200% such as at least 500%. The extent of the production of secretory granules can be monitored by methods well known in the art including microscopy (more specifically confocal microscopy or electron microscopy), measurement of the expression of one or more components of the secretory granules at the protein or the mRNA level, and determining the amount of insulin, e.g. using Western blotting, a radio-immuno assay (RIA) or an ELISA assay. Components of the secretory granules are further detailed herein below. The Examples and the Figures enclosed herewith exemplify the measurement of the expression of components of the secretory granules at the protein and the mRNA level.

The term "peptide hormone-secreting endocrine cells or neurons" refers to any mammalian and preferably human cell that naturally produces and secretes peptide hormones or neuropeptides. Also included are precursors of such cells as well as cells which in their normal context in the body do not secrete such peptides but which have been manipulated to secrete such peptides, for example by genetic engineering. In principle, one option in this regard envisages reprogramming a somatic cell to transdifferentiate into a peptide-secreting endocrine cell or a neuron. It is preferred in accordance with the present invention that peptide hormone-secreting endocrine cells or neurons are human cells that naturally produce and secrete peptide hormones or neuropeptides.

The term "promoting the presence" in general means that a certain level of PTB or a biologically active fragment or derivative thereof is maintained or produced in the cytoplasm of the recited cells. Accordingly, the invention envisages that, in one alternative, an existing level of PTB or a biologically active fragment or derivative thereof in the cytoplasm is maintained or essentially maintained, such as maintained to at least 70%, preferably to at least 80%, more preferred to at least 90% and most preferred to at least 95% such as 100%. Important options to maintain an existing level of PTB or a biologically active fragment or derivative thereof in the cytoplasm are described in connection with preferred embodiments of the method of the invention further below. In a second alternative, PTB or a biologically active fragment or derivative thereof is actively produced inside the cell and/or shifted from the nucleus into the cytoplasm. In this alternative, several options are possible how the level of PTB or a biologically active fragment or derivative thereof may be enhanced in a cell or in the cytoplasm. For example, if the cell naturally does not produce PTB, production may be stimulated by introducing a vector capable of expressing PTB into said cell. Of course, the levels of PTB may also be enhanced in a cell that naturally produces PTB by way of expression from an introduced vector. Important options how the level of PTB may be enhanced by production of PTB or a biologically active fragmen or derivative thereof are, again, discussed in connection with preferred embodiment further below. Further to the above, it is to be understood that the term "enhancing" in connection with the production of PTB relates both to the enhancement of levels of PTB already existing in the cytoplasm and to the de novo production of PTB (or a biologically active fragment or derivative thereof) in a cell. The level of PTB required in a cell that is necessary for stimulating production of secretory granules may vary from cell type to cell type.

The term "a biologically active fragment" of PTB refers to a portion of PTB that maintains the function of stimulation SG production. In one preferred embodiment, the fragment comprises (i.e. preferably extending not more than 10 amino acids beyond) or consists of PTB RNA binding domains 3 and 4, encompassing amino acid 361-563.

The term "derivative" of PTB refers to a molecule that is modified in its primary amino acid sequence as compared to the naturally occurring PTB molecule or a fragment thereof as indicated above such as a mutein but retains the function of SG stimulation. As a reference of the human PTB sequence that can be mutated, see accession number 26294 in Unigene database at http://www.ncbi.nlm.nih.gov/entrez, and references cited therein, including: Gil.A., Sharp.P.A., Jamison, S.F. and Garcia- Blanco.M.A. (1991) Characterization of cDNAs encoding the polypyrimidine tract- binding protein. Genes Dev. 5, 1224-1236. It is well known in the art that amino acids in certain positions of an amino acid sequence may be exchanged by different amino acids expected not to (essentially) change the higher order structure of the corresponding peptide or polypeptide. Such an exchange includes the exchange of alanine and valine, for example. The skilled artisan would primarily consider to exchange amino acids of the same polarity. Usually, the active center of a peptide or polypeptide will not be affected by such an exchange. The establishment or availability of the three-dimensional structure will assist the choice of options for exchanging certain amino acids without the concomitant expectation of a change in structure and/or function. Tests based on the teachings of this invention can be used to check for a change in the function of the peptide or polypeptide. Alternatively, a derivative of PTB may be produced by peptidomimetics; see for further teaching in this regard, for example in Ostresh, Methods in Enzymology 267 (1996), 220-234 and Dorner, Bioorg. Med. Chem. 4 (1996), 709-715.

In accordance with the present invention, a factor has been identified for the first time that induces the stimulation of the production of secretory granules in peptide hormone-secreting endocrine cells or neurons. Surprisingly, it could be demonstrated that the well-known molecule PTB, for which a number of functions have been described in the art, is necessary for the stimulation of secretory granules in secretory granule producing cells.

As regards the underlying mechanism, it was, inter alia, found that PTB promotes the expression of mRNA even in resting conditions. Most surprisingly, it was found that PTB appears not only to increase mRNA stability but also mRNA translation by binding to 5-UTRs. On the basis of this finding, the art will now be enabled to develop cures for diseases associated with the lack of production, diminished production or retarded production of secretory granules. The cures will have to focus on the enhancement of the level of cytoplasmic PTB or a fragment or derivative thereof, as has been described above, or its activity in affected cells. In addition, the invention allows for the development of cures associated with the overproduction of granules.

Therefore, the present invention also relates to a method for reducing production of secretory granules in peptide hormone-secreting endocrine cells or neurons comprising the step of reducing the presence and/or activity of polypyrimidine tract binding protein (PTB) or a biologically active fragment or derivative thereof in the cytoplasm of said cells. The term "reducing production of secretory granules (SGs) in peptide hormone- secreting endocrine cells or neurons" refers to the reduction or stop of a process in peptide hormone-secreting endocrine cells or neurons that leads to reduction of production of such granules of at least 10%, such as at least 20%, preferably at least 30%, more preferred at least 50%, even more preferred at least 75% and most preferred at least 95% as compared to a physiological, normal and optionally non- pathogenic status of corresponding endocrine cells or neurons.

Diseases can now be treated by blocking PTB activity or by reducing PTB levels in the cytoplasm of appropriate cells. Examples of such diseases include hyperprolactinemia or acromegalia as well as depression or sleeping disorders. The first two diseases result from tumors of endocrine cells that oversecrete certain hormones, such as prolactin or growth hormone in the case of the two diseases mentioned above. In accordance with the invention, it is intended to pursue the selective down-regulation of PTB in these cells to reduce hormone production. This measure will provide an assistant or alternative means for surgery, which is presently the therapy of choice. Reducing the level or activity of PTB or a biologically active fragment or derivative thereof may be accomplished by a variety of means all of which are to be considered as preferred embodiments in accordance with the invention. These means preferably include inhibition of transcription, for example by using antisense constructs, RNAi, intercellular blocking by antibodies, inactivation of the PTB gene, to name some options. The term "reducing", as already indicated above, includes a reduction of at least 10%, preferably at least 30%, more preferred at least 50%, even more preferred at least 80% and particularly preferred at least 90% of the normal level. Most preferred is that the reduction of intracellular level is achieved to at least 95% such as at least 98%. Reduction of the levels includes down-regulation of the gene by appropriate means as well as complete blockage of expression in further alternatives. Prevention of the nucleoplasmic transport of PTB is another means of reducing the cytoplasmic levels of PTB. Further included are enhancement of degradation or prevention of phophorylation of PTB. In principle, for many embodiments the reverse means that have been explained or will be explained in connection with the promotion of the presence/activity of PTB will have to be effected. More generally, the present invention thus also relates to a method for reducing or blocking production of secretory granules in peptide hormone-secreting endocrine cells or neurons comprising the step of reducing the presence and/or the activity of polypyrimidine tract binding protein (PTB) or a biologically active fragment or derivative thereof in the cytoplasm of said cells.

The PTB or a fragment or derivative thereof or an agent promoting/reducing the presence/activity will, for these purposes, preferably be formulated into a pharmaceutical composition.

The pharmaceutical composition may, in general, be in solid, liquid or gaseous form and may be, inter alia, in a form of (a) powder(s), (a) tablet(s), (a) solution(s) or (an) aerosol(s).

The pharmaceutical composition may be particularly useful for the treatment of any disease that is causally interrelated with lack of production, diminished production or retarded production of secretory granules (or, in other recited embodiments, with overproduction).

The pharmaceutical composition may further comprise pharmaceutically acceptable carriers, excipients and/or diluents. Examples of suitable pharmaceutical carriers, excipients and/or diluents are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising such carriers can be formulated by well known conventional methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial administration. It is particularly preferred that said administration is carried out by injection and/or delivery, e.g., to a site in the pancreas or into a brain artery or directly into brain tissue. The compositions may also be administered directly to the target site, e.g., by biolistic delivery to an external or internal target site, like the brain. The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, dosages for any one patient depend upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Proteinaceous pharmaceutically active matter may be present in amounts between 1 ng and 10 mg/kg body weight per dose; however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. If the regimen is a continuous infusion, it should also be in the range of 1 μg to 10 mg units per kilogram of body weight per minute.

Progress can be monitored by periodic assessment. The compositions of the invention may be administered locally or systemically. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of 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. 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, and inert gases and the like. Furthermore, the pharmaceutical composition may comprise further agents depending on the intended use of the pharmaceutical composition.

Additionally, a nucleic acid molecule and preferably a DNA molecule encoding said PTB or fragment or derivative thereof or (poly) peptides used in the enhancement or reduction of presence or activity of PTB may formulated into a pharmaceutical composition. The nucleic acid molecule is eventually to be introduced into the desired cells. Appropriate formulations include those wherein 10⁶ to 10¹² copies of the DNA molecule, advantageously comprised in an appropriate vector are administered per dose. The vector may be, for example, a phage, plasmid, viral or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host/cells. The nucleic acid molecules (or polynucleotides) may be joined to a vector containing selectable markers for propagation in a host. Generally, a plasmid vector is introduced in a precipitate such as a calcium phosphate precipitate or rubidium chloride precipitate, or in a complex with a charged lipid or in carbon-based clusters, such as fullerens. Should the vector be a virus, it may be packaged in vitro using an appropriate packaging cell line prior to application to host cells; see, for general information in this regard, Ausubel et al., Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (2001).

Advantageously, the polynucleotide is operatively linked to expression control sequences allowing expression in prokaryotic or eukaryotic cells or isolated fractions thereof.

Expression of said polynucleotide comprises transcription of the polynucleotide, preferably into a translatable mRNA. Regulatory elements ensuring expression in eukaryotic cells, preferably mammalian cells, are well known to those skilled in the art. They usually comprise regulatory sequences ensuring initiation of transcription and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers. Possible regulatory elements permitting expression in prokaryotic host cells (a less preferred embodiment) comprise, e.g., the lac, trp or tac promoter in E. coli, and examples for regulatory elements permitting expression in eukaryotic host cells (the more preferred embodiment) are the AOX1 or GAL1 promoter in yeast or the CMV-, SV40- , RSV-promoter (Rous sarcoma virus), CMV- enhancer, SV40-enhancer or a globin intron in mammalian and other animal cells. Preferred promoters are the natural promoter of PTB as well as promoters allowing the tissue specific expression of PTB such as promoters active in pancreatic β-cells or neuronal cells. Besides elements which are responsible for the initiation of transcription such regulatory elements may also comprise transcription termination signals, such as the SV40-poIy-A site or the tk-poly-A site, downstream of the polynucleotide. In this context, suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pCDMδ, pRc/CMV, pcDNAI , pcDNA3 (Invitrogen), pSPORTI (GIBCO BRL). Preferably, said vector is an expression vector and/or a gene transfer or targeting vector. Expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpes viruses, or bovine papilloma virus, may be used for delivery of the polynucleotides or vector into targeted cell population. Methods which are well known to those skilled in the art can be used to construct recombinant viral vectors; see, for example, the techniques described in Sambrook, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (2001). Alternatively, the polynucleotides or vectors can be reconstituted into liposomes for delivery to target cells.

The term "isolated fractions thereof" refers to fractions of eukaryotic or prokaryotic cells or tissues which are capable of transcribing or transcribing and translating RNA from the vector. Said fractions comprise proteins which are required for transcription of RNA or transcription of RNA and translation of said RNA into a polypeptide. Said isolated fractions may be, e.g., nuclear and cytoplasmic fractions of eukaryotic cells such as of reticulocytes. Kits for transcribing and translating RNA which encompass the said isolated fractions of cells or tissues are commercially available, e.g., as TNT reticulolysate (Promega).

