Patent Application
20150204850
The present invention relates to in vitro and in vivo methods for the production of pancreatic β-cells as well as to methods for the screening of compounds inducing a pancreatic β-cell phenotype.
In humans, plasma glucose levels are regulated by the action of insulin, a peptide hormone that is produced and secreted by the pancreatic β-cells in response to nutrition. Diabetes mellitus, which comprises a heterogeneous group of hyperglycaemic disorders, results from inadequate mass and function of pancreatic β-cells. Worldwide prevalence figures give an estimate of 151 million cases in 2000 and an extrapolated number of 221 million in 2010 (Simmet, 2001, for the list of references see the example section). There are two forms of diabetes, namely type I diabetes linked to a selective autoimmune destruction of pancreatic β-cells and type II diabetes which is a severe disease of intermediary metabolism usually caused by β-cell dysfunction and/or resistance to the biological actions of insulin on its main target tissues, i.e. liver, muscle and fat. Current therapy for type I diabetes usually requires the treatment with insulin while the current therapy for type II diabetes may additionally include the modification of lifestyle such as diet and exercise and the use pharmacological agents that stimulate insulin secretion. However, to date, there is no cure available for the treatment of diabetes.
The endocrine pancreas is organized in islets of Langerhans comprising five original cell subtypes, α-, β-, δ-, ε-, and PP-cells, secreting glucagon, insulin, somatostatin, ghrelin, and pancreatic polypeptide (PP), respectively. The identification and characterization of the genetic determinants underlying endocrine pancreas morphogenesis and regeneration may potentially aid designing cell replacement therapies to treat type 1 and 2 diabetes. In this context, and as also described in detail in the example by reference to numerous scientific publications, a number of studies have demonstrated that, during development, the cooperation of several transcription factors successively specifies progenitor cells towards the pancreatic-, endocrine- and ultimately islet-cell fates, indicating that the formation of the different cell types of the pancreas is due to the activities of a complex network of transcription factors.
Furthermore, it has been shown in the art that Pax-4 is able to induce proliferation of pancreatic β-cells (WO 06/015853). However, this document is silent about the transdifferentiation of α-cells or precusur cells into β-cells.
There is an urgent need for providing further means and methods for the treatment of diabetes.
The present invention relates to an in vitro method for the generation of pancreatic β-cells, comprising the step of providing at least one pancreatic α-cell or at least one precursor cell with a pancreatic β-cell phenotype-inducing compound.
The present invention relates to an in vitro method for the generation of pancreatic β-cells, comprising the step of providing at least one pancreatic α-cell or at least one precursor cell with Pax-4 or a nucleic acid encoding Pax-4.
In a preferred embodiment, said method includes providing at least one pancreatic α-cell or at least one precursor cell as the starting material.
In a preferred embodiment, the precursor cell is a pancreatic precursor cell, a small intestine precursor cell, a liver precursor cell or a precursor cell contained within the duct population.
In a preferred embodiment, said nucleic acid is a DNA.
In a preferred embodiment, the DNA is comprised in a vector.
In a preferred embodiment, said nucleic acid is an mRNA.
In a preferred embodiment, said method does not include providing other proteins involved in cellular development or nucleic acids encoding proteins involved in cellular development.
In a preferred embodiment, said method does not include providing other proteins apart from marker proteins or other genes apart from genes encoding marker proteins.
The present invention further relates to a pancreatic β-cell phenotype inducing compound for use in a method for the generation of pancreatic β-cells in vivo, wherein said method includes providing at least one pancreatic α-cell or at least one precursor cell with a pancreatic β-cell phenotype-inducing compound in vivo.
The present invention further relates to Pax-4 or a nucleic acid encoding Pax-4 for use in a method for the generation of pancreatic β-cells in vivo, wherein said method includes providing at least one pancreatic α-cell or at least one precursor cell with Pax-4 or a nucleic acid encoding Pax-4 in vivo.
The invention further relates to a method for the generation of pancreatic β-cells in vivo, wherein said method includes providing at least one pancreatic α-cell or at least one precursor cell with Pax-4 or a nucleic acid encoding Pax-4 in vivo.
In a preferred embodiment, said nucleic acid is a DNA.
In a preferred embodiment, the DNA is comprised in a vector.
In a preferred embodiment, said nucleic acid is an mRNA.
In a preferred embodiment, said method does not include providing other proteins involved in cellular development or nucleic acids encoding proteins involved in cellular development.
In a preferred embodiment, said method does not include providing other proteins apart from marker proteins or other genes apart from genes encoding marker proteins.
In a preferred embodiment, said method is for the treatment of a pancreatic disease.
In a preferred embodiment, said pancreatic disease is diabetes.
The invention further relates to a method for the screening of a pancreatic-β-cell phenotype-inducing compound, comprising the step of
In a preferred embodiment, said testing includes determining whether after the contacting the cell is capable of producing insulin.
In a preferred embodiment, said testing includes determining whether after the contacting the expression of Pax-4 is increased in the cell.
In a preferred embodiment, said method is performed in vitro.
In the context of the present invention, it has been surprisingly found that induction of the expression of Pax-4 in pancreatic adult α-cells or pancreatic precursor cells results in the generation of pancreatic β-cells in vivo (cf. the example). This enables in vitro and in vivo methods for the generation of pancreatic β-cells as well as screening methods for pancreatic β-cell phenotype inducing compounds.
“Pancreatic β-cells” or “β-cells” are well known in the art (for the respective literature see the example) and are, in the context of the present invention, cells capable of producing insulin upon stimulation with glucose.
In the context of the present invention, a pancreatic β-cell phenotype-inducing compound is provided to α-cells or precursor cells. After said provision, the level of said compound is increased in cell, with the consequence that the cell differentiates to a pancreatic β-cell phenotype. In the context of the present invention, the term “pancreatic β-cell phenotype” means that the cell is capable of producing insulin upon stimulation with glucose.
Method for determining whether a cell has a pancreatic β-cell phenotype are known in the art and include incubating the cell with glucose and testing whether insulin expression in the cell is increased or started (Nolan, 2009). Other methods include the testing whether β-cell specific transcription factors are expressed, the detection of β-cell specific gene products with the help of RNA quantitative PCR, the transplantation of a candidate cell in diabetic mice, and subsequent testing of the physiologic response following said transplantation as well analysing the cells with electron microscopy. These methods are all known in the art.
In principle, the pancreatic β-cell phenotype-inducing compound may be a compound of any type. This includes a protein, a nucleic acid, but also other compounds like small organic molecules, especially in the context of the screening claims of the invention (see below.)
In a preferred embodiment of the invention, Pax-4 or a nucleic acid encoding Pax-4 are provided to the cell.
The functional, wild-type Pax-4 to be particularly used in context of this invention is the wildtype Pax-4 of mouse, rat or human. Most particularly, the functional, wild-type Pax-4 to be employed is human Pax-4. The corresponding sequences (i.e., nucleotide sequences and amino acid sequences) are known in the art (see below and the example section).