Furthermore, the vector may be a gene transfer or gene targeting vector. Gene therapy, which is based on introducing therapeutic genes into cells by ex-vivo or in- vivo techniques is one of the most important applications of gene transfer. Suitable vectors, methods or gene-delivering systems for in-vitro or in-vivo gene therapy are described in the literature and are known to the person skilled in the art; see, e.g., Giordano, Nature Medicine 2 (1996), 534-539; Schaper, Circ. Res. 79 (1996), 911- 919; Anderson, Science 256 (1992), 808-813, Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res. 77 (1995), 1077-1086; Onodua, Blood 91 (1998), 30-36; Verzeletti, Hum. Gene Ther. 9 (1998), 2243-2251 ; Verma, Nature 389 (1997), 239- 242; Anderson, Nature 392 (Supp. 1998), 25-30; Wang, Gene Therapy 4 (1997), 393-400; Wang, Nature Medicine 2 (1996), 714-716; WO 94/29469; WO 97/00957; US 5,580,859; US 5,589,466; US 4,394,448 or Schaper, Current Opinion in Biotechnology 7 (1996), 635-640, and references cited therein. In particular, said vectors and/or gene delivery systems are also described in gene therapy approaches in neurological tissue/cells (see, inter alia Blδmer, J. Virology 71 (1997) 6641-6649) or in the hypothalamus (see, inter alia, Geddes, Front Neuroendocrinol. 20 (1999), 296-316 or Geddes, Nat. Med. 3 (1997), 1402-1404). Further suitable gene therapy constructs for use in neurological cells/tissues are known in the art, for example in Meier (1999), J. Neuropathol. Exp. Neural. 58, 1099-1110. The above described modes for introducing the vector into a desired target tissue or cell also find application if the introduction is for the purposes of gene therapy. Thus, the nucleic acid molecules and vectors may be designed for direct introduction or for introduction via liposomes, viral vectors (e.g. adenoviral, retroviral), electroporation, ballistic (e.g. gene gun) or other delivery systems into the cell. Additionally, a baculoviral system can be used as eukaryotic expression system for the nucleic acid molecules of the invention. The introduction and gene therapeutic approach should, preferably, lead to the expression of a functional PTB or fragment or derivative thereof.

As may be envisaged in view of the above, the method of the invention may be put into practice in vivo, ex vivo or in vitro (this holds also true for further embodiments of the invention discussed further below). In vivo applications have been discussed above. Ex vivo applications refer, inter alia, to embodiments wherein cells or tissues are treated outside the body in order to stimulate the production of SGs in accordance with the invention and wherein said cells or tissues, upon successful stimulation or successful modification allowing the stimulation in vivo (for example, by way of an inducible promoter) subsequently are re-introduced into the body; In vitro applications include those that allow the further investigation of the mechanism by which PTB stimulates SG production. These investigations will allow the identification of further molecules interacting with PTB and resulting in the production of SGs. Such further molecules also include members of a protein cascade that only indirectly interact with PTB.

Preferably or alternatively, the method further comprises promoting/reducing the activity of PTB or said biologically active fragment or derivative thereof in the cytoplasm of said cells.

Whereas the promotion of the presence of PTB or the fragment or derivative thereof in the cytoplasm is one necessary feature in accordance with the main embodiment of the invention, the invention also envisages to alternatively or additionally promote the activity of PTB or said biologically active fragment or derivative thereof in the cytoplasm of said cells. Promoting the activity of PTB may be achieved by a variety of means wherein preferred means are addressed herein below and include the meaning of promoting PTB from an inactive or less active into an active or more active form. Additional means include the mutagenesis of the protein (e.g. on the DNA level by site-directed mutagenesis) followed by a test for an enhanced activity (which may be based on tests performed in the appended examples) thus enhancing the intrinsic activity of PTB. It is of note that the term PTB when used alone, in accordance with the invention would optionally also have the meaning of a fragment or derivative of PTB as defined elsewhere.

In an alternative embodiment of the invention, if prima facie sufficient amounts of PTB or said fragment or derivative are present in the cell, then the invention contemplates solely the enhancement of the activity by any of the means discussed herein below or above. In another alternative embodiment of the invention, if reduction of PTB levels is desired, then it may be sufficient not to reduce the levels of PTB but to solely reduce the activity of the molecule. Similarly, if a reduction of overproduction of SGs is envisaged, then either the presence or the activity of PTB or said fragment or derivative thereof or both may be reduced.

Also preferred in accordance with the invention is a method, wherein the promotion/reduction (of the presence and optionally the activity, also in connection with the further preferred embodiments recited herein below) comprises the promotion/reduction of the nucleocytoplasmic transport of PTB or of a biologically active fragment or derivative thereof.

As has been found in accordance with the present invention, promotion, i.e., for example, enhancement of or instigation of nucleocytoplasmic transport is a particularly suitable means for stimulating production of SGs. Similarly, if a reduction of SG production is envisaged in accordance with another preferred embodiment of the invention, the nucleocytoplasmic transport of PTB or of said biological active fragment or derivative thereof will be reduced or abolished.

The promotion/reduction of the nucleocytoplasmic transport may be achieved by different means.

In a preferred embodiment of all pertaining previously cited embodiments of the invention, the promotion of the presence, activity and preferably nucleocytoplasmic transport comprises the activation of phosphorylation of PTB (or, if reduction is envisaged, activation of phosphorylation should be avoided or stalled.) Whereas protein kinase A is a prime example of a phosphorylating agent and will thus separately be addressed below, the invention envisages the phophorylation by different kinases, alternatively or in addition to the phosphorylation by protein kinase A. In principle, the means to effect phosphorylation by these different enzymes can be based on the same principles that are described in detail for protein kinase A below. Activation of phosphorylation includes the de novo synthesis of any of these enzymes, the activation of inactive molecules, the prevention of degradation, the enhancement of stability and the enhancement of the intrinsic activity, e.g. by providing muteins of such enzymes. PTB phosphorylation can also be enhanced by inhibiting the activity of a dephosphorylating agent, such as a phosphatase. Reduction of PTB presence or activity in the cytosol or its nucleocytoplasmic translocation could e.g. be induced by a) inhibiting the kinase activity; b) activating PTB dephosphorylation. This could be accomplished using similar strategies as for its phosphorylation, i.e through a specific dephosphorylating agent such as a phosphatase.

It is most preferred that PKA phosphorylates serine residue 16 of PTB. It was further demonstrated in accordance with the invention that cAMP-dependent phosphorylation of PTB is ERK1/2 independent.

In a particularly preferred embodiment of the method of the invention, said promotion of the nucleocytoplasmic transport of PTB or of said biologically active fragment or derivative thereof is effected by promoting the presence or activity of protein kinase A (PKA) in said cell. Alternatively, said presence or activity is reduced.

Protein kinase A has been shown in the art to activate PTB. The term "promotion of the presence" has the same meaning in accordance with this invention as explained above and elsewhere in connection with the promotion of the presence of PTB, with the exception, of course, that here the promotion of the presence of PKA is addressed. Thus, promoting the presence of PKA includes the prevention or reduction of PKA degradation, or enhancing the stability of PKA. It is of note that the term PKA does not only include naturally occurring PKA but, in addition, molecules that retain the kinase function and substrate specificity of naturally occurring PKA. Such molecules include fragments or derivatives such as muteins of naturally occurring PKA. It is particularly preferred in accordance with the invention that the promotion of the presence of protein kinase A in said cell is effected by overexpression or activation of protein kinase A. Promotion of the activity includes enhancement of the intrinsic activity of the enzyme. Again, if reduction is desired instead of promotion, the presence or activity of protein kinase A should be diminished or abolished.

Most preferred are embodiments wherein said overexpression of protein kinase A is effected by overexpressing PKA from a vector, by increasing cAMP levels in the cell or by contacting the cell with a drug that induces the overexpression of PKA or the increase of cAMP levels in said cell. As has been found in accordance with the present invention, cAMP influences phosphorylation of PTB in INS-1 cells and induces ICA512 expression said neuroendocrine cell line. If reduction of the presence of PTB is desired, then cAMP levels should be decreased or the cell should be contacted with a drug that decreases or abolishes the expression of PKA in said cell.

Here and elsewhere in the specification the term "cAMP" also refers to derivatives of cAMP having the same or essentially the same biological function and that are preferably well known in the art.

Overexpression of PTB protein may be effected using the vectors as described above. cAMP levels in the cell may be increased by conventional means. Drugs that induce the overexpression of PTB or lead to in increase of cAMP levels include cAMP analogs that are commercially available. cAMP levels can also be increased by activators of adenylate cyclase, which produces cAMP, or by ligands of the G- protein coupled receptors, which, in turn, stimulate adenylate cyclase activity. Another way to increase cAMP levels is to inhibit the activity of phosphodiesterase, which hydrolizes cAMP into AMP. In addition, PKA activity may be favored by reducing agents, such as DTT or β-mercaptoethanol, while oxidative agents inhibit PKA activity by promoting its glutathionylation (see: Humphries KM, Juliano C, Taylor SS. 2002. Regulation of cAMP-dependent protein kinase activity by glutathionylation. J Biol Chem. 277:43505-11 ; and Kopperud R, Krakstad C, Selheim F, Doskeland SO. 2003 cAMP effector mechanisms. Novel twists for an 'old' signaling system. FEBS Lett. 3;546:121-6). All these possibilities are included within the scope of the present invention. For achieving reduction of the presence or activity, the reverse means as outlined above may be employed.

Direct proof for the interrelation between phosphorylation and translocation comes from a series of findings that were made in accordance with this invention. Thus, it was found that a mutant of PTB where in position 16 the serine residue that can phosphorylated by, for example, PKA, is altered into an aspartate residue, the PTB molecule stays in the cytoplasm (see Example 6). It is of note that this mutant cannot be dephosphorylated. Similarly, a peptidomimetic that mimics the phosphorylated protein (but also cannot be desphosphorylated) stays in the cytoplasm. These proteins have been shown to be responsible for SG formation, to promote insulin expression and in particular the translation machinery pertaining to insulin expression.

Another preferred embodiment of the present invention relates to a method wherein the promotion comprises the transient or stable expression of PTB or of said biologically active fragment or derivative thereof from an exogenously introduced vector.

This preferred embodiment of the invention may be put into practice according to conventional techniques of molecular biology and requires, optionally after cloning of the PTB gene and insertion into a vector, the introduction into the cell (see above) and the expression of PTB or of said biologically active fragment or derivative thereof from an expression vector. Expression vectors useful in this embodiment of the invention and options for the introduction of such vectors into suitable cells have been described herein above.

In a further preferred embodiment of the method of the invention, the promotion comprises enhancing the stability of PTB or of said biologically active fragment or derivative thereof or reducing degradation of PTB or of said biologically active fragment or derivative thereof in said cell. If reduction is required, then the corresponding embodiment comprises decreasing the stability of PTB or of said biologically active fragment or derivative thereof or stimulating degradation or said biologically active fragment or derivative thereof in said cell. The enhancement of the stability of the protein may be achieved by agents that inhibit proteasome activity. Reduction of the degradation comprises methods wherein PTB degrading proteins are inhibited such as by RNAi or antisense oligonucleotides [see, for example, 25]. Preferably, the enhancement and reduction, respectively, amount to at least 25% of the amount of PTB present in the cytoplasm, preferably at least 50%, more preferred at least 75% and most preferred at least 90%. In the case of enhancement of the stability, an increase of more than 100% is advantageously envisaged.

A further preferred embodiment of the invention relates to a method wherein the promotion comprises introducing PTB or a biologically active fragment or derivative thereof into the cell.

In this preferred embodiment, the protein or peptidomimetic is directly introduced into the desired cell or tissue. Available processes for such an introduction include microinjection techniques or protein transduction procedures. See, for example, Lindsay MA. Peptide-mediated cell delivery: application in protein target validation. 2002. Curr Opin Pharmacol. 2:587-94.

In an additional preferred embodiment of the method of the invention, the promotion of the presence or activity of polypyrimidine tract binding protein (PTB) or a biologically active fragment or derivative thereof in the cytoplasm of said cells comprises the inhibition of dephosphorylation of PTB (or in the case of reduction the stimulation of dephosphorylation of PTB) or said fragment or derivative (as long as said derivative is a (poly)peptide, i.e. a peptide (up to 30 amino acids) or a polypeptide, the term "polypeptide" having the same meaning therein as the term "protein). Since, as was explained above, phosphorylation of PTB by PKAs is a means to promote the formation of SGs, the same effect is expected to be achieved by inhibiting the dephosphorylation of said molecule. A large number of general as well as specific serine/threonine phosphatase inhibitors is commercially available.

It is further preferred in accordance with the present invention that said PTB is the 59 kD isoform having four RNA recognition motif domains of PTB. This is the largest isoform arising from several alternatively spliced variants [44,45]. Particularly preferred is a method wherein said endocrine cells to be employed in accordance with the invention are pancreatic β-cells.

As newly-synthesized SGs are preferentially recruited for exocytosis [34-36], alterations in the ability of rapidly promoting SG biosynthesis is expected to affect the pattern of insulin secretion. This will be relevant in type-1 and type-2 diabetes, in which insulin secretion is typically impaired because of the inadequate release on newly-synthesized SGs [37]. This particularly preferred embodiment of the invention allows for the triggering of SG production and is thus expected to form a cure for type-1 and type-2 diabetes.

In this connection, it is further preferred that said promotion is effected by glucose or glucagon-like peptide-1 (GLP-1). Glucagon-like peptide-1 (GLP-1) is produced by intestinal L-cells, which secrete it in response to food intake. The hormone affects the physiology of multiple organs, including pancreatic islets. Specifically, it rapidly potentiates insulin secretion and its biosynthesis. It also promotes islet cell growth and islet neogenesis. Most of these effects can be ascribed, directly or indirectly, to the elevation of intracellular cAMP levels, since binding of GLP-1 to its receptor stimulates adenylate cyclase activity See: MacDonald PE, El-Kholy W, Riedel MJ, Salapatek AM, Light PE, Wheeler MB. 2002. The multiple actions of GLP-1 on the process of glucose-stimulated insulin secretion. Diabetes 51 Suppl 3:S434-42.

In another preferred embodiment of the method of the invention, said method requires that the secretory peptide contained in said secretory granules is insulin, amylin or a peptide hormone or neuropeptide derived from one of the following precursors: ADM precursor, Agouti switch protein precursor, Agouti-related protein precursor, Apelin precursor, Atrial natriuretic factors, Beta-neoendorphin-dynorphin precursor, Brain natriuretic peptide precursor, Calcitonin gene-related peptide I precursor, Calcitonin gene-related peptide, II precursor, Calcitonin precursor, Cholecystokinin precursor, Chromogranin A precursor, Cocaine- and amphetamine- regulated transcript protein precursor, Corticoliberin precursor, Corticotropin- lipotropin precursor, Cortistatin precursor, FMRFamide-related peptides precursor, FMRFamide-related peptides precursor, Follistatin precursor, Follitropin beta chain precursor, Galanin precursor, Galanin-like peptide precursor, Gastric inhibitory polypeptide precursor, Gastrin precursor, Gastrin-releasing peptide precursor, Ghrelin precursor, Glucagon precursor, Glycoprotein hormones alpha chain precursor, Growth hormone variant precursor, Insulin precursor, Insulin-like growth factor binding protein 3 precursor, Insulin-like peptide INSL6 precursor, Islet amyloid polypeptide precursor, Leydig insulin-like peptide precursor, Morphogenetic neuropeptide, Motilin precursor, Neurexophilin 2 precursor, Neurexophilin 3 precursor, Neurexophilin 4 precursor, Neuroendocrine protein 7B2 precursor, Neurokinin B precursor, Neuromedin B-32 precursor, Neuromedin U-25 precursor, Neuropeptide B precursor, Neuropeptide W precursor, Neuropeptide Y precursor, Neurotensin precursor, Nociceptin precursor, Orexin precursor, Oxytocin- neurophysin 1 precursor, Pancreatic hormone precursor, Parathyroid hormone precursor, Parathyroid hormone-related protein precursor, Peptide YY precursor, Pituitary adenylate cyclase activating polypeptide precursor, Proenkephalin A precursor, Progonadoliberin I precursor, Progonadoliberin II precursor, Prokineticin 2 precursor, Prolactin precursor, Pro-MCH precursor, Prorelaxin H1 precursor, Protachykinin 1 precursor, Protein-tyrosine phophatase-like N precursor, Receptor- type protein-tyrosine phophatase N2 precursor, Regulated endocrine specific protein 18 precursor, Resistin precursor, Resistin-like beta precursor, Secretin precursor, Secretorygranin I precursor, Secretorygranin II precursor, Secretorygranin III precursor, Somatoliberin precursor, Somatostatin precursor, Somatotropin precursor, Stanniocalcin 1 precursor, Stanniocalcin 2 precursor, Urocortin II precursor, Urocortin precursor, Vasoactive intestinal peptide precursor, Vasopressin-neurophysin 2- copeptin precursor.

It is of note and well known in the art that various hormones or neuropeptides are derivable from the precursors cited above. Important examples of such precursors and hormones or neuropeptides derived therefrom include the following:

1) Glucacagon precursor (P01275)

MKSIYFVAGL FVMLVQGSWQ RSLQDTEEKS RSFSASQADP LSDPDQMNED KRHSQGTFTS DYSKYLDSRR AQDFVQWLMN TKRNRNNIAK RHDEFERHAE GTFTSDVSSY LEGQAAKEFI AWLVKGRGRR DFPEEVAIVE ELGRRHADGS FSDEMNTILD NLAARDFINW LIQTKITDRK

Derived peptides: a) Glicentin-related polypeptide (residues 21-50): RSLQDTEEKS RSFSASQADP LSDPDQMNED b) Glucagon (residues 53-81): HSQGTFTS DYSKYLDSRR AQDFVQWLMN T c) Glucagon-like peptide 1 (residues 98-127): HAE GTFTSDVSSY LEGQAAKEFI AWLVKGR d) Glucagon-like peptide 2 (residues 146-178): HADGS FSDEMNTILD NLAARDFINW LIQTKITD

2) Corticoliberin precursor (P06850)

MRLPLLVSAG VLLVALLPCP PCRALLSRGP VPGARQAPQH PQPLDFFQPP PQSEQPQQPQ ARPVLLRMGE EYFLRLGNLN KSPAAPLSPA SSLLAGGSGS RPSPEQATAN FFRVLLQQLL LPRRSLDSPA ALAERGARNA LGGHQEAPER ERRSEEPPIS LDLTFHLLRE VLEMARAEQL AQQAHSNRKL MEIIGK

Derived peptide: a) Corticoliberin (residues 154-194 ): SEEPPIS LDLTFHLLRE VLEMARAEQL AQQAHSNRKL MEII

3) Corticotropin-lipotropin precursor (P01189)

MPRSCCSRSG ALLLALLLQA SMEVRGWCLE SSQCQDLTTE SNLLECIRAC KPDLSAETPM FPGNGDEQPL TENPRKYVMG HFRWDRFGRR NSSSSGSSGA GQKREDVSAG EDCGPLPEGG PEPRSDGAKP GPREGKRSYS MEHFRWGKPV GKKRRPVKVY PNGAEDESAE AFPLEFKREL TGQRLREGDG PDGPADDGAG AQADLEHSLL VAAEKKDEGP YRMEHFRWGS PPKDKRYGGF . MTSEKSQTPL VTLFKNAIIK NAYKKGE Derived peptides: a) NPP (residues 27-102): WCLE SSQCQDLTTE SNLLECIRAC KPDLSAETPM FPGNGDEQPL TENPRKYVMG HFRWDRFGRR NSSSSGSSGA GQ b) Melanotropin gamma (residues 77-87): YVMG HFRWDRF c) melanotropin alpha (residues 138-150): SYS MEHFRWGKPV d) Corticotropin (residues 138-176): SYS MEHFRWGKPV GKKRRPVKVY PNGAEDESAE AFPLEF e) Lipotropin beta (residues 179-267): EL TGQRLREGDG PDGPADDGAG AQADLEHSLL VAAEKKDEGP YRMEHFRWGS PPKDKRYGGF MTSEKSQTPL VTLFKNAIIK NAYKKGE f) Lipotropin gamma (residues 179-234): EL TGQRLREGDG PDGPADDGAG AQADLEHSLL VAAEKKDEGP YRMEHFRWGS PPKD g) Melanotropin beta (residues 217-234) : DEGP YRMEHFRWGS PPKD h) Beta endorphin (residues 237-267): YGGF MTSEKSQTPL VTLFKNAIIK NAYKKGE i) Met-enkephalin (residues 237-241): YGGF M

4) Orexin precursor (043612)

MNLPSTKVSW AAVTLLLLLL LLPPALLSSG AAAQPLPDCC RQKTCSCRLY ELLHGAGNHA AGILTLGKRR SGPPGLQGRL QRLLQASGNH AAGILTMGRR AGAEPAPRPC LGRRCSAPAA ASVAPGGQSG I

Derived peptides: a) Orexin A (residues 34-66): QPLPDCC RQKTCSCRLY ELLHGAGNHA AGILTL b) Orexin B (residues 70-97): R SGPPGLQGRL QRLLQASGNH AAGILTM

5) Pituitary adenylate cyclase activating polypeptide precursor (P 18509) MTMCSGARLA LLVYGIIMHS SVYSSPAAAG LRFPGIRPEE EAYGEDGNPL PDFGGSEPPG AGSPASAPRA AAAWYRPAGR RDVAHGILNE AYRKVLDQLS AGKHLQSLVA RGVGGSLGGG AGDDAEPLSK RHSDGIFTDS YSRYRKQMAV KKYLAAVLGK RYKQRVKNKG RRIAYL a) PACAP-related peptide (residues 82-129): DVAHGILNE AYRKVLDQLS AGKHLQSLVA RGVGGSLGGG AGDDAEPLS b) Pituitary adenylate cyclase activating peptide-27 (residues 132-158): AHSDGIFTDS YSRYRKQMAV KKYLAAVL c) Pituitary adenylate cyclase activating peptide- 38 (residues 132-169): HSDGIFTDS YSRYRKQMAV KKYLAAVLGK RYKQRVKNK

6) Ghrelin precursor (Q9UBU3)

MPSPGTVCSL LLLGMLWLDL AMAGSSFLSP EHQRVQQRKE SKKPPAKLQP RALAGWLRPE DGGQAEGAED ELEVRFNAPF DVGIKLSGVQ YQQHSQALGK FLQDILWEEA KEAPADK

Derived peptide: a) Ghrelin (residues 24-51 ): GSSFLSP EHQRVQQRKE SKKPPAKLQP R

On the basis of this embodiment, a number of cures to pertinent diseases are feasible; see for further guidance: Harrison's Principles of Internal Medicine. By Eugene Braunwald M.D. (Editor), Anthony S. Fauci M.D. (Editor), Dennis L. Kasper M.D. (Editor), Stephen L. Hauser M.D. (Editor), Dan L. Longo M.D. (Editor), J. Larry Jameson M.D. (Editor). McGraw-Hill Professional; 15th edition (February 16, 2001) ISBN: 0070072728; Williams Textbook of Endocrinology, by Robert Hardin Williams (Editor), P. Reed Larsen (Editor), Jean D. Wilson, Melmed Shlomo, Daniel W. Foster, Henry M. Kronenberg. W B Saunders; 10th edition (December 20, 2002) ISBN: 0721691846. Peptides and Non Peptides in Neuroendocrinology and Oncology: From Basic to Clinical Research, by E. E. Muller (Editor) Springer-Verlag Telos (August 2003) ISBN: 8847002958.

A comprehensive list of accession numbers associated with the above hormones, neuropeptides and precursors thereof is provided by Table 3. Further information about the hormones/peptides/precursors can be obtained from these sources. A list of diseases associated with the hormones/peptides/precursors is provided by Table 4. It is to be understood in accordance with the present invention, that the invention comprises the treatment of any of the diseases associated with any of these hormones/peptides/precursors by manipulating PTB presence/activity as described for the treatment of type-1 and type-2 diabetes, sleeping disorders or depression elsewhere in this specification with the exception that different cells are targeted, if appropriate. The skilled artisan is well aware which type of cell is to be targeted for each disease; see also the above cited textbooks for further guidance.

The present invention also relates to a method of treating or preventing type-1 or type-2 diabetes comprising stimulating production of insulin-containing secretory granules in pancreatic β-cells wherein said stimulation comprises the step of promoting the presence or activity of polypyrimidine tract binding protein (PTB) or of a biologically active fragment or derivative thereof in the cytoplasm of said β-cells. Preferred is in accordance with this embodiment of the invention that the promotion of the presence is mandatory whereas the promotion of the activity is optional. Alternatively, if sufficient amounts of PTB or said fragment or derivative are present in the cell, then the invention contemplates solely the enhancement of the activity by any of the means discussed herein above. The same holds true for any of the further embodiments referred to in this specification wherein the feature "promoting the presence or activity" occurs, also in connection with other compounds such as protein kinase A.

In an additional aspect, the present invention relates to the use of an agent of the promotion of the presence or activity of polypyrimidine tract binding protein (PTB) or of a biologically active fragment or derivative thereof in ^β-cells for the preparation of a pharmaceutical composition for treating or preventing type-1 or type-2 diabetes.

Formulations of compounds into pharmaceutical compositions, regimens of treatment and modes of administration have been discussed herein above in connection with the method of the invention and apply equally here.

It is of note and has been mentioned above that the principle underlying the present invention can be applied for curing or preventing a variety of diseases, wherein type- 1 and type-2 diabetes are prominent and advantageous examples. In accordance with this notion, the invention further relates to the use of an agent of the reduction of the presence or activity of polypyrimidine tract binding protein (PTB) or of a biologically active fragment or derivative thereof in hypothalamic neurons for the preparation of a pharmaceutical composition for treating or preventing sleep disorders or depression.

The secretory granules of hypothalamic neurons in the medial parvocellular portion of the paraventricular nucleus contain and release cortocoliberin, also known as corticotropin-releasing hormone (CRH), CRH in turn stimulates the biosynthesis and release from the anterior, pituitary of corticotropin-lipotropin derived peptide hormones, such as ACTH and β-endorphin. Excess release of CRH has been implicated in sleep disorders and depression. Reducing the presence/activity of PTB in the cytoplasm of hypothalamic neurons is therefore expected to be beneficial for the treatment of these common disorders, by downregulating CHR expression and release from SGs. Likewise, the generation of a transgenic mouse in which the presence /activity of PTB or part thereof in the cytoplasm of hypothalamic neurons is enhanced is expected to establish an animal model for sleep disorder and depression. These animal models could be useful for the screening of drugs for the therapy of sleep disorders and depression in humans.

In a preferred embodiment of the use of the invention, said agent is GLP-1 (for diabetes) or CRH (for sleeping disorders or depression).