In accordance with this invention, also Pax-4 molecules may be employed which are highly homologous to the functional, wild-type Pax-4 molecules known in the art and disclosed herein. Yet, these molecules have to be functional in the sense that these molecules do stimulate the generation of β-cells as documented herein. Accordingly, the invention also provides for a “read-out System” whether a given Pax-4 molecule is “functional”, i.e., capable of generating β-cells starting from α-cells or precursor cells.
Functional, wild-type Pax-4 molecules are known in the art, and comprise, but are not limited to the sequences disclosed in U.S. Pat. No. 6,071,697. Further “functional, wild-type Pax4” molecules are encoded by nucleic acid molecules as provided in the GeneBank under accession numbers: (rat Pax-4) NM—031799 (mousePax-4) NM—011038 and (humanPax-4): AF043978 or NM—006193.
In a preferred embodiment, these Pax-4 molecules may have a sequence identity of at least 40%, particularly at least 50%, more particularly at least 60%, even more particularly at least 70%, particularly at least 80%, more particularly at least 90%, even more particularly at least 95%>>, 97% or 98% and most particularly at least 99% identity with a nucleic acid sequence as described above encoding a wild-type Pax-4.
In accordance with the present invention, the term “identical” or “percent identity” in the context of two or more nucleic acid or amino acid sequences, refers to two or more sequences or subsequences that are the same, or that have a specified percentage of amino acid residues or nucleotides that are the same (e.g., 70-95% identity, more particularly at least 95%, 97%, 98% or 99% identity), when compared and aligned for maximum correspondence over a window of comparison, or over a designated region as measured using a sequence comparison algorithm as known in the art, or by manual alignment and visual inspection. Sequences having, for example, 95% or greater sequence identity are considered to be substantially identical. Such a definition also applies to the complement of a test sequence. Those having skill in the art will know how to determine percent identity between/among sequences using, for example, algorithms such as those based on CLUSTALW Computer program (Thompson, 1994; or FASTDB (Brutlag, 1990, as known in the art)).
In case that a nucleic acid encoding Pax-4 is used in the context of the present invention, this nucleic acid is preferably a DNA, which is preferably comprised in a vector, especially an expression vector. However, it is also envisaged that the Pax-4 encoding nucleic acid is provided per se to the cell or cells, and is then integrated into the cellular DNA e.g. by homologous recombination.
Consequently, “recombinant vectors” or “expression vectors” or “expression constructs” form important further embodiments of the present invention. The terms “expression vector”, “recombinant vector” or “expression construct” are used interchangeably in the context of the present invention and mean any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed, and later on translated, either entirely due to information provided on the vector or construct or also partially due to information provided by the host cell.
Particularly useful vectors are contemplated to be those vectors in which the coding portion of the DNA segment, whether encoding a full length protein or smaller peptide, is positioned under the transcriptional control of a promoter. A “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The phrases “operatively positioned”, “under control” or “under transcriptional control” means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression of the gene. The promoter may be in the form of the promoter that is naturally associated with a particular gene, as may be obtained by isolating the 5′ non-coding sequences located upstream of the coding segment or exon, for example, using recombinant cloning and/or polymerase chain reaction (PCR™) technology.
In other embodiments, it is contemplated that certain advantages will be gained by positioning the coding DNA segment under the control of a recombinant, or heterologous, promoter. As used herein, a recombinant or heterologous promoter is intended to refer to a promoter that is not normally associated with a particular gene in its natural environment. Such promoters may include promoters normally associated with other genes, and/or promoters isolated from any other bacterial, viral, eukaryotic, or mammalian cells.
Naturally, it will be important to employ a promoter that effectively directs the expression of the DNA segment in the cell type chosen for expression. The use of promoter and cell type combinations for protein expression is generally known to those of skill in the art of molecular biology, for example, see Sambrook (2001, which can be used as a reference for all molecular biology methods described herein). The promoters employed may be constitutive, or inducible, and can be used under the appropriate conditions to direct high level expression of the introduced DNA segment, such as is advantageous in the large-scale production of recombinant proteins or peptides.
The promoter is required to express the transforming genetic construct to a degree sufficient to effect transformation of a target cell type amongst a population of different cell types such that the transformed target cell results in the generation of a stable human regulated secretory cell. Promoters can be classified into two groups, ubiquitous and tissue- or cell-specific. Ubiquitous promoters activate transcription in all or most tissues and cell types. Examples of ubiquitous promoters are cellular promoters like the histone promoters, promoters for many metabolic enzyme genes such as hexokinase I and glyceraldehyde-3-phosphate dehydrogenase, and many viral promoters such as the cytomegalovirus promoter (CMVp) and the Rous sarcoma virus promoter (RSVp).
Tissue- or cell-specific promoters activate transcription in a restricted set of tissues or cell types or, in some cases, only in a single cell type of a particular tissue.
Alternatively, the nucleic acid encoding Pax-4 may be an mRNA.
As it can be taken from the examples, the inventors of the present invention have been able to show that the transfection of Pax-4 encoding nucleic acids is sufficient for the generation of a pancreatic β-cell phenotype, i.e. for the differentiation of pancreatic α-cells or precursor cells into pancreatic β-cells. Consequently, in a preferred embodiment of the invention, Pax-4 or a nucleic acid encoding Pax-4 are the only functional agents provided to the cells.
In this context, the term “no other functional agent” means that no other agent is added which is involved in the differentiation into a pancreatic β-cell phenotype.
In a further preferred embodiment, this means that said method does not include providing other proteins involved in cellular development or nucleic acids encoding proteins involved in cellular development or that said method does not include providing other proteins apart from marker proteins or protein conferring resistance against antibiotics or other genes nucleic acid sequences apart from genes encoding marker proteins or proteins conferring resistance against antibiotics or regulatory sequences. It will be appreciated by the person skilled in the art that when a nucleic acid encoding Pax-4 is transfected, other nucleic acids may also be transfected which includes marker genes, antibiotic resistance genes or regulatory sequences. These nucleic acids, however, do not have any impact on the differentiation into the pancreatic β-cell phenotype.
In the context of the methods of the invention, compounds and in particular Pax-4 or nucleic acid encoding Pax-4 are provided to at least one pancreatic α-cell or at least one precursor cell.
Methods for providing a protein, and especially a transcription factor like Pax-4, to cells are well known in the art and include incubation of the respective cell with a liposome-containing protein, electroporation (Amaxa, Lonza, Nucleofector) as well as delivery through the HIV TAT system (Zhao, 2007).
Methods for providing the respective cell with a nucleic acid encoding Pax-4 are also well known in the art and include preferably transfection methods and viral infections.
In order to effect expression, the nucleic acid must be delivered into a cell. As described below, a possible mechanism for delivery is via viral infection, where the nucleic acid is encapsidated in an infectious viral particle. However, several non-viral methods for the transfer of the nucleic acid into cultured mammalian cells (transfection methods) also are contemplated by the present invention. In one embodiment of the present invention, the nucleic acid may consist only of naked recombinant DNA or plasmids. Transfer of the vector may be performed by any of the methods mentioned which physically or chemically permeabilize the cell membrane.