Other preferred embodiments for said agents are disclosed throughout this specification. If reduction is desired, then, also siRNA technology, antibodies, ribozymes, antisense molecules, derivatives or fragments of antibodies such as Fab fragments, F(ab')2, scFvs, protease, etc. and other means described herein may be used. Similarly, if promotion of the presence or activity is desired, further means have been described throughout this specification.

In a further preferred embodiment of the latter method of the invention or the use of the invention, the promotion (in the case of diabetes treatment, also for the following preferred embodiments) or reduction (for sleeping disorders or depression, also for the following preferred embodiments) comprises the promotion/reduction of the nucleocytoplasmic transport of PTB or of a biologically active fragment or derivative thereof.

This preferred embodiment and the following preferred embodiment of this method and the use of the invention have been described in some detail in connection with the main embodiment of the invention. These explanations apply mutatis mutandis here.

In an additionally (more) preferred embodiment of the method or use of the invention, the promotion/reduction comprises the activation/reduction or prevention of phosphorylation of PTB or said fragment or derivative thereof (in the case of sleeping disorders and depression, dephosphorylation is also considered).

In a more preferred embodiment of the method or use of the invention, said promotion/reduction of the nucleocytoplasmic transport of PTB or of said biologically active fragment or derivative thereof is effected by promoting/reducing the presence or activity of protein kinase A in said cell.

In a particularly preferred embodiment of the method or use of the invention, the promotion/reduction of the presence or activity of protein kinase A in said cell is effected by the oyerexpression or activation of protein kinase A/reduction of expression of protein kinase A.

In another more preferred embodiment of the method or use of the invention, said overexpression of protein kinase A is effected by overexpressing PKA from a vector, by increasing cAMP levels in the cell or by contacting the cell with a drug that induces the overexpression PKA or the increase of cAMP levels in said cell. In the case of reducing PTB levels, preferably cAMP levels will be decreased.

Still another preferred embodiment of the latter method of the invention or the use of the invention requires that the promotion comprises the transient or stable expression of PTB or of said biologically active fragment or derivative thereof from an exogenously introduced vector.

An additional preferred embodiment of the method of the invention or the use of the invention necessitates that the promotion/reduction comprises enhancing/reducing the stability of PTB or of said biologically active fragment or derivative thereof in said cell or reducing/enhancing degradation of PTB or of said biologically active fragment or derivative thereof in said cell.

In still another alternative, the promotion comprises introducing PTB or a biologically active fragment or derivative thereof into the cell.

A further preferred embodiment of the invention requires a method or the use wherein the promotion/reduction comprises the inhibition/activation of dephosphorylation of PTB.

In another embodiment, the present invention relates to a method of screening for an agent capable of stimulating production of secretory granules in peptide hormone- secreting endocrine cells or neurons comprising the steps of (a) contacting a cell capable of forming secretory granules and expressing polypyrimidine tract binding protein (PTB) or a biologically active fragment or derivative thereof with one or more compounds; and (b) assessing whether said one or more compounds promote the presence or activity of said polypyrimidine tract binding protein (PTB) or said biologically active fragment or derivative thereof in the cytoplasm of said cell. Contacting of the cell in step (a) will be done, for example, under conditions that allow the uptake of the one or more compounds into the cell. Alternatively, conditions are provided where the compounds are allowed to interact with the surface receptors of the plasma membrane. For both embodiments, the conditions may be the same. Appropriate conditions include physiological conditions such as incubation in physiological saline. If more than one compound is contacted with the cell and the number of compounds tests positive in step (b), it is advantageous to repeat the experiment by incubating the cell with only one compound at the time. In so far, a direct relation between the compound tested and its capability to promote the presence of PTB can be established.

The assessment in step (b) can be established by a variety of means. For example, the presence of PTB in the cytoplasm can be observed by microscopic means or biochemical subcellular fractionation. If microscopic assessment is envisaged, it is advantageous to label PTB or said biologically active fragment or derivative thereof. Means of labeling proteins and cells are well known in the art and include labeling by tags such as myc-tag, His-tag or Flag-tag. Labels further include phosphorescent or fluorescent labels. Activity can be measured, e.g by the increase of SGs in the cells. An additional means envisaged by the present invention is to test the binding activity of PTB to mRNA encoding secretory granule components, such as ICA512, and stabilization or translation of mRNA encoding secretory granule components, such as ICA512, upon PTB binding.

Said method of screening can be accomplished in a high-throughput manner. Robotic equipment for that purpose is known in the art and available from a number of suppliers. Cells are grown in wells of plates containing arrays of 96, 384, 1536 or more wells. Transfer of the well-plates from incubators, addition of test compounds, optional washing steps as well determining the read-out is performed in an automated fashion without requiring user interference using hundreds of thousands to millions of compounds in days to weeks.

Once a compound of interest has been identified, it can be further developed into a pharmaceutical agent such as by reducing its toxicity, prolonging shelf life and so on. In an alternative embodiment, the present invention relates to a method of screening for an agent capable of reducing production of secretory granules in peptide hormone-secreting endocrine cells or neurons comprising the steps of (a) contacting a cell capable of forming secretory granules and expressing polypyrimidine tract binding protein (PTB) or a biologically active fragment or derivative thereof with one or more compounds; and (b) assessing whether said one or more compounds reduce the presence or activity of said polypyrimidine tract binding protein (PTB) or said biologically active fragment or derivative thereof in the cytoplasm of said cell.

Additionally, the invention pertains to a method for screening for an agent useful as a cure for sleeping disorders or depression comprising the steps of , (a) contacting an animal carrying a transgene encoding polypyrimidine tract binding protein (PTB) or a biologically active fragment or derivative thereof under the control of a promoter that is active in hypothalamic neurons with one or more compounds; and (b) assessing whether said one or more compounds reduce the presence or activity of said polypyrimidine tract binding protein (PTB) or said biologically active fragment or derivative thereof in the cytoplasm of said neurons.

Monitoring of presence or activity, respectively, of PTB or of said biologically active fragment or derivative thereof may be effected by determining the level of protein expression of PTB or of said biologically active fragment or derivative thereof or by determining the phosphorylation status thereof, respectively, in a sample taken from the transgenic animal by using, for example, methods disclosed herein and exemplified in the Examples.

In this and other embodiments pertaining to screening methods, the agent to be screened can be contained in libraries of small molecules, such as organic or inorganic small molecules which may be commercially available. In addition, libraries comprising antibodies or functional fragments or derivatives thereof (i.e. fragments or derivatives maintaining the binding specificity of the original antibody) may be used as a starting point in the screening process. Also, libraries of aptamers or peptides might be employed. The skilled artisan is of course free to use any other starting point of desired compounds for use in the screen assays described throughout the specification. The animal to be employed is a non-human animal.

For example, rodent hypothalamic neurons carrying the transgene can be isolated and grown in culture. Their response to drugs in term of PTB presence in the cytoplasm, expression of CRH and secretory granules may be monitored by microscopy or biochemically.

Additionally, the invention pertains to a method for screening for an agent useful as a cure for type-1 or type-2 diabetes comprising the steps of (a) contacting an anirrial carrying a transgene encoding polypyrimidine tract binding protein (PTB) or a biologically active fragment or derivative thereof under the control of a promoter that is active in pancreatic β- cells with one or more compounds; and (b) assessing whether said one or more compounds increase the presence or activity of said polypyrimidine tract binding protein (PTB) or said biologically active fragment or derivative thereof in the cytoplasm of said cells.

In a preferred embodiment of this method of the invention said promoter is an inducible promoter.

The term "inducible promoter" is well known in the art and denotes a promoter the activity of which can be influenced by external parameters such as upon the addition of a drug.

Particularly preferred is a method wherein said promoter is the vasopressin promoter. To selectively target the hypothalamic neurons releasing CRH, this vasopressin gene promoter may be used (see: Murphy, D. & Wells, S. 2003. In Vivo Gene Transfer Studies on the Regulation and Function of the Vasopressin and Oxytocin Genes. Journal of Neuroendocrinology 15, 109-125). CRH and vasopressin are produced by the same hypothalamic neurons (see: Whitnall MH, Mezey E, Gainer H. 1985. Co- localization of corticotropin-releasing factor and vasopressin in median eminence neurosecretory vesicles. Nature 317 :248-50).

In another preferred embodiment of the method of the invention, said derivative of PTB is a fusion protein of PTB or a biologically active fragment thereof and a detectable marker.

The making of fusion proteins is well known in the art and requires the availability of the coding sequence of PTB and the fusion partner. Using standard means of molecular biology (such as described in Sambrook et al., loc. cit.) a nucleic acid molecule encoding a fusion protein may be prepared. PTB may be fused, for example, to a marker such as a tag. Appropriate tags have been discussed above, and include FLAG, Myc, HIS and others. Markers that can be used in accordance with the invention also include radioactive markers or phosphorescent markers.

Particularly preferred is a method wherein said detectable marker is a fluorescent compound. Appropriate fluorescent compounds include Green Fluorescent Protein (GFP) and derivatives thereof.

Further preferred is a method wherein the assessment in step (b) comprises the assessment of nucleocytoplasmic transport of PTB.

It is also preferred that said endocrine cells are pancreatic β-cells. A particularly preferred embodiment is a method wherein the peptide is insulin.

In another preferred embodiment of the method of the invention, said method further comprises the step of

(c) comparing the promotion/reduction effected by said compound , on the presence or activity of said polypyrimidine tract binding protein (PTB) or said biologically active fragment or derivative thereof in the cytoplasm of said cell with the promotion effected by glucose or GLP-1 and CRH, respectively.

This embodiment of the invention advantageously allows the quantitative assessment of the tested compound with respect to its capability to induce/reduce SG formation.

Performing this step will allow a first assessment in how far the compound investigated will be useful for the development of a pharmaceutical compound. In the method of the invention, it is additionally preferred that said one or more compounds are members of a library of compounds.

Useful libraries include those libraries of small molecules, either organic or inorganic or of peptides. Suitable libraries are commercially available, for example from

ChemBridge Corp., San Diego, US.

The method of the invention thus also allows for testing whether the compound may affect not only the presence of PTB in the cell but also whether it can (de)activate

PTB.

The method of the invention in a further preferred embodiment additionally or in the alternative to step (c)) comprises the step of

(d) testing the efficacy of a compound assessed as being capable of promoting or reducing the presence or activity of PTB in step (b) in the stimulating of the production of secretory granules in peptide hormone-secreting endocrine cells or neurons in an animal model.

This additional step will allow significant insights in the in vivo applicability of the compound that tested positive in an in vitro assay. Parameters like toxicity, half-life etc. that have to be tested in pre-clinical trials may be assessed in this step. Suitable

(non-human) animals foremost include laboratory mice and rats such as available from Charles River Laboratories etc.

Additionally, the invention envisages that the method comprises the further step of improvement or of refining the pharmacological properties of the identified promoting or reducing agent (in the following also referred to as "compound" or "drug") , by the method as described herein above, said method comprising the optionally the steps of said methods and:

(1) identification of the binding sites of the compound and PTB molecule by site- directed mutagenesis or chimeric protein studies;

(2) molecular modeling of both the binding site of the compound and the binding site of the PTB molecule; and

(3) modification of the compound to improve its binding specificity for PTB.

All techniques employed in the various steps of the method of the invention are conventional or can be derived by the person skilled in the art from conventional techniques without further ado. Thus, biological assays based on the herein identified nature of the PTB may be employed to assess the specificity or potency of the drugs wherein the increase of one or more activities of PTB may be used to monitor said specificity or potency. Steps (1) and (2) can be carried out according to conventional protocols. A protocol for site directed mutagenesis is described in Ling MM, Robinson BH. (1997) Anal. Biochem. 254: 157-178. The use of homology modeling in conjunction with site-directed mutagenesis for analysis of structure-function relationships is reviewed in Szklarz and Halpert (1997) Life Sci. 61 :2507-2520. Chimeric proteins are generated by ligation of the corresponding DNA fragments via a unique restriction site using the conventional cloning techniques described in Sambrook (1989), loc. cit.. A fusion of two DNA fragments that results in a chimeric DNA fragment encoding a chimeric protein can also be generated using the gateway- system (Life technologies), a system that is based on DNA fusion by recombination. A prominent example of molecular modeling is the structure-based design of compounds binding to HIV reverse transcriptase that is reviewed in Mao, Sudbeck, Venkatachalam and Uckun (2000). Biochem. Pharmacol. 60: 1251-1265. For example, identification of the binding site of said drug by site-directed mutagenesis and chimerical protein studies can be achieved by modifications in the PTB primary sequence that affect the drug affinity; this usually allows to precisely map the binding pocket for the drug.

As regards step (2), the following protocols may be envisaged: Once the effector site for drugs has been mapped, the precise residues interacting with different parts of the drug can be identified by combination of the information obtained from mutagenesis studies (step (1)) and computer simulations of the structure of the binding site provided that the precise three-dimensional structure of the drug is known (if not, it can be predicted by computational simulation). If said drug is itself a peptide, it can be also mutated to determine which residues interact with other residues in the (poly)peptide of interest.

Finally, in step (3) the drug can be modified to improve its binding affinity or its potency and specificity. If, for instance, there are electrostatic interactions between a particular residue of the PTB of interest and some region of the drug molecule, the overall charge in that region can be modified to increase that particular interaction. Identification of binding sites may be assisted by computer programs. Thus, appropriate computer programs can be used for the identification of interactive sites of a putative inhibitor and the (poly)peptide by computer assisted searches for complementary structural motifs (Fassina, Immunomethods 5 (1994), 114-120). Further appropriate computer systems for the computer aided design of protein and peptides are described in the prior art, for example, in Berry, Biochem. Soc. Trans. 22 (1994), 1033-1036; Wodak, Ann. N. Y. Acad. Sci. 501 (1987), 1-13; Pabo, Biochemistry 25 (1986), 5987-5991. Modifications of the drug can be produced, for example, by peptidomimetics and other inhibitors can also be identified by the synthesis of peptidomimetic combinatorial libraries through successive chemical modification and testing the resulting compounds. Methods for the generation and use of peptidomimetic combinatorial libraries are described in the prior art, for example in Ostresh, Methods in Enzymology 267 (1996), 220-234 and Dorner, Bioorg. Med. Chem. 4 (1996), 709-715. Furthermore, the three-dimensional and/or crystallographic structure of activators of the expression of PTB can be used for the design of peptidomimetic activators (Rose, Biochemistry 35 (1996), 12933-12944; Rutenber, Bioorg. Med. Chem. 4 (1996), 1545-1558).

In accordance with the above, in a preferred embodiment of the method of the invention said pharmacological properties of the identified inhibitor or antagonist is further improved or refined by peptidomimetics.

The method of the invention can further include modifying a compound identified, improved of refined by the method as described herein above as a lead compound to achieve (i) modified site of action, spectrum of activity, organ specificity, and/or (ii) improved potency, and/or (iii) decreased toxicity (improved therapeutic index), and/or (iv) decreased side effects, and/or (v) modified onset of therapeutic action, duration of effect, and/or (vi) modified pharmakinetic parameters (resorption, distribution, metabolism and excretion), and/or (vii) modified physico-chemical parameters (solubility, hygroscopicity, color, taste, odor, stability, state), and/or (viii) improved general specificity, organ/tissue specificity, and/or (ix) optimized application form and route by (i) esterification of carboxyl groups, or (ii) esterification of hydroxyl groups with carbon acids, or (iii) esterification of hydroxyl groups to, e.g. phosphates, pyrophosphates or sulfates or hemi succinates, or (iv) formation of pharmaceutically acceptable salts, or (v) formation of pharmaceutically acceptable complexes, or (vi) synthesis of pharmacologically active polymers, or (vii) introduction of hydrophylic moieties, or (viii) introduction/exchange of substituents on aromates or side chains, change of substituent pattern, or (ix) modification by introduction of isosteric or bioisosteric moieties, or (x) synthesis of homologous compounds, or (xi) introduction of branched side chains, or (xii) conversion of alkyl substituents to cyclic analogues, or (xiii) derivatisation of hydroxyl group to ketales, acetales, or (xiv) N-acetylation to amides, phenylcarbamates, or (xv) synthesis of Mannich bases, imines, or (xvi) transformation of ketones or aldehydes to Schiff's bases, oximes, acetales, ketales, enolesters, oxazolidines, thiozolidinesor combinations thereof; said method optionally further comprising the steps of the above described methods. The various steps recited above are generally known in the art. They include or rely on quantitative structure-action relationship (QSAR) analyses (Kubinyi, "Hausch- Analysis and Related Approaches", VCH Verlag, Weinheim, 1992), combinatorial biochemistry, classical chemistry and others (see, for example, Holzgrabe and Bechtpld, Deutsche Apotheker Zeitung 140(8), 813-823, 2000).

The invention moreover contemplates in another preferred embodiment a method wherein the compound identified and optionally further modified as indicated above is formulated into a pharmaceutical composition.

The figures show:

Figure 1 Glucose-stimulated induction of pro-ICA512 in insulinoma and rat islet cells. a-d, Western blots for pro-ICA512 and g-tubulin on Triton X-100 extracts of INS-1 cells (a, b) or purified pancreatic islets (c, d) kept either in resting buffer or stimulated with 25mM glucose for the indicated times, with (b) or without 5μg/ml actinomycin D (AmD) (a, c, d). Cells were incubated at 37 °C (a, b, c) or at 19 °C (d).

Figure 2 Stabilization of rat ICA512 mRNA by PTB. a, Quantitative RT-PCR for ICA512 and insulin with 1mg total RNA from resting or glucose-stimulated INS-1 cells. Amounts were normalized against b-actin. b, c, Autoradiographies (b) and quantification by phosphoimaging (c) of ³²P-labeled ICA512 mRNA 3'-UTR incubated for the indicated times with cytosolic extracts from INS-1 cells or islets which were either kept in resting buffer or glucose-stimulated for 60 or 120 min. d, Electrophoretic mobility shift assays (EMSA) of biotinylated RNA-oligonucleotides including either the wild-type (wt1 , wt2) or mutated (ml , m2) first or second consensus sites for PTB binding in the 3'- UTR of ICA512 mRNA. Prior to EMSA, oligonucleotides were incubated with cytosolic extracts from resting (-) or glucose- stimulated (+) INS-1 cells, e, Binding of wt1 or wt2 oligonucleotides to PTB as measured by ELISA. PTB was captured with a specific antibody from cytosolic extracts of resting or 120 min glucose-stimulated INS-1 cells, f, Immunoblots for PTB and D-tubulin in INS-1 cell cytosolic extracts: lane 1 , 120 min resting cells; lane 2: 120 min stimulated cells; lane 3: 120 min stimulated cells + PTB-immunodepletion; lane 4: 120 min stimulated cells + mock-immunodepletion. g, Binding of ³²P-labeled full-length ICA512 mRNA 3'-UTR to the cytosolic extracts 1-4 shown in (f). h, Decay assays as in (c) upon incubation of ICA512 mRNA 3'-UTR with cytosolic extracts 1-4 shown in (f).

Figure 3 Stability of PC1/3, PC2 and CPE mRNAs. Quantification by phosphoimaging of ³²P-labeled PC1/3, PC2 and CPE mRNA 3'-UTRs incubated for the indicated times with cytosolic extracts from INS-1 cells which were either kept in resting buffer or glucose-stimulated for 60 or 120 min.

Figure 4 Depletion of SGs in INS-1 cells upon RNA interference for PTB. a, b, Western blotting for PTB, various SG components (a) or housekeeping proteins (b) in INS-1 cells treated (T) or untreated (U) by RNAi for PTB. c, Insulin in the medium and in the total protein extracts of INS-1 cells treated or untreated by RNAi for PTB, as measured by RIA. d, Immunofluorescence for PTB (red), insulin (green) and ICA512 (red) in INS-1 cells treated or untreated by RNAi for PTB. Nuclei were counterstained in blue with DAPI. e, Electron micrographs of INS-1 cells untreated (U) and treated (T) by RNAi for PTB. In untreated INS-1 cells secretory granules (arrows) are mostly aligned along the plasma membrane.

Figure 5 Nucleocytoplasmic translocation of PTB upon glucose stimulation of b-cells. a-c, Low (a, c) and high power (b) immunofluorescence images for PTB (red) in INS- 1 cells (a, b) and islets (c) incubated either in resting buffer or glucose stimulated for 120 min. Nuclei were counterstained in blue with DAPI. d, e, Western blotting for PTB and g-tubulin on both nuclear and cytosolic fractions (d) or cytosolic fractions only (e) from islets that were incubated in resting buffer or glucose-stimulated for 120 min. Islets were cultured in vitro for 1 day (d) or 3, 6 and 9 days (e). Figure 6 Glucose-regulated luciferase expression in INS-1 cells upon inclusion of PTB binding sites. a, Schematic drawing of firefly luciferase cDNA constructs in pGL3-Basic. pGL3 PC2 3'-UTR and pGL3 PC2 5'-UTR included the corresponding UTRs from rat PC2. Two additional pGL3 constructs were generated with mutations in the PTB binding sites of PC2 (pGL3 PC2 3'-UTR mut and pGL3 PC2 5'-UTR mut). b, RLU in resting or 120 min stimulated INS-1 cells transfected with the pGL3 constucts shown in (a). RLU in resting or stimulated INS-1 cells transfected with pGL3-Basic was equal to 100 %.

Figure 7 Electrophoretic mobility shift assay (EMSA) of the biotinylated RNA- oligonucleotide wt1 following incubation with nuclear extracts from INS-1 cells kept at rest or glucose stimulated for 30, 60 or 120 min. There was no electrophoretic mobility shift of the oligo wt1 , indicating that nuclear PTB, even from stimulated cells, was not competent for binding the corresponding consensus motif in ICA512 mRNA 3'-UTR.

Figure 8a, Western blotting for cytosolic and nuclear PTB, chromogranin A and γ- tubulin in_. INS-1 cells untreated (-) or treated with siRNA oligos 1+2, 3 or 4 for PTB. Left and right panels are from different experiments. The additional siRNA oligos 3 and 4 for rat PTB were selected by Cenix Biosciences with a proprietary algorithm and chemically synthesized by Ambion. siRNA oligo 3: sense oligo, 5'- GGUGAUAACAGGAGCACAGdTdT; antisense oligo, 5'-

CUGUGCUCCUGUUAUCACCdTdT. siRNA oligo 4: sense oligo, 5'-

GGCUUCAAGUUCUUCCAGAdTdT; antisense oligo, 5'-

UCUGGAAGAACUUGAAGCCdTdT. b, Quantitation of PTB, SG and control proteins upon transfection of INS-1 cells with siRNA oligos 3 and 4 for PTB. Cells were transfected with either 1 μg siRNA oligo 3 or 4. All proteins were quantified by western blotting and normalized against γ-tubulin, except insulin, which was measured by RIA. The values are from two independent experiments, c, Quantitation of SG proteins and luciferase activity upon treatment of INS-1 cells with the indicated siRNA oligos. Control scrambled 21-mer siRNA oligos were synthesized with the Silencer siRNA Construction Kit (Ambion) using the following cDNA primers: sense primer, 5'-AATGCTCGACATGACAGACGGCCTGTCTC; antisense primer, 5'- AACCGTCTGTCATGTCGAGCACCTGTCTC. Firefly luciferase (F-Luc) was knockdown by RNAi with the following chemically synthesized siRNA oligo (kind gift from Cenix Bioscience): sense oligo, 5'-CUUACGCUGAGUACUUCGA-dTdT; antisense oligo, 5'-UCGAAGUACUCAGCGUAAG-dTdT. Two days before transfection of siRNA oligo for firefly luciferase, INS-1 cells were co-transfected by electroporation (Amaxa) with pGL3-Basic and phRL vectors for co-expression of firefly and renilla luciferase, respectively. Insulin content was measured by RIA, while the other proteins were quantified by immunoblotting and normalized against γ- tubulin. The results shown are from six (siRNA oligos 1+2 for PTB or control scrambled siRNA oligos) or four (siRNA oligo for firefly luciferase) independent experiments, d, Western blotting for the ER markers calnexin and PDI in INS-1 cells incubated with resting (R) or stimulating (S) buffer for 120 min. Unlike most SG proteins, calnexin and PDI are not up-regulated by glucose stimulation, despite the presence of a consensus motif for PTB binding in their mRNA 3'-UTR. Equal loading of proteins was verified by western blotting for γ-tubulin. e, Immunofluorescence for PTB (red), insulin (green) and ICA512 (red) in INS-1 cells untransfected or transfected with siRNA oligos 3 or 4. Nuclei were counterstained in blue with DAPI.

Figure 9 RT-PCR for PTB on 1μg total RNA from INS-1 cells, rat islets and Jurkat cells. In the case of INS-1 cells and rat islets RNA templates were obtained from cells kept at rest or glucose-stimulated for the indicated times. For PCR the following specific primers flanking the entire open reading frame of rat PTB were used: forward, 5'-ATGGACGGCATCGTCCCAG; reverse, 5'-

CTAGATGGTGGACTTGGAAAAG. Annealing temperature: 55 °C; amplification: 32 cycles. The 1592 bp cDNA encoding the 57 kD PTB isoform was readily amplified from INS-1 cell and islet RNA, whereas no shorter PTB variants, including the 700 bp cDNA corresponding to PTB-T, were detected. However, the 700 bp cDNA for PTB-T could be amplified in the same conditions from Jurkat cells, which instead expressed almost no mRNA for the full-length PTB. This expression pattern of PTB in Jurkat cells is comparable to that shown in Fig. 3B in ref. 17. These results indicate that both resting and 120 min. stimulated INS-1 and islet cells do not express PTB-T. Figure 10. cAMP-dependent induction of ICA512 and other secretory granule markers in INS-1 cells. A, mRNA expression of insulin, ICA512, PC1 and CPE. B to D, protein expression of ICA512.

Figure 11. Phosphorylation of PTB is in β-cells by PKA.

Figure 12. Phosphorylation on Ser16 controls the translocation of PTB.

Figure 13. PTB translocation induces the expression of insulin secretory granule marker.

The examples illustrate the invention.

Example 1

Glucose-stimulated activation of PTB promotes the stability of ICA512 mRNA and upregulates ICA512 expression.