In a further embodiment of the invention, the nucleic acid may be entrapped in a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution.
In certain embodiments of the present invention, the nucleic acid is introduced into the cell via electroporation. Electroporation involves the exposure of a Suspension of cells and DNA to a high-voltage electric discharge. Transfection of eukaryotic cells using electroporation has been quite successful in the art (see e.g. Sambrook (2001)).
In other embodiments of the present invention, the nucleic acid is introduced to the cells using calcium phosphate precipitation, as generally in the art. Other transfection methods include the use of DEAE-dextran followed by polyethylene glycol or particle bombardment.
The vector expressing a nucleic acid encoding Pax-4 as defined above to be used in an in vitro or an in vivo method of the invention in accordance with this invention may also be a viral vector. In this context, the vector may be selected from the group consisting of an adenoviral vector, an adeno-associated virus vector, a vaccinia virus vector, a herpes virus vector, a retroviral vector or an lentiviral vector, but also other viral vectors known in the art may be used. Such vectors are known in the art (Gene Transfer, Delivery and Expression of DNA and RNA, A Laboratory manual, Edit by T. Friedmann, J. Rossi, Cold Spring Harbor Laboratory Press, New York 2006), Hitt (2006), Salmon (2006)). Viral vector are especially preferred in the in vivo methods of the present invention.
According to the invention, the compound is provided to at least one pancreatic α-cell or at least one precursor cell. Consequently, in a preferred embodiment, first at least one pancreatic α-cell or at least one precursor cell are provided as the starting material. In a further preferred embodiment, the starting material is at least one isolated α-cell or at least one isolated precursor cell.
“α-cells” or “pancreatic α-cells” are known in the art (see also the example for literature) and are capable of expressing glucagon as a consequence of decreased blood sugar levels. Consequently, in the context of the present invention, an α-cell is preferably a cell capable of expressing glucagon as a consequence of decreased blood sugar levels.
Alternatively, also other adult cells, especially liver cells, duct cells or intestinal cells can be used as the starting material of the method of the present invention.
In the context of the present invention, the term “isolated” means that the respective cell has been at least partially purified.
Methods for providing at least one α-cell are known in the art and include isolating pancreatic tissue and isolating the cell e.g. with the help of FACS (cell sorter) as known in the art or providing a α-cell-line (Powers, 1990).
Methods for providing precursor cells are also known in the art and include either providing tissue containing said precursor cells and isolating the cells by methods known in the art, e.g. with the help of cell surface specific antibodies and using a FACS (cell sorter) or cultivation of the cells under specific conditions allowing the growth of precursor cells. Furthermore, also suitable cell lines are known in the art (Lieber (1975)).
Every precursor cell being capable of differentiating into pancreatic β-cells can be used as the starting cell of the method of the invention. Especially, this includes all precursor cells derived from a human or animal tissue, in particular the endoderm. Preferably, the precursor cell is a native precursor cell, i.e it contains the same genetic information as a precursor cell present in the respective tissue. In particular, the precursor cell has not been genetically modified, in particular it has not been genetically modified by transfection with a nucleic acid. In a preferred embodiment, said precursor cell is selected from the group consisting of a pancreatic precursor cell, a small intestine precursor cell, a liver precursor cell, a precursor cell derived from the duct population, and a pancreatic stem cell.
After the cells have been provided, said cell or cells may be present in any in vitro cell culture system useful for the context of the present invention. This includes standard cell culture systems like tissue culture dishes as well as 6-well, 24-well or 96-well plates. Culture conditions will depend on the respective cell. The person skilled in the art will know how to cultivate the cells.
In the context of the present invention, it has been shown that an increase of functional Pax-4 in a pancreatic α-cell or a precursor cell results in the formation of pancreatic β-cells. In the examples of the present invention, this increase in functional Pax-4 has been obtained by providing a nucleic acid encoding Pax-4 in vivo. However, as outlined above, it is equally feasible to provide Pax-4 protein itself. Furthermore, it is feasible to provide any other compound which is able to induce a pancreatic β-cell phenotype. Consequently, the finding of the inventors of the present invention that it is possible to generate pancreatic β-cells out of pancreatic α-cells or precursor cells enables also a screening method for pancreatic β-cell phenotype inducing compounds.
Consequently, in another aspect of the present invention, the invention relates to a method for the screening of a pancreatic β-cell phenotype inducing compound comprising the steps of
In principle, with the method of the invention, it is possible to screen for every compound being capable of inducing a β-cell phenotype.
Preferably, said compound is selected from the group consisting of synthetic or naturally occurring chemical compounds or organic synthetic drugs, more preferably small molecule organic drugs or natural small molecule compounds. Preferably, said compound is identified starting from a library containing such compounds. Then, in the course of the present invention, such a library is screened.
Such small molecules are preferably not proteins or nucleic acids. Preferably, small molecules exhibit a molecular weight of less than 1000 Da, more preferred less than 750 Da, most preferred less than 500 Da.
A “library” according to the present invention relates to a (mostly large) collection of (numerous) different chemical entities that are provided in a sorted manner that enables both a fast functional analysis (screening) of the different individual entities, and at the same time provide for a rapid identification of the individual entities that form the library. Examples are collections of tubes or wells or spots on surfaces that contain chemical compounds that can be added into reactions with one or more defined potentially interacting partners in a high-throughput fashion. After the identification of a desired “positive” interaction of both partners, the respective compound can be rapidly identified due to the library construction. Libraries of synthetic and natural origins can either be purchased or designed by the skilled artisan. Examples of the construction of libraries are provided in, for example, Breinbauer R, (2002), wherein natural products are described that are biologically validated starting points for the design of combinatorial libraries, as they have a proven record of biological relevance. This special role of natural products in medicinal chemistry and chemical biology can be interpreted in the light of new insights about the domain architecture of proteins gained by structural biology and bioinformatics. In order to fulfill the specific requirements of the individual binding pocket within a domain family it may be necessary to optimise the natural product structure by chemical variation. Solid-phase chemistry is said to become an efficient tool for this optimisation process, and recent advances in this field are highlighted in this review article. The current drug discovery processes in many pharmaceutical companies require large and growing collections of high quality lead structures for use in high throughput screening assays. Collections of small molecules with diverse structures and “drug-like” properties have, in the past, been acquired by several means: by archive of previous internal lead optimisation efforts, by purchase from compound vendors, and by union of separate collections following company mergers. Although high throughput/combinatorial chemistry is described as being an important component in the process of new lead generation, the selection of library designs for synthesis and the subsequent design of library members has evolved to a new level of challenge and importance. The potential benefits of screening multiple small molecule compound library designs against multiple biological targets offers substantial opportunity to discover new lead structures.
The compound to be screened is added to at least one α-cell or at least on precursor cell as described above for the method of the invention for the generation of β-cells. Furthermore, also the embodiments described above with respect to the providing of α-cells or precursor cells including the respective cell lines, especially with respect to their isolation, also apply to the screening method of the invention.