The findings made in accordance with the invention originated from the study of ICA512/IA-2, a receptor tyrosine phosphatase-like protein associated with insulin SGs, and neurosecretory granules in general [21]. Cleavage by a furin-like convertase of pro-ICA512 leads to the generation of a transmembrane fragment (ICA512 TMF) of 65 kD, which is enriched in SGs [21 ,22]. Glucose stimulation of rat insulinoma INS-1 cells induces the biosynthesis of pro-ICA512 [23]. This induction was already apparent in cells stimulated for 30 minutes and persisted upon stimulation for 120 minutes. Rapid induction of pro-ICA512 biosynthesis escaped detection when islet stimulation was performed at 37° C (fig. 1c), but it was clearly revealed upon stimulation at 19° C (fig. 1d), a temperature that prevents the exit of secretory proteins from the Trans Golgi Network (TGN). This was not unexpected, since ICA512 has a very rapid turnover [22,24] and maturation of secretory proteins is more efficient in β-cells than in insulinoma cells. Since pro-ICA512 levels were still affected by glucose stimulation in the presence of actinomycin D (fig. 1 b), which blocks transcription, rapid induction of ICA512 biosynthesis must be due to post- transcriptional mechanisms.

Quantitative real time PCR showed that ICA512 mRNA was increased 1.46, 1.33 and 2.31 folds in INS-1 cells stimulated with 25 mM glucose for 30, 60 and 120 minutes, respectively. This increase exceeded that of insulin mRNA, which after 120 minutes stimulation was enhanced 1.43 fold (Fig. 2a). We investigated therefore whether glucose stimulation promoted the stability of ICA512 mRNA. To this aim, the labeled 3'-UTR of rat ICA512 mRNA was incubated with cytosolic extracts from INS-1 cells that have been either incubated in resting buffer (no glucose) or with 25 mM glucose for 30, 60 or 120 minutes. Equivalent amounts of intact 1CA512 mRNA 3'-UTR were recovered following its incubation with extracts from INS-1 cells kept in resting buffer or stimulated for 60 minutes (half-life = 105 min) (fig. 2b and c). Glucose stimulation for 120 minutes, however, stabilized ICA512 mRNA 3'-UTR by 1.43 fold (half-life = 150 min). An even greater and quicker stabilization effect was observed upon incubation of ICA512 mRNA 3'-UTR with extracts from glucose-stimulated pancreatic islets (2.46 fold increase after glucose stimulation for 60 min) (fig. 2b and c). The combined results of the quantitative PCR and decay assays indicated that stabilization of ICA512 mRNA accounts, at least in part, for the rapid glucose- dependent stimulation of pro-ICA512 biosynthesis.

ICA512 mRNA 3'-UTR contains two evolutionary conserved consensus binding sites for PTB (fig. 2d). We asked then whether glucose stimulation stabilizes ICA512 mRNA by promoting the binding of PTB to its 3'-UTR. Two biotinylated oligonucleotides, each including one of the two putative PTB binding sites in rat ICA512 mRNA, were independently incubated with cytosolic extracts from INS-1 cells that were either non-stimulated or glucose-stimulated for 30, 60 or 120 minutes. The cytosolic extracts of cells stimulated for 120 minutes displayed a binding activity that caused a similar retardation of both oligonucleotides in electrophoretic mobility shift assays (fig. 2d, arrows). This retardation was not detected upon oligonucleotide incubation with nuclear extracts from cells stimulated for 120 minutes ( fig. 7) or when cytosolic extracts were tested for binding to oligonucleotides lacking the PTB consensus sequence (fig. 2d). To unequivocally identify PTB as the binding factor, we developed a solid phase ELISA in which PTB from cytosolic extracts of INS-1 cells was captured with a specific antibody. Stimulation of cells for 120 minutes enhanced the binding of PTB to the first and second biotinylated oligonucleotides by 6.9 and 3.8 folds, respectively, compared to non-stimulated cells (fig. 2e). Stimulation also increased the binding of PTB to the full ICA512 mRNA 3'-UTR by 2.1 fold (Fig. 2g). Immunodepletion of PTB from cytosolic extracts of stimulated cells (Fig. 2f), on the other hand, drastically reduced the recovery of ICA512 mRNA 3'-UTR (Fig. 2g) and its stability (Fig. 2h), whereas mock-immunodepletion with mouse IgGs had no effect. These results confirm that sustained glucose stimulation promotes the binding of cytosolic PTB to the 3'-UTR of ICA512 mRNA and could account for its stabilization.

Expression of ICA512 mRNA as measured by quantitative real-time PCR (Fig. 10A) is induced by 1mM IBMX, which inhibits phosphodiesterase activity and therefore leads to an increase of intracellular cAMP levels. IBMX induction was also observed for another SG protein (prohormone convertase 1/3, PC1/3), but not in case of carboxypeptidase E, another SG marker. IBMX induction of ICA512 and PC1/3 mRNAs was prevented by treatment with H89, which inhibits cAMP-dependent PKA. Furthermore, it has been found that the glucose- and cAMP-induced expression of ICA512 are additive. Specifically, Panel B in Fig. 10 shows that incubation of INS-1 with a 10 mM H89 abolishes glucose-induced expression of pro-ICA512 protein. Conversely, as shown in panel C, treatment of INS-1 cells with 1mM IBMX alone induces pro-ICA512 protein expression. Further enhancement of pro-ICA512 expression is seen upon treatment of INS-1 cells with 25 mM glucose and 1mM IBMX (panel D).

Example 2

Glucose-stimulation enhances the stability of mRNAs encoding secretory granule proteins

Numerous other components of SGs, in addition to insulin and ICA512, include conserved putative PTB binding sites in their mRNA 3'-UTR (table 1). As test cases, the stability of the 3'-UTRs of mRNAs encoding PC1/3 and PC2 was analyzed, which are up-regulated by glucose stimulation [4]. RNA decay assays showed that glucose stimulation stabilizes rat PC1/3 (85% increase) and PC2 (90% increase) mRNA 3'- UTRs even more effectively than ICA512 mRNA 3'-UTR (fig. 3), while it had no effect on the stability of carboxypeptidase E (CPE) mRNA 3'-UTR. CPE is a protease that is partly responsible for the processing of insulin within SGs, but whose expression is not glucose-regulated. Notably, the 3'-UTR of CPE mRNA does not contain any consensus for PTB binding and was significantly more stable than the 3'-UTR of ICA12, PC1/3 and PC2 mRNAs.

Example 3

PTB is necessary for secretory granule biogenesis

In view of these findings it was further investigated whether PTB could have a general effect on the biogenesis of insulin SGs. To test this hypothesis, the expression of PTB in INS-1 cells was down-regulated by RNA interference (RNAi) [25]. The transfection efficiency of INS-1 cells with CY3-conjugated double-stranded RNA oligonucleotides was found to be 50%-60% by fluorescence microscopy (not shown). Four days after transfection, the expression of PTB was reduced by 44-45% compared to control cells (fig. 4a). A more dramatic decrease was observed for SG components that contain consensus sites for PTB binding in their mRNA 3'-UTR, including ICA512, PC1/3, PC2, chromogranin A, secretogranin II, synaptobrevin 2 and synaptophysin (table 1 and fig. 4a and fig. 8a) and by 40% in the cytosol (fig. 8a and 8b). Cell insulin content, as measured by RIA, was reduced by 69% (0,683 ± 0,040 μg/mg protein, control cells vs. 0.209 ± 0,017 μg/mg protein, treated cells) while secreted insulin was reduced by 48% (2,97 ± 0,65 ng/ml, control cells vs. 1 ,56 ± 0,32 ng/ml, treated cells) (fig. 4c and 8b and 8c). Conversely, treatments with control siRNA oligos did not significantly alter the expression of these SG components (fig. 8c). The expression of CPE was not affected (fig. 4b, 8b and 8c). There was also no significant change in the expression of widely expressed housekeeping proteins, such as markers of the endoplasmic reticulum (calnexin and protein disulfide isomerase), the Golgi complex (p58 and mannosidase II), or in the cytosol (tubulin). Intriguingly, both rat calnexin and PDI mRNA 3'-UTRs contain a consensus for PTB binding, and yet their expression was not stimulated by glucose (fig. 8d). Consistent with the immunoblot results, light immunomicroscopy on RNAi- treated cells showed a significant reduction of PTB and even more dramatically of insulin and ICA512 (fig. 4d and 8e). These data suggested that PTB may affect the biogenesis of SGs. Quantification by electron microscopy confirmed the virtual absence of SGs in 60% of the cells treated by RNAi for PTB (fig. 4e and table 2). The number of granules / cell profile was reduced from 18.8 ± 1.1 in control cells to 6.6 ± 1.1 (N= 140) in RNAi-treated cells. Cells treated by RNAi for PTB could clearly be distinguished in two categories. One group of cells (60%) contained up to 2 granules, with an average number of 0.3 ± 0.1 granules. The other group (40%) contained at least 7 granules / cell profile and had an average number of granules (16.6 ± 1.1) that was not different from the control cells. As these numbers correlate well with the transfection efficiency measured by fluorescence microscopy, cells with < 2 granules are likely to represent cells in which PTB expression was affected by RNAi. Conversely, treatments with siRNA oligos that were either scrambled or targeting the mRNA of transfected firefly luciferase had no impact on SG content (Table 2). Despite the loss of SGs, cells treated by RNAi for PTB were otherwise normal, with neither changes in size (table 2) and shape nor in the general appearance of organelles such as the nucleus, the endoplasmic reticulum, the Golgi complex or mitochondria. These data clearly demonstrate that PTB is a key factor for the biogenesis of SGs.

Example 4

Glucose-stimulation induces the nucleocytoplasmic translocation of PTB

The question was then addressed how glucose stimulation activates PTB and promotes the stabilization of mRNAs for SG components. As shown in other cell types [26,27], PTB was mainly localized in the nucleus of resting INS-1 cells, while immunoreactivity in the cytosol was low and mostly concentrated in particles whose identity remains to be clarified (fig. 5a and b). Glucose stimulation for 120 minutes reduced PTB positive nuclei by 72%, while cytosolic immunoreactivity was enhanced, albeit cytosolic PTB-positive particles were decreased (fig. 5a and b). In stimulated cells cytosolic PTB appeared more diffuse and reticulated, suggesting its targeting to the endoplasmic reticulum. Similar changes in PTB distribution were observed between resting and stimulated pancreatic islet cells (fig. 5c). Western blot on islet extracts confirmed that glucose stimulates the nucleocytoplasmic translocation of PTB (Fig. 5d). Together with the translocation of the 59 kD PTB isoform, glucose enhanced the levels, primarily in the nucleus, of a 27 kD PTB species. This represents most likely a C-terminal proteolytic fragment of the 59 kD form ²⁵, since no other spliced variants were detected by RT-PCR in INS-1 and islet cells (fig. 9). Notably, glucose stimulated translocation of PTB in purified pancreatic islets was drastically decreased with time (Fig. 5e).

In summary, PTB was identified in accordance with the invention as a factor required for the biogenesis of β-cell SGs. The 3'-UTR of many SG components and other proteins associated with the neuroendocrine phenotype contains one or more consensus sites for PTB binding and at least in the case of PC2 also in the 5'-UTR. Glucose stimulation of β-cells promotes the nucleocytoplasmic translocation of PTB as well as the stabilization of mRNAs encoding SG components by the cytoplasmic pool of PTB. Thus, it is concluded that mRNA stabilization is a mechanism by which PTB up-regulates the expression of β -cell SG proteins in response to glucose. Since most of these proteins are found in SGs of other peptide-secreting endocrine cells and neurons, it is concluded in accordance with the invention that also in these cells PTB supports SG biosynthesis in response to various stimuli. Stabilization of mRNAs for secretory proteins by cytosolic PTB in response to treatments that increase intracellular Ca²⁺ levels, such as phorbol esters and ionomycin, has been reported in T-lymphocytes [28]. Ca²⁺ alone, however, may not be sufficient to activate PTB in β - cells, since stimulations with high potassium or sulphonylureas increase intracellular Ca²⁺ levels and insulin secretion, but do not induce ICA512 [23] and insulin [29] biosynthesis. PTB activation must depend therefore on other second messengers, such as cAMP, whose increased levels upon glucose metabolism have already been proposed to promote insulin biosynthesis [30,31]. In addition to mRNA stabilization, other post-transcriptional mechanisms should participate in the rapid stimulus- dependent up-regulation of insulin and peptide hormone expression, even before stabilization of their mRNAs by PTB. Such mechanisms may include, for instance, the phosphorylation and dephosphorylation of factors implicated in cap-dependent mRNA translation [32,33].

Example 5:

PKA phosphorylates PTB in β-cells

Since induction of cAMP potentiates glucose stimulation of ICA512 expression (see Example 1 , Fig. 10D), it was investigated whether PTB in β-cells is phosphorylated by PKA, whose activity is cAMP-dependent. PTB includes a consensus site for phosphorylation by PKA on Ser16 within its nuclear export signal. To address whether PTB is phosphorylated by PKA in β-cells, INS-1 cells were labeled with ³²P-orthophosphoric acid. ³²P-labelled PTB was detected by autoradiography (Fig. 11A, top) and immunoblot (Fig. 11 A, middle) following PKA activation with IBMX, which increases cAMP levels. This phosphorylation was inhibited by H89, a specific inhibitor for PKA. The anti-phospho PTB antibody specifically reacts with the protein phosphorylated on Ser16. Fig. 11 A, bottom, shows the total amount of PTB detected with a pan-PTB antibody.