In the context of the screening claims of the present invention, it is particularly preferred that cells of a given suitable cell line, e.g. an α-cell line or a duct cell line, are cultivated and contacted with the compound. A suitable readout system could then be whether the cells are then capable of producing insulin. Another readout system could be to transfect the cells with a reporter gene under the control of a β-cell specific promoter, e.g. the insulin promoter, and testing whether the compounds are capable of inducing the expression of the reporter gene.
In the context of the screening method of the invention, it is tested whether a given compound is able to induce a pancreatic β-cell phenotype. In a preferred embodiment, said testing includes determining whether after the contacting the cells are capable of producing insulin, in particular after incubation of the cells with glucose. This readout of the screening of the invention is especially suitable because the capacity of producing insulin is the major feature of pancreatic β-cells.
However, since it has been shown that Pax-4 is able to induce a pancreatic β-cell phenotype if more functional Pax-4 is present in the cell (see above), said testing includes in a preferred embodiment also whether after the contacting the expression of Pax-4 is increased in the cell. In a preferred embodiment, the level of Pax-4 is compared to the level of Pax-4 in a cell not contacted with the compound.
In an especially preferred embodiment, said testing includes both the capacity of producing insulin as well determining the expression of Pax-4.
In a further preferred embodiment of this aspect of the invention, said screening method is performed in vitro, preferably in the context of a medium- or high-throughput system. However, it is equally envisaged to use animal models for the testing of compounds. In this context, animal models of diabetes as known in the art are especially suitable and include NOD mice, streptozotocin treated mice, as well as mice treated with diphtheria toxin.
As shown in the examples of the present invention, with the help of Pax-4 or nucleic acid encoding Pax-4, it is also possible to generate pancreatic β-cells in vivo. Consequently, the present invention also relates to the in vivo application of a pancreatic β-cell phenotype-inducing compound, especially Pax-4 or a nucleic acid encoding Pax-4. This compound may be preferably contained in a pharmaceutical composition.
In general, the pharmaceutical compositions of the present invention comprise a therapeutically effective amount of a therapeutic, and a pharmaceutically acceptable carrier. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeias for use in animals, and more particularly, in humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, including but not limited to peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered orally. Saline and aqueous dextrose are preferred carriers when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions are preferably employed as liquid carriers for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Such compositions will contain a therapeutically effective amount of the therapeutic, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.
In a preferred embodiment, the composition is formulated, in accordance with routine procedures, as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anaesthetic such as lidocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water or saline for injection can be provided so that the ingredients may be mixed prior to administration.
The therapeutics of the invention can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free carboxyl groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., those formed with free amine groups such as those derived from isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc., and those derived from sodium, potassium, ammonium, calcium, and ferric hydroxides, etc.
The amount of the therapeutic of the invention which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. However, suitable dosage ranges for intravenous administration are generally about 20-500 micrograms of active compound per kilogram body weight. Suitable dosage ranges for intranasal administration are generally about 0.01 pg/kg body weight to 1 mg/kg body weight. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. In general, suppositories may contain active ingredient in the range of 0.5% to 10% by weight; oral formulations preferably contain 10% to 95% active ingredient.
Various delivery systems are known and can be used to administer a therapeutic of the invention, e.g., encapsulation in liposomes, microparticles, and microcapsules: use of recombinant cells capable of expressing the therapeutic, construction of a therapeutic nucleic acid as part of a retroviral or other vector, etc. Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compounds may be administered by any convenient route, for example by infusion, by bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral, rectal and intestinal mucosa, etc.), and may be administered together with other biologically active agents. Administration can be systemic or local.
In a specific embodiment, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment. This may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, nonporous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. In one embodiment, administration can be by direct injection at the site (or former site) of a malignant tumor or neoplastic or pre-neoplastic tissue.
In another embodiment, the therapeutic can be delivered in a vesicle, in particular a liposome (Langer, (1990)).
In a further preferred embodiment of the aspect of the invention where Pax-4 or nucleic acid encoding Pax-4 is provided in vivo, said method is for the treatment of a pancreatic disease, preferably diabetes.
The invention is further illustrated by the following figures and examples which are intended to explain, but not to limit the scope of the present invention.
(A) Characterization of the lifespan and glycemia (in mg/dl) of POE::Pdx1cre and POE::Pax6cre mice 1 day and 6 weeks postpartum, as well as short before death (SBD). Note that initially, these animals are hypo- and subsequently hyperglycemic. (B-P) Immunohistochemical analysis of the islets of double-transgenic mice. A dramatic increase in islet size and insulin-expressing cell mass is evident 6 weeks postpartum (C, F, I, L, O) and is even more pronounced SBD (D, G, J, M, P) as compared to representative control islets (B, E, H, K, N). Concurrently, a loss of α- (E-M), δ- (H-J), PP- (K-M), and Arx-labeled (N-P) cells is evident, but, interestingly, few of these remain detectable at one pole of the islet, some co-expressing the glucagon and somatostatin (arrow in I) or PP (arrow in L) hormones. (Q) A quantification of the endocrine cell alterations ascertains these observations (also see Table 2). For the purpose of clarity, the magnification of double-transgenic islets is twice (C, F, I, L, O) or eight times (D, G, J, M, P) reduced compared to controls. (n>11, * P<0.05, ** P<0.01, *** P<0.001, all values expressed as means±standard error of the mean).
(A-T) Characterization of POE::Pdx1cre and POE::Pax6cre hormone-expressing cells. Sections of double-transgenic pancreata (of the indicated ages) were stained using the mentioned antibodies, the number of labeled cells counted and reported to the count obtained in POE control animals. The values are expressed in percentage of change in labeled cell number compared to controls. All values are statistically significant with P values lower than or equal to 0.05. Note that the numbers of insulin-expressing cells are dramatically increased in double-transgenic animals at all examined stages (A-P, S-T) and this, as early as E13 (A-B), compared to controls. Concurrently, the mean contents of α- (A-B, G-J, Q-R), δ- (C-D), PP(E-F) and Arx- (I-J) marked cells appear drastically reduced. A thorough analysis of endocrine cell-specific markers demonstrates that, all insulin-producing cells clearly express Pax4 (G-H), as well as the β-cell markers Nkx6.1 (K-L), Glut-2 (M-N) and Pdx1 (O-P). These are negative for the α-cell specific factor Brn-4 (Q-R), whereas, all endocrine cells express Pax6 (S-T). (U) An improved glucose tolerance and insulin release are highlighted in 3-week-old double-transgenic mice as compared to controls POE mice (n>3, ***P<0.001, **P<0.01, * P<0.05). Each picture is representative of at least 4 independent animals; note that, for the purpose of clarity, a POE::Pax6cre islet is displayed in H.
(A-C) Quantification of the endocrine cell content alterations upon ectopic expression of the Pax4 gene in glucagon-producing cells in 1-week-old animals. A clear increase in the number of insulin-/Pax4-labeled cells at the expense of glucagon-expressing cells is highlighted (A), whereas the δ- and PP-cell contents are found unchanged (B-C). Note the accumulation of the remaining glucagon-marked cells at one pole of the islet. (D-F) The detection of insulin- and β-galactosidase-expressing cells on serial sections demonstrates that numerous insulin-labeled cells do express the β-galactosidase gene that normally marks glucagon-positive cells. (G-I) The same observation is made in 6-week old animals using co-immunofluorescence. All reported values are statistically significant (P<0.05, n=5). See Table 2 for a detailed analysis.
Six-week old pancreata of the indicated genotypes were sectioned and stained using anti-insulin (A, C, E, G, I, K) or glucagon (B, D, F, H, J, L) antibodies. The values at the top right corner of each photograph represent the number of hormone-producing cells per square centimeter of pancreas. The counts reported underneath each picture set represent variations in islet surface (estimated in silico) using POE::Pax6cre/Pdx1cre pancreata (C-D) as reference for photographs A-H and POE::Glucre pancreata (I-J) as reference for photographs I-L. Note the overall diminution in islet size and glucagon+ cell content, as well as the drastic decrease in insulin-expressing cell number (E-H compared to C-D and K-L compared to I-J) in animals supplemented with glucagon for 3 weeks compared to untreated ones (*P<0.05, **P<0.01, n=3).
Next to a pulse of BrdU at three weeks of age and examination a week later, a quantitative analysis established a 2.35 increase in the number of proliferating cells in POE::Glucre pancreas compared to POE controls (A-B) Importantly, most BrdU-labeled cells are not detected within the islets, but rather near or within the duct epithelium (B). It is worth noticing that this location corresponds to the cluster of glucagon-producing cells consistently observed in this genotype. Within the duct epithelium, scattered endocrine cells could be detected, some co-expressing the glucagon and sometimes somatostatin hormones (arrowheads in C-D). Along the same line, islet cells positive for the duct marker genes CK19 and SPP1 are observed adjacent to the duct epithelium (arrowheads in E-F). Notably, the duct lining also appears to contain numerous cells positive for the proendocrine marker gene Ngn3 (G-H), but negative for Pdx1 (I). (J-L) Infection of controls (K) and double-transgenic (L-O) pancreata using a construct containing a CMV promoter driving the constitutive expression of a c-Myc tag (the latter being not expressed in wild-type tissues—J). Due to the method used, two weeks post-infection, most exocrine cells are labeled by the virus, whereas only very few islet cells are (islet underlined in K) Importantly, a majority of double-transgenic endocrine cells appear marked by the virus, suggesting their ductal or acinar origin (L). These cells express the insulin hormone (M) and are β-galactosidase-positive (N-O), indicating that they once expressed the glucagon hormone.
(A-H) Infection of POE::Glucre animals using lentiviruses producing either an shRNA targeting Ngn3 transcripts (E-H—Xu et al., 2008) or producing a scrambled shRNA (based on the former; A-D). Two weeks post-infection, Ngn3 knockdown pancreata (at the indicated magnifications) display an efficient 61% diminution in Ngn3 production (Xu et al., 2008, A, E and Table 6), but also a 69% decrease in insulin- (B, F, D, H) or glucagon- (C, G) expressing cell numbers (Table 6) compared to scramble-infected counterparts.
(A) Following streptozotocin injection at the indicated age (starting age), the glycemia and survival of the treated animals was followed for two months. Note that animals older than four weeks of age become diabetic and die as a consequence of the obliteration of β-cells, the same being true for all controls (data not shown) Importantly, in younger animals, a steady recovery leading to normoglycemia is observed next to a peak in glucose levels (all values are expressed as means±standard error of the mean). (B-J) Islet cell contents in 4-week-old POE::Glucre streptozotocin- (D-J) or sham- (B-C) treated animals were quantified (see at the bottom of the concerned pictures) 3 days (B-E), 10 days (F-G), 16 days (H) and 60 days (I-J) days post-injection. Three days post-injection, the β-cell mass present in controls (B-C) is almost entirely lost in streptozotocin-treated mice (D-E), the only insulin labeling being observed in areas devoid of nuclei, suggesting a detection of hormone released from killed β-cells. A 15% increase in glucagon-producing cells is highlighted 10 days post-injection compared to controls, most of these cells neighboring duct structures (G) Importantly, few insulin-labeled cells are detected (F). This trend is ascertained at day 16 (H) with a major increase in the insulin-expressing cell number. A co-detection with the glucagon hormone does not indicate any co-expression. Finally, at day 60, the β-cell mass appears statistically normal as compared to control animals (I compared to B-C), most of the cells present in the islet expressing the β-galactosidase enzyme marking glucagon-producing cells (J).
(A) Schematics depicting the targeting vector prior to (top) and following (bottom) homologous recombination of the two LoxP sites induced by the phage P1 cre recombinase. β-Galactos., β-galactosidase. (B-C) Visual examination under fluorescent light of an E10.5 embryo and adult pancreas, both representative of Cre-negative animals. (D-G) POE exocrine and endocrine (note the islet outlined in D-E) cells do not express β-galactosidase as demonstrated through LacZ staining (whereas GFP staining is present in a majority of pancreatic cells; D-E) Importantly, β-galactosidase activity is found restricted to the islet of Langerhans in age-matched POE::Pax6cre pancreata (F), whereas almost all pancreatic cells are β-galactosidase-positive in POE::Pdx1cre pancreata (G).
Representative islets of POE (A and
Representative sections of POE (A) and POE::Pax6cre/Pdx1cre (B) pancreata stained with anti-insulin antibodies and observed using the same magnification. Note the remarkable islet size augmentation in POE::Pax6cre/Pdx1cre pancreata (B) as a result of a drastic increase in β-cell numbers.
Using immunohistochemistry, an anti-Pax4 antibody (Wang et al, 2008) raised in rabbit was tested in wild-type (A-B) and Pax4-depleted (C-D) adult islets. In the wild-type situation, Pax4 is clearly detected in most β-cells (A), but not in α-cells (B), nor in 6- or PP-cells (see
An anti-Pax4 antibody (Wang et al., 2008) was used to study Pax4 expression in adult pancreata together with the indicated endocrine hormones. Pax4 is clearly detected in most insulin-expressing cells both in POE (A-D) and POE::Pax6cre islets (E-H), whereas α-, δ- and PP-cells are found devoid of Pax4 protein (B-D, F-H, respectively). Interestingly, the vast majority of POE::Pdx1cre pancreatic cells are found positive for Pax4 (I-L). For the purpose of clarity, the magnification of double-transgenic islets is five times reduced compared to controls. Each picture is representative of at least 4 independent animals.
Due to conflicting reports concerning the expression of Pax4 in adult β-cells, we attempted to amplify Pax4 mRNA using classical RT-PCR. Such an approach was unsuccessful, most likely due to the presence of numerous secondary structures within the Pax4 mRNA (
The putative presence of secondary structures within the Pax4 mRNA was revealed using the RNAfold program (http://mobyle.pasteur.fr/cgi-bin/MobylePortal/portal.py?form=rnafold). The plot of the predicted secondary structures calculated based on minimum free energy demonstrates that Pax4 mRNA is exceptionally structured through matching of complementary nucleotides. This may be the cause of the difficulties encountered with classical RT-PCR, but also explains the lack of working antisense probe for in situ hybridization purposes. Note that this plot allows online close-up viewing.