Panel B of Fig. 11 shows that detection of phospho-PTB by Western blot is sensitive to alkaline phosphatase (AP) treatment. Treatment of INS-1 extracts with alkaline phosphatase was performed as described previously [23].

PKA can activate Erk1/2 through MEK1/2. These kinases, however, are not involved in PTB phosphorylation, because the phospho-PTB bands were still detected following treatment with PD98057 which inhibits MEK1/2 (Fig. 11C).

Example 6: Phosphorylation on Ser16 controls the translocation of PTB

As shown in Fig. 12A, in resting INS-1 cells, PTB is mostly found in the nucleus, as shown by the. co-localization with DAPI, a fluorescence dye which binds DNA. Stimulation with IBMX leads to the disappearance of PTB immunoreactivity in the nucleus and its increased detection in the cytosol. The nucleocytoplasmic translocation by IBMX can be prevented by co-treatment with H89. Panel B shows that a PTB allele fused with GFP and in which Ser16 was converted into Ala is restricted to the nucleus. Conversely, a PTB mutant in which Ser16 was converted into Asp was mostly enriched in the cytosol. Site-directed mutagenesis was performed using the QuickChange mutagenesis kit by Stratagene and following the manufacturer's manual. Panel C shows that conversion of Ser16 into Ala correlates with inability of PTB to translocate from the nucleus into the cytosol upon IBMX treatment of INS-1 cells. The first bar in Panel C is indicative of the transfection efficiency in INS-1 cells (around 50%). Panel D shows that in resting INS-1 cells transfected PTB-GFP is restricted to the nucleus. Upon stimulation with IBMX, the protein translocates to the cytosol, similarly to endogenous PTB (as shown in Panel A). This translocation is not inhibited by RNA interference with a scrambled oligo. However, knock-down with a mixture of siRNA oligos for the regulatory subunit II of PKA, which binds cAMP effectively prevents the IBMX-induced nucleocytoplasmic translocation of PTB-GFP. This result further corroborates the conclusion that cAMP- dependent phosphorylation and nucleocytoplasmic translocation of PTB is PKA- dependent.

Example 7:

Increased translation of mRNAs binding PTB

To verify whether PTB acts as a glucose-dependent regulator of mRNA stability and translation in vivo, INS-1 cells were transiently transfected with firefly luciferase constructs, which either carried the original 5'- and 3'-UTRs or also included the corresponding regions of rat PC2 mRNA or rat ICA512 mRNA (Fig. 6a and Fig. 13). The choice of rat PC2 UTRs was convenient because its 3'-UTR and 5'-UTR both contain a single putative PTB binding site. Inclusion of PC2 3'-UTR increased luciferase activity by 8.2 and 28.8 fold in resting and stimulating conditions, respectively (Fig. 6b). Luciferase activity was further enhanced by including PC2 5'- UTR (increase of 14.7 fold at rest and 56.2 fold upon stimulation). Mutagenesis of the PTB binding sites in the 3'- or 5'-UTR of PC2, on the other hand, was sufficient to reduce luciferase activity at rest (PC2 3'-UTR mut = 4.29 fold decrease; PC2 5'-UTR mut = 7.16 fold decrease) and even more following stimulation (PC2 3'-UTR mut = 6.44 fold decrease; PC2 5'-UTR mut = 11.29 fold decrease). These data demonstrated that PTB promotes the expression of mRNAs even in resting conditions and that its activity is glucose-regulated. Furthermore, they suggest that PTB may not only increase mRNA stability but also mRNA translation by binding to 5'-UTRs, as already shown in the case of its interaction with viral internal ribosome entry sites [16, 21].

Furthermore, expression of firefly luciferase constructs including the 3' UTR PTB consensus binding site of ICA512 or PC2 was enhanced by treatment of INS-1 cells with 1 mM IBMX, as measured by luciferase activity (Fig. 13). Induction by IBMX of transfected luciferase construct including the 3' UTR of either ICA512 or PC2 was inhibited by co-treatment with 10 mM H89.

Example 8: Methods employed

Islet preparations and cell culture

Pancreatic islets were isolated from female Wistar rats by collagenase digestion, purified by density gradient centrifugation, and cultured (400/60mm culture dish) as described previously [38]. INS-1 cells were grown as described [39].

Cell extracts and immunoblotting

Stimulation of INS-1 cells (7 x 10⁵/35 mm well) and pancreatic islets (400-1000) with 25 mM glucose for 0-120 minutes was performed as described [23]. After stimulation cells were washed with cold PBS and then extracted in lysis buffer (20mM TRIS/HCI, pH 8.0, 140mM NaCI, 1mM EDTA, 1 % Triton X-100 and 1 % protease inhibitor cocktail (Sigma). Cytosolic and nuclear extracts were prepared using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce) according to the manufacturer's instructions. For immunodepletion experiments, cytosolic extracts of INS-1 cells were incubated overnight at 4 °C with 5 μg monoclonal antibody 1 directed against the C-terminal region of PTB (Zymed Lab) or mouse IgGs (BioRad) prior to the addition of protein G Sepharose (Pharmacia) and centrifugation. Protein concentration in the detergent soluble material was measured using the BCA assay (Pierce). Cell extracts were separated by SDS-PAGE and immunoblotted as described [23] using the following antibodies: mouse monoclonals anti-PTB, anti- ICA512 ²³, anti-calnexin (Transduction Laboratories), anti-chromogranin A (Immunon), anti-PDI (Stressgen), anti-γ-tubulin and anti-insulin (Sigma), anti- synaptophysin and anti-synaptobrevin 2 (Synaptic Systems); rabbit polyclonals anti- secretogranin 2 (gift from Dr. W. Huttner), anti-mannosidase II ⁴², anti-p58 ⁴³, anti- PC1/3, anti-PC2, and anti-CPE (Chemicon). Chemiluminescence was performed using the Supersignal West Pico Substrate (Pierce) as substrate and detected with a LAS 3000 Bioimaging System (Fuji).

mRNA decay assay

The 3'-UTRs of rat ICA512, PC1, PC2, and CPE mRNAs were subcloned into pCRII- TOPO with both T7 and SP6 promoters (Invitrogen). [a-³²P]-UTP labeled RNA was synthesized with the T7-MEGA script kit (Ambion). Decay assays were performed as described [40] with minor modifications by incubating 10⁴ cpm labeled in-vitro transcripts with 10μg cytosolic extracts from resting or stimulated cells for 0-240 minutes. Reactions were stopped with 6x gel loading buffer and separated on a 6% polyacrylamide-7M urea gel. Dried gels were exposed and quantified with a BAS 180011 phosphoimager (Fuji) using the Image Gauge v3.45 software.

RNA-EMSA

For electrophoretic mobility shift assays (EMSA) the following biotin-conjugated oligonucleotides were used: 5'-ACUCUUCAGCCCCUACCCAUCUGCC (1° PTB binding site in rat ICA512, wt1); 5'- ACUCUUCAGCAAAAAGGGAUCUGCC (mutated l° PTB binding site, ml); 5'-UGUACCUCCCCACUCCCACCAGCCUA (II⁰ PTB binding site in rat ICA512, wt2); 5'-UGUACAUAGGAACUAGGACCAGCCUA (mutated IT PTB binding site, m2). The binding reaction was carried out using the LightShift Chemoluminescent EMSA Kit (Pierce). 10mg cytosolic extracts from resting or stimulated INS-1 cells were incubated with 5pmol RNA oligonucleotides in binding buffer including 100mM KCI and 1.5mM MgCI₂. After 30 min incubation, 5U RNAse T1 were added to each reaction and incubated for 10 min. After additional 10 min incubation with 110mg heparin, the reaction was stopped with 5μl loading buffer. Electrophoresis, blotting, UV cross-linking and detection were performed according to manufacturer's instructions.

PTB binding assay

Ninety-six well plates were coated with monoclonal anti-PTB antibody (Zymed Lab). Binding reactions between biotinylated RNA oligonucleotides and INS-1 cells cytosolic extracts were carried out as above and UV cross-linked prior to the incubation with anti-PTB coated plates for 2 hrs. Following incubation with horseradish-peroxidase conjugated streptoavidin for 1 hr, the bounded peroxidase activity was measured at 405nm with ABTS (Sigma) as substrate. Bound radioactivity was measured by β -counting.

Real Time PCR

Total RNA from 6x10⁵ INS-1 cells was isolated with TRIZOL (Invitrogen) according to the manufacturer's protocol. 1mg of total RNA was used for the reverse transcriptase reaction with 2mM gene specific antisense primers for b-actin, ICA512 and insulin. The expression of mRNA was analyzed by quantitative real-time PCR using the LightCycler system (Roche Diagnostics) as described [41].

Luciferase assays

Subcloning of rat PC2 3'-UTR and 5'-UTR into pGL3-Basic (Promega) encoding firefly luciferase and multiple site-directed mutagenesis of PTB binding sites were performed using standard protocols. pGL3 constructs and phRL (Promega) encoding renilla luciferase were co-transfected into INS-1 cells with Lipofectamine (Invitrogen). Firefly luciferase activity was measured 4 days after transfection and normalized with that of renilla luciferase using the dual luciferase system (Promega) according to manufacturer's instructions.

RNA interference

INS-1 cells (6x10⁵/35 mm well) were grown for 2 days before transfection. 21-mer dsRNA oligonucleotides for rat PTB mRNA were synthesized with the Silencer siRNA Construction Kit (Ambion) (for further details see [47]) using the following primers: sense primer 1 , 5'-AAGATACCTAGTGATGTCACTCCTGTCTC; sense primer 2, 5'- AAGGACCGCAAGATGGCACTGCCTGTCTC; antisense primer 1 , 5'- AAAGTGACATCACTAGGTATCCCTGTCTC; antisense primer 2, 5'- AACAGTGCCATCTTGCGGTCCCCTGTCTC). For silencing of rat regulatory subunit II of PKA (sequence accession number M 12492 as published in [46]), the following primers were used: sense primer 1 5'- AAGGGTGTCAACTTCGCGGAGCCTGTCTC, sense primer 2 5'- AATGTGATGGCGTTGGAAGATCCTGTCTC, sense primer 3 5'- AATGCTTAGCCATGGATGTGCCCTGTCTC, antisense primer 1 5'- AACTCCGCGAAGTTGACACCCCCTGTCTC, antisense primer 2 5'- AAATCTTCCAACGCCATCACACCTGTCTC, antisense primer 3 5'- AAGCACATCCATGGCTAAGCACCTGTCTC. Cells were transfected with 1μg of dsRNA/well using Lipofectamin (Invitrogen). 4 days after transfection cells were harvested in lysis buffer for immunoblotting or processed for immunocytochemistry.

Imrnunocytochemistry

INS-1 cells and islets were fixed with 4% paraformaldehyde. After embedding in gelatin, 5μm islet sections were prepared. INS-1 cells and islet sections were then permeabilized with 0.2% saponin or 0.3% Triton X-100 and incubated with monoclonal antibodies against PTB, insulin (Sigma) or ICA512 [21] for 1 h. After washing and incubation with goat-anti-mouse Alexa488 or Alexa568 conjugated secondary antibodies (Molecular Probes) nuclei were counterstained with DAPI (Sigma). Images were acquired with a CoolSnap-HQ CCD camera (Roper Scientific) attached to an Olympus BX61 microscope and processed with Metamorph 4.65 (Universal Imaging).

Electronmicroscopy

INS-1 cells grown on glass coverslips were fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer and processed for standard Epon embedding. Surface areas of cell sections were analyzed using a SIS Megaview camera with analysis software installed on a Tecnai12 electron microscope (FEI).

Insulin RIA

Total insulin content in the cells and in the medium was measured with the Sensitive Rat Insulin RIA Kit (Linco Research). Tables and Table Legends

Table 1 Consensus for PTB binding in the 3'-UTR of mRNAs of secretory granule proteins. Sequences with asterisks are from reference 12.