Assessment of Cre recombinase activity in glucagon promoter-Cre (Glucre-Herrera, 2000) mice crossed with Rosa26 promoter-LoxP-STOP-LoxP-β-Galactosidase reporter animals (Soriano, 1999). Cre activity was visualized using LacZ staining (A-B) at the indicated magnifications and compared to glucagon expression assayed using immunohistochemistry on a consecutive section (C). A similar expression pattern was observed for glucagon and β-galactosidase thereby confirming previously published results (Herrera, 2000). Each picture is representative of at least 3 independent animals.
Serial sections of POE::Glucre animals treated with BrdU and examined a week later (F) or not were stained using the indicated antibody combinations. While Ngn3-expressing cells were found mostly located adjacent to the islet (A), Pax4- (B), Pdx1- (C) and 3-galactosidase(E) producing cells were detected exclusively within the islet of Langerhans. Only few glucagon+/Pax4+ (inlet in B) or glucagon+/β-galactosidase+ cells (inlet in E), and even fewer glucagon+/insulin+ (inlet in G), were observed in POE::Glucre pancreata, indicating an extremely rapid transition between glucagon+ and insulin+ phenotypes Importantly, most proliferating cells were detected within the duct epithelium (D, F) or the islet domain adjacent to ducts (F). The duct epithelium and lining were found uniformly positive for GFP (H-K), suggesting that Pax4 ectopic expression mostly occurs within the islet (B, E).
Endocrine cell counts from adult wild-type and POE::Inscre animals were assessed after immunostaining against all four hormones. No statistically significant difference could be found between both genotypes (n=7, all values are expressed as means±standard error of the mean).
Ngn3 expression was determined using a mouse anti-Ngn3 antibody (Zahn et al., 2004; A-B, E-H) and a goat anti-Ngn3 antibody (provided by M. Sander's laboratory (Seymour et al., 2007; C-D, G-H). Staining of POE (Cre−, G-H), POE::Pdx1cre/POE::Pax6cre/POE::Glucre (Cre+, A-F) pancreata suggest Ngn3 expression in the pancreatic duct lining from animals ectopically expressing Pax4.
Ngn3 expression was assayed using two antisense probes (constructed by G. Gradwohl and P. Ravassard). (A-F) Albeit extremely difficult on adult tissues, this approach allowed us to detect few labeled cells in ductal structures in A and D (serial 18 μm sections). Photographs of the same sections counterstained with propidium iodide are provided in B and E, and merged pictures in C and F, respectively. Note the expression of Ngn3 in the cytoplasm of ductal cells (inlets in C, F). Bear in mind that due to thickness of the section (18 μm) and curved nature of the duct tubes, the lumen of the latter is not always distinguishable (D-F). (G-I) Using a sense probe, we did not detect any labeled cells.
Lentiviruses encompassing a 5566-bp long fragment of the mouse Ngn3 promoter (orthologous region of the human Ngn3 promoter characterized in Lee et al., 2001) driving the expression of a DsRED2::c-Myc cassette (see supplementary experimental procedures) were used to infect the pancreata of POE::Glucre (C-N) or POE (A-B) animals as previously described (Xu et al., 2008). Due to the technical limitations previously discussed, the transgene was detected using an anti-c-Myc antibody. Control pancreata were found negative for c-Myc detection (A-B), whereas variable results were obtained within the same POE::Glucre pancreas, ranging from ducts negative for c-Myc (C-D), to ducts displaying different numbers of c-Myc-labeled cells (E-N), such discrepancies most likely originating from different levels of infection. Note that the specificity of the Ngn3 promoter employed was also tested (
The duct displayed in
(A-L) To determine whether the 5566-bp long fragment of the mouse Ngn3 promoter was sufficient to recapitulate Ngn3 expression in the pancreas, a lentiviral vector expressing the eGFP reporter gene under the control of this promoter element was injected in the perivitelline space of mouse fertilized eggs. The resulting eggs were reimplanted in pseudo pregnant females and the derived embryos isolated 14.5 days later. Eleven transgenic founder embryos were obtained and co-stained to detect both Ngn3 and eGFP expression. Briefly, using TSA amplification, 12 μm cryosections were stained with the anti-Ngn3 mouse monoclonal antibody (red, B-C, E-F, H-I, K-L) from and according to Zahn et al. (2004). Concomitantly, eGFP expression was revealed with a rabbit polyclonal anti-GFP antibody (A, C, D, F, G, I, J and L) as described by Castaing et al. (2005). (A-C) Pancreata of wild-type embryos derived from re-implanted injected eggs, are expectedly eGFP-negative (A), whereas scattered Ngn3-labelled positive cells are visualized (B-C). (D-L) The analysis of representative sections of 3 pancreata of eGFP-expressing founders (6 out of 11) indicates that the vast majority of Ngn3-expressing cells are eGFP positive thereby demonstrating the specificity of the 5566-bp long mouse Ngn3 promoter fragment (F, I and L).
Representative islet of adult glucagon-receptor-deficient animals assayed for Ngn3 (A) and counterstained with DAPI (B). A merged picture is displayed in C. Note the detection of few Ngn3-labelled cells in this genotype, whereas control age-matched counterparts are negative for Ngn3 (
(A) Through a combination of glucose challenge and circulating insulin measurements, an impairment in β-cell function is outlined in 10-week old POE::Glucre/POE::Pdx1cre/POE::Pax6cre animals: these exhibit a worsened and extended response time to glucose injection that may be explained by a decreased insulin secretion as compared to POE control counterparts. Note that 600 mg of glucose per dl of blood correspond to the maximal value detectable by our glucose monitoring system. (B) For the purpose of comparison, a copy of the graph displayed in
The endocrine pancreas is organized in islets of Langerhans comprising five original cell subtypes, α-, β-, δ-, e-, and PP-cells, secreting glucagon, insulin, somatostatin, ghrelin, and pancreatic polypeptide (PP), respectively (Collombat et al., 2006). The identification and characterization of the genetic determinants underlying endocrine pancreas morphogenesis and regeneration may potentially aid designing cell replacement therapies to treat type 1 and 2 diabetes. In this context, a number of studies have demonstrated that, during development, the cooperation of several transcription factors successively specifies progenitor cells towards the pancreatic-, endocrine- and ultimately islet-cell fates. Hence, Pdx1 is required for pancreatic epithelium determination (Ahlgren et al., 1996; Ahlgren et al., 1998; Grapin-Botton et al., 2001; Jonsson et al., 1994; Offield et al., 1996) and subsequently Neurogenin3 (Ngn3) for endocrine lineage specification (Gradwohl et al., 2000; Gu et al., 2002; Jensen et al., 2000; Johansson et al., 2007). Next to Ngn3 induction, a complex network of transcription factors progressively and differentially promotes the particular endocrine fates, including Arx and Pax4 (Collombat et al., 2003; Sosa-Pineda et al., 1997). In mice lacking Arx, the β- and δ-cell fates were found favored at the expense of α-cell genesis, while the total endocrine cell content remained normal (Collombat et al., 2003). Conversely, in the absence of Pax4, the opposite phenotype was observed (Sosa-Pineda et al., 1997), indicating an inhibitory, cross-regulatory circuit between Arx and Pax4 (Collombat et al., 2005). Additional findings supported these conclusions and suggested that, firstly, Arx and Pax4 instruct endocrine precursor cells towards either an α-cell or a β-/δ-cell fate, respectively. Next, through the analysis of double-mutant mice, a secondary function of Pax4 in specifying the β-cell lineage in β-/δ-precursor-cells was uncovered (Collombat et al., 2005). Recent evidence have demonstrated that the forced expression of Arx in early pancreatic cells drives endocrine progenitors towards either an α- or, surprisingly, a PP-cell fate (Collombat et al., 2007). It was therefore concluded that Arx is not only necessary, but also sufficient to instruct the α- and PP-cell lineages.
Of particular interest was the finding that the forced expression of Arx triggered into adult β-cells induced their conversion into cells exhibiting α- or PP-cell phenotypes (Collombat et al., 2007). This discovery was of fundamental importance in the context of β-cell-based therapy and implied that the opposite conversion might be achieved, that is, to generate β-cells from other endocrine cells. To test this hypothesis, we generated mice conditionally and ectopically expressing the Pax4 gene. Our data indicates that the ectopic expression of Pax4 in early pancreatic cells, but also in α-cells, induces their respecification towards a β-cell fate or identity. As a consequence of the ensuing glucagon deficiency, an ongoing neogenesis of α-cells occurs. However, such α-cells are continuously converted into β-cells upon Pax4 ectopic expression, resulting in the development of oversized islets of Langerhans Importantly, a prominent expression of the proendocrine transcription factor Ngn3 in the pancreas of such animals is highlighted. Our results are consistent with the recently reported notion of facultative adult stem cells that reactivate Ngn3 expression in injured pancreas (Xu et al., 2008). Finally, following streptozotocin-induced depletion of β-cells in young mice ectopically expressing Pax4, an α-cell-mediated regeneration of the β-cell mass, a progressive normalization of the glycemia and an extended lifespan are observed.
Taking advantage of the Cre-LoxP system, we generated transgenic mice able to conditionally express the Pax4 gene (POE). Briefly, the construct used included the cytomegalovirus enhancer/human β-actin (CAG) promoter driving the expression of the eGFP gene followed by translation stop codons in all three frames and flanked by LoxP sites (
POE animals were bred with Pdx1cre or Pax6cre animals to target Pax4 expression to the pancreatic epithelium or the endocrine tissue, respectively (Ashery-Padan et al., 2004; Gu et al., 2002). Double-transgenic POE::Pdx1cre and POE::Pax6cre mice were born and developed normally. However, animals of both genotypes eventually died at the age of 3 to 12 weeks, a majority after 9 weeks (
To assess the consequences of the forced expression of Pax4 in Pdx1 and Pax6 expression domains, immunohistochemical analyses of double-transgenic pancreata were performed. As similar results were obtained in POE::Pax6cre and POE::Pdx1cre mice, pictures representative of both genotypes are displayed for 6-week-old animals (
Pax4 expression was assayed with a newly-developed antibody that detected Pax4 in wild-type embryonic pancreata (Wang et al., 2008) and specifically in adult β-cells (
Further examination of POE::Pax6cre and POE::Pdx1cre mice during the development of the endocrine pancreas indicated that α-, 8, and PP-cell lineages were disfavored to the benefit of an insulin-producing cell fate (
Conversion from an α- to a β-Cell Phenotype
As Pax4 specifically favors the β-cell fate and identity throughout the morphogenesis of the endocrine tissue, we wondered whether this might also apply to mature endocrine cells. Therefore, animals conditionally expressing Pax4 in glucagon-producing cells (Herrera, 2000—
Pax4 Ectopic Expression in β-Cells does not Induce their Proliferation
To further characterize the origin of the supernumerary β-cells, we assayed proliferating cells combining bromodeoxyuridine (BrdU) incorporation and Ki67 staining. Following a BrdU pulse in 5-week old POE::Glucre mice, pancreata were quantitatively analyzed a week later: a significant 2.35-fold increase in the number of BrdU-labeled cells was evidenced as compared to controls (
The cell-autonomous effect of an augmentation of the Pax4 dose in β-cells was analyzed by crossing POE with Inscre mice expressing the cre recombinase under the control of the insulin promoter (Herrera, 2000). The quantification of the endocrine cell numbers in 3-week old POE::Inscre pancreata did not reveal any increase in β-cell numbers, nor in islet size (Figure 17 and data not shown), despite the use of the POE mouse line inducing the highest Pax4 expression (Table 5, line 5). Thus, although Pax4 may promote a modest proliferation of β-cells in rat islet cultures (Brun et al., 2004), alternative mechanisms clearly act to boost the robust expansion of the β-cell mass consistently observed in POE::Pdx1cre, POE::Pax6cre and POE::Glucre mice (from hereon termed “double-transgenic animals”). One issue of concern to our approach was related to the overexpression of Pax4. However, through the use of different POE transgenic founder mice, we demonstrated that the ectopic expression of Pax4 at a dose lower than found in normal β-cells, is sufficient to induce a loss of the α-cell phenotype to the benefit of β-cell features (Table 5).
As lineage-tracing experiments demonstrated that glucagon-expressing cells were converted into cells displaying a β-cell phenotype, a depletion of α-cells was expected. However, we consistently detected clusters of glucagon+ cells in our double-transgenic mice (
The location of endocrine non-β-cells at one pole of the islet of Langerhans of double-transgenic animals was remarkable. This observation was confirmed through z-stack analysis of POE::Pdx1Cre, POE::Glucre and POE::Pax6cre whole islets stained with anti-glucagon antibodies (data not shown). Most importantly, these glucagon-labeled cell clusters were consistently found adjacent to the ductal lining (data not shown). The very detection of such glucagon+ cells was intriguing as their conversion into β-cells was expected upon Pax4 expression. A likely explanation may be the continuous generation of glucagon-labeled cells, their detection indicating a transitional state prior to their conversion into insulin-expressing cells. This hypothesis was supported by the active cell proliferation observed near and within the duct epithelium (
To test whether glucagon signaling deficiency may also induce the reactivation of Ngn3, in addition to POE::Glucre animals, glucagon receptor-deficient mice (characterized by an α-cell hyperplasia—Gelling et al., 2003) were analyzed. Combining Real-time RT-PCR and counts of immunostained sections (Table 3-4,
Thus far, our data suggested that the forced expression of Pax4 in either Pax6, Pdx1 or glucagon expression domains ultimately led to the development of oversized islets of Langerhans mostly composed of seemingly differentiated and functional β-cells. We reasoned that such cells might be capable of replacing lost β-cells in diabetic mice. Hence, POE::Glucre mice of different ages were injected with a single dose of the β-cell toxin streptozotocin (STZ), and both blood glucose levels as well as viability were monitored for 8 weeks (
In this study, we report that the forced expression of the Pax4 gene in the mouse endocrine pancreas results in oversized islets composed mainly of cells displaying a β-cell phenotype. Our findings are consistent with the induction of progenitor cells that adopt an α-cell identity as a consequence of decreased glucagon levels, and subsequently acquire β-cell features upon Pax4 ectopic expression, Ngn3 reactivation being instrumental in this processes. The resulting β-cells are functional at least at an early age, and can repopulate the islets of diabetic mice to normalize blood sugar levels.