Gene PTB consensus Accession Nr. binding sequence CYYYY CYYYY G

Secretory granule proteins

ICA512, human CCUAC CCAUC UG 18983 CACUC CCACC AG

ICA512, rat CCUAC CCAUC UG U40652 CCACTJ CCCAC CA insulin, human CGCCG CCUCC UG NM000207 (* insulin I, rat CCCAC CCCUC UG J00747 (*) insulin I, mouse CCCAC CCCUC UG NM08386 (*) insulin, rabbit CACCC ACCCC UG M61153 (*) insulin, sheep CCUGG CCCGC CG U00659 (*) insulin, chicken CUTJAC UCUAU CG X58993(*) prohormone convertasel, rat CCCUC CUUUU CC M76705 prohormone convertasel, mouse CCCUC CUUUU CC M58589 prohormone convertasel, bos Tc CCUUC CCCCU UA AF186405 prohormone convertase2, rat CCUUC CUCCC UG M76706 ccccc CUCCC CC • AF186406 chromogranin A, rat cuucc CUUCU cu ^' NM021655 chromogranin A, mouse CUUUG CCCCU CA BC026554 chromogranin A, bos taurus cuuuu ccucc UG NM181005 secretogranin II, rat ccccu cccuu uc AF111115 secretogranin II, human ccucc CACCC CA NM003469 secretogranin II, mouse cccuu CUUUC CC BC048249 synaptobrevin 1 (Vampl) , human ccuuu CCUCC CA NM016830 synaptobrevin 1 (Vampl) , rat cuucc cuuuu UG NM013090 synaptobrevin 2 (Vamp2), human cuuuu cuucc UG NM014232 synaptobrevin 2 (Vamp2), rat cuccu CUCCC UC NM012663 ccccc CUCCC uu synaptophysin 1, rat ccccc CUUUU CC NM012664 ccucu CCCUC UG ccccc CUUUC UC cucu CCCUC CA synaptophysin, mouse ccccc CUUCC CA NM009305 ccuuc CUUCU CU ccucu CCCUC CA cuuuu CCCUC CA synaptophysin, human ccccc CUUCC CA X06389 ccucu CUCUC CC ccccu cuucc CC cuccc ucccu CC Table 2 Morphometry in RNAi-treated INS-1 cells. The total number of cells per group (N) was from 3 independent experiments. As no differences between these experiments were found by ANOVA, all data were pooled. Statistical analysis was performed using a t-test or, in case variances were not equal, a Welch test. Cells in the RNAi-treated group were compared for granule content and size (p values in brackets) with cells in the untreated group. Each of the two distinct pools of cells in the RNAi-treated group was also independently compared to the untreated group (p values in brackets) and to each other (p values marked with an asterisk).

Table 3

Table 4

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Claims

1. A method for stimulating production of secretory granules in peptide hormone-secreting endocrine cells or neurons comprising the step of promoting the presence of polypyrimidine tract binding protein (PTB) or a biologically active fragment or derivative thereof in the cytoplasm of said cells.

2. A method for stimulating production of secretory granules in peptide hormone-secreting endocrine cells or neurons, preferably of claim 1 , comprising or further comprising promoting the activity of PTB or said biologically active fragment or derivative thereof in the cytoplasm of said cells.

3. The method of claim 1 or 2 wherein the promotion comprises the promotion of the nucleocytoplasmic transport of PTB or of a biologically active fragment or derivative thereof.

4. The method of any one of claims 1 to 3 wherein the promotion comprises the activation of phosphorylation of PTB.

5. The method of claim 3 or 4 wherein said promotion of the nucleocytoplasmic transport of PTB or of said biologically active fragment or derivative thereof is effected by promoting the presence or activity of protein kinase A in said cell.

6. The method of claim 5 wherein the promotion of the presence or activity of protein kinase A in said cell is effected by overexpression or activation of protein kinase A.

7. The method of claim 6 wherein said overexpression of protein kinase A is effected by overexpressing PKA from a vector, by increasing cAMP levels in the cell or by contacting the cell with a drug that induces the overexpression of PKA or the increase of cAMP levels in said cell.

8. The method of claim 1 or 2 wherein the promotion comprises the transient or stable expression of PTB or of said biologically active fragment or derivative thereof from an exogenously introduced vector.

9. The method of claim 1 or 2 wherein the promotion comprises enhancing the stability of PTB or of said biologically active fragment or derivative thereof or reducing degradation of PTB or of said biologically active fragment or derivative thereof in said cell.

10. The method of claim 1 or 2 wherein the promotion comprises introducing PTB or a biologically active fragment or derivative thereof into the cell.

11. The method of claim 1 or 2 wherein the promotion comprises the inhibition of dephosphorylation of PTB.

12. The method of any one of claims 1 to 11 wherein said endocrine cells are pancreatic β-cells.

13. The method of claim 12 wherein said promotion is effected by glucose or GLP-1.

14. The method of any one of claims 1 to 13 wherein the peptide hormone or neuropeptide contained in said secretory granules is insulin, amylin or a peptide hormone or neuropeptide derived from one of the following precursors: ADM precursor, Agouti switch protein precursor, Agouti-related protein precursor, Apelin precursor, Atrial natriuretic factors, Beta- neoendorphin-dynorphin precursor, Brain natriuretic peptide precursor, Calcitonin gene-related peptide I precursor, Calcitonin gene-related peptide, II precursor, Calcitonin precursor, Cholecystokinin precursor, Chromogranin A precursor, Cocaine- and amphetamine-regulated transcript protein precursor, Corticoliberin precursor, Corticotropin- lipotropin precursor, Cortistatin precursor, FMRFamide-related peptides precursor, FMRFamide-related peptides precursor, Follistatin precursor, Follitropin beta chain precursor, Galanin precursor, Galanin-like peptide precursor, Gastric inhibitory polypeptide precursor, Gastrin precursor, Gastrin-releasing peptide precursor, Ghrelin precursor, Glucagon precursor, Glycoprotein hormones alpha chain precursor, Growth hormone variant precursor, Insulin precursor, Insulin-like growth factor binding protein 3 precursor, Insulin-like peptide INSL6 precursor, Islet amyloid polypeptide precursor, Leydig insulin-like peptide precursor, Morphogenetic neuropeptide, Motilin precursor, Neurexophilin 2 precursor, Neurexophilin 3 precursor, Neurexophilin 4 precursor, Neuroendocrine protein 7B2 precursor, Neurokinin B precursor, Neuromedin B-32 precursor, Neuromedin U-25 precursor, Neuropeptide B precursor, Neuropeptide W precursor, Neuropeptide Y precursor, Neurotensin precursor, Nociceptin precursor, Orexin precursor, Oxytocin-neurophysin 1 precursor, Pancreatic hormone precursor, Parathyroid hormone precursor, Parathyroid hormone- related protein precursor, Peptide YY precursor, Pituitary adenylate cyclase activating polypeptide precursor, Proenkephalin A precursor, Progonadoliberin I precursor, Progonadoliberin II precursor, Prokineticin 2 precursor, Prolactin precursor, Pro-MCH precursor, Prorelaxin H1 precursor, Protachykinin 1 precursor, Protein-tyrosine phophatase-like N precursor, Receptor-type protein-tyrosine phophatase N2 precursor, Regulated endocrine specific protein 18 precursor, Resistin precursor, Resistin-Iike beta precursor, Secretin precursor, Secretorygranin I precursor, Secretorygranin II precursor, Secretorygranin III precursor, Somatoliberin precursor, Somatostatin precursor, Somatotropin precursor, Stanniocalcin 1 precursor, Stanniocalcin 2 precursor, Urocortin II precursor, Urocσ fiff precursor, Vasoactive intestinal peptide precursor, Vasopressin-neurophysin 2-cόpeptin precursor.

15. A method of treating or preventing type-1 or type-2 diabetes comprising stimulating production of insulin-containing secretory granules in pancreatic β-cells wherein said stimulation comprises the step of promoting the presence or activity of polypyrimidine tract binding protein (PTB) or of a biologically active fragment or derivative thereof in the cytoplasm of said β- cells.

16. The method of claim 15, further comprising promoting the activity of PTB or said biologically active fragment or derivative thereof in the cytoplasm of said cells.

17. Use of an agent for the promotion of the presence or activity of polypyrimidine tract binding protein (PTB) or of a biologically active fragment or derivative thereof in β-cells for the preparation of a pharmaceutical composition for treating or preventing type-1 or type-2 diabetes.

18. Use of an agent for the reduction of the presence or activity of polypyrimidine tract binding protein (PTB) or of a biologically active fragment or derivative thereof in hypothalamic neurons for the preparation of a pharmaceutical composition for treating or preventing sleep disorders or depression.

19. The use of claim 17 wherein said agent is GLP-1.

20. The use of claim 18 wherein said agent is corticotropin-releasing hormone (CRH).

21. The method of claim 15 or 16 or the use of any one of claims 17 to 20 wherein the promotion/reduction comprises the promotion/reduction of the nucleocytoplasmic transport of PTB or of a biologically active fragment or derivative thereof.

22. The method of claim 15 or 16 or the use of any one of claims 17 to 21 wherein the promotion/reduction comprises the activation/reduction or prevention of phosphorylation of PTB or the biologically active fragment or derivative thereof or the dephosphorylation of PTB or the biologically active fragment or derivative thereof.

23. The method or use of claim 21 or 22 wherein said promotion/reduction of the nucleocytoplasmic transport of PTB or of said biologically active fragment or derivative thereof is effected by promoting/reducing the presence or activity of protein kinase A in said cell.

24. The method or use of claim 23 wherein the promotion/reduction of the presence or activity of protein kinase A in said cell is effected by the overexpression or activation of protein kinase A/reduction of expression of protein kinase A.

25. The method or use of claim 24 wherein said overexpression of protein kinase A is effected by overexpressing PKA from a vector, by increasing cAMP levels in the cell or by contacting the cell with a drug that induces the overexpression of PKA or the increase of cAMP levels in said cell.

26. The method of claim 15 or 16 or the use of any one of claims 17 to 19 wherein the promotion comprises the transient or stable expression of PTB or of said biologically active fragment or derivative thereof from an exogenousiy introduced vector.

27. The method of claim 15 or 16 or the use of any one of claims 17 to 19 wherein the promotion/reduction comprises enhancing/decreasing the stability of PTB or of said biologically active fragment or derivative thereof in said cell or reducing/enhancing degradation of PTB or of said biologically active fragment or derivative thereof in said cell.

28. The method of claim 15 or 16 or the use of any one of claims 17 to 19 wherein the promotion comprises introducing PTB or a biologically active fragment or derivative thereof into the cell.

29. The method of claim 15 or 16 or the use of any one of claims 17 to 19 wherein the promotion/reduction comprises the inhibition/activation of dephosphorylation of PTB.

30. A method of screening for an agent capable of stimulating production of secretory granules in peptide hormone-secreting endocrine cells or neurons comprising the steps of (a) contacting a cell capable of forming secretory granules and expressing polypyrimidine tract binding protein (PTB) or a biologically active fragment or derivative thereof with one or more compounds; and (b) assessing whether said one or more compounds promote the presence or activity of said polypyrimidine tract binding protein (PTB) or said biologically active fragment or derivative thereof in the cytoplasm of said cell .

31. A method of screening for an agent capable of reducing production of secretory granules in peptide hormone-secreting endocrine cells or neurons comprising the steps of (a) contacting a cell capable of forming secretory granules and expressing polypyrimidine tract binding protein (PTB) or a biologically active fragment or derivative thereof with one or more compounds; and (b) assessing whether said one or more compounds reduce the presence or activity of said polypyrimidine tract binding protein (PTB) or said biologically active fragment or derivative thereof in the cytoplasm of said cell.

32. A method for screening for an agent useful as a cure for sleeping disorders or depression comprising the steps of (a) contacting an animal carrying a transgene encoding polypyrimidine tract binding protein (PTB) or a biologically active fragment or derivative thereof under the control of a promoter that is active in hypothalamic neurons with one or more compounds; (b) assessing whether said one or more compounds reduce the presence or activity of said polypyrimidine tract binding protein (PTB) or said biologically active fragment or derivative thereof in the cytoplasm of said neurons.

33. A method for screening for an agent useful as a cure for typel or type-2 diabetes comprising the steps of (a) contacting an animal carrying a transgene encoding polypyrimidine tract binding protein (PTB) or a biologically active fragment or derivative thereof under the control of a promoter that is active in β- cells with one or more compounds; (b) assessing whether said one or more compounds increase the presence or activity of said polypyrimidine tract binding protein (PTB) or said biologically active fragment or derivative thereof in the cytoplasm of said cells.

34. The method of claim 32 or 33 wherein said promoter is an inducible promoter.

35. The method of claim 32 or 34 wherein said promoter is the vasopressin promoter.

36. The method of any one of claims 30 to 35 wherein said derivative of PTB is a fusion protein of PTB or a biologically active fragment thereof and a detectable marker.

37. The method of claim 36 wherein said detectable marker is a fluorescent compound.

38. The method of any one of claims 30 to 37 wherein the assessment in step (b) comprises the assessment of nucleocytoplasmic transport of PTB.

39. The method of any one of claims 30, 33 and 36 to 38 wherein said endocrine cells are pancreatic β-cells.

40. The method of claim 39 wherein the secretory-peptide contained in said secretory granules is insulin. i

41. The method of any one of claims 30 to 40 further comprising the step of (c) comparing the promotion/reduction effected by said compound on the presence or activity of said polypyrimidine tract binding protein (PTB) or said biologically active fragment or derivative thereof in the cytoplasm of said cell with the promotion effected by glucose or GLP-1 or CRH.

42. The method of any one of claims 30 to 41 wherein said one or more compounds are members of a library of compounds.

43. The method of any one of claims 30 to 42 further comprising the step of (d) testing the efficacy of a compound assessed as being capable of promoting/reducing the presence or activity of PTB in step (b) in the stimulating of the production of secretory granules in peptide hormone- secreting endocrine cells or neurons in an animal model.

44. Use of an agent reducing or blocking the presence or activity of polypyrimidine tract binding protein (PTB) or of a biologically active fragment or derivative thereof in endocrine cells for the preparation of a pharmaceutical composition for treating or preventing hyperprolactinemia or agromegalia.

45.The method or use of any of the pending claims wherein said PTB is the 59 kD isoform of PTB.