The forced expression of Pax4, either in Pdx1+ pancreatic progenitor cells (and ultimately in all pancreatic cells) or in Pax6+ endocrine precursor cells (and ultimately in all islet cells) results in the nearly exclusive specification of cells exhibiting a β-cell identity at the expense of the α-, δ- and PP-cell lineages. At birth, these animals display normal pancreas morphology, unaffected exocrine tissue and well-sized islets of Langerhans, indicating that the main alterations observed are solely related to the allocation of the different endocrine cell lineages during embryonic development. This also suggests that Pax4 does not alter the pancreatic exocrine differentiation program, but rather acts on the specification of endocrine progenitor cells by promoting the acquisition of the β-cell fate. Interestingly, following birth, an age-dependent increase in islet size is evidenced in pancreata ectopically expressing Pax4 that was attributed to the continuous generation of cells displaying a β-cell phenotype. In an effort to ascertain the origin of such cells, lineage-tracing experiments were performed using POE::Glucre mice. Our initial goal was to trigger the ectopic expression of Pax4 in adult glucagon-producing cells, but an inducible glucagon-cre line is hitherto unavailable. Hence, the classical glucagon-cre mouse line (Herrera, 2000) was used to induce Pax4 expression in glucagon-producing cells. It is important to notice that during embryogenesis, the glucagon gene is initially expressed in early endocrine cells often co-expressing additional hormones, including insulin. However, by irreversibly tagging the progeny of cells using the Cre/LoxP system, Herrera (2000) demonstrated that mature glucagon- and insulin-producing cells do not derive from cells that previously expressed insulin or glucagon, respectively. Interestingly, lineage-tracing experiments performed in POE::Glucre mice revealed that the vast majority of newly formed β-galactosidase+/insulin+ cells originate from cells that previously expressed glucagon (note that endogenous β-galactosidase−/insulin+ cells remain detectable). Based on these results, but also on the age-dependent increase of β-galactosidase+ β-cell numbers and the concomitant decrease in α-cell contents, the dramatic expansion of the β-cell mass throughout the postnatal lifespan of double transgenic mice was attributed to a continuous neo-formation of β-cells through α-cell redifferentiation rather than to the slow self-renewal capacity of β-cells (Dor et al., 2004). Although our findings are in agreement with a putative neogenesis/conversion of somatostatin- or PP-positive cells into β-cells, testing this hypothesis will have to await the generation of somatostatin-cre and PP-cre mice to allow lineage-tracing experiments.
We demonstrate that glucagon supplementation reduces the β-cell hyperplasia in double-transgenic mice, most likely by compensating the deficiency in circulating glucagon resulting from the loss of α-cells through Pax4-induced redifferentiation. Interestingly, compromised glucagon signaling has previously been associated with α-cell neogenesis: both Glucagon receptor (Gcgr)- and prohormone convertase 2 (Pcsk2)-deficient animals display oversized islets, mainly composed of glucagon-producing cells (Furuta et al., 1997; Gelling et al., 2003). Similar to the present report, exogenous glucagon treatment significantly reduced the endocrine cell hyperplasia in Pcsk2-deficient animals (Blume et al., 1995; Petersson and Hellman, 1963; Webb et al., 2002). Pcsk2 mutants were also found to contain glucagon+ cells near the duct epithelium and their contribution to the neogenesis of the supernumerary glucagon+ cells was suggested. Accordingly, our findings indicate that, upon Pax4 ectopic expression, a physiologically significant glucagon deficiency activates a continuous compensatory response resulting in α-cell neogenesis, as seen in Gcgr and Pcsk2 mutant mice. However, these cells are subsequently converted into β-cells upon Pax4 ectopic expression. In animals ectopically expressing Pax4, but also in Gcgr mutants, a reactivation of Ngn3, a gene normally exclusively expressed during embryonic development of the pancreas and required for the endocrine differentiation program, was observed. However, due to the difficulties encountered to detect Ngn3 transcripts or protein, the determination of the origin of such Ngn3+ cell will require more elaborated lineage-tracing experiments and/or more specific antibodies/in situ probes. Interestingly, lentivirus-mediated cell tracing and knockdown experiments showed that Ngn3 re-expression is in fact crucial for the α-cell-mediated β-cell neogenesis. As important was a recent report demonstrating that, upon pancreatic duct ligation, facultative adult stem cells are activated along the lining of duct epithelium (Dor and Melton, 2008; Xu et al., 2008). It was also established that these cells reactivate Ngn3, differentiate into endocrine cells and contribute to the formation of oversized islets. In mice ectopically expressing Pax4, our results suggest that duct-lining cells may represent the source of the neogenerated glucagon-expressing cells. Although we cannot exclude the contribution of acinar cells to this process, additional evidence favor a duct-to-islet cell conversion mechanism: (1) the continuous detection of neo-generated glucagon+ islet cells adjacent to duct structures, (2) the active proliferation within the duct epithelium and near the islet pole where non-β endocrine cells accumulate, (3) the presence of glucagon+ endocrine cells in the ductal lining, and (4) the detection of cells expressing duct markers within the islet. Our findings are supported by recent reports demonstrating the plasticity of pancreatic cells. For instance, D. Melton and coworkers proved that acinar cells could be reprogrammed into β-cells upon the ectopic expression of selected genes in vivo (Zhou et al., 2008), whereas Inada et al. (2008) provided evidence that duct cells may give rise to endocrine and acinar cells in the adult pancreas. In agreement with these, our analysis underlines that hypoglucagonemia activates compensatory mechanisms provoking the conversion of progenitor cells into α-cells that subsequently acquire a β-cell phenotype upon Pax4 ectopic expression, these Ngn3-dependent processes ultimately leading to the generation of oversized islets of Langerhans.
Pax4 Ectopic Expression Rescues from Streptozotocin-Induced Diabetes.
We reasoned that the continuous β-cell neogenesis observed in double-transgenic animals might be able to rescue experimentally-induced diabetes. Therefore, the function of the newly formed β-cells was assessed in diabetic mice with more than 95% β-cell loss following STZ treatment. No insulin supplement was used to counter the sudden β-cell loss, as the consequences of exogenous insulin treatment on islet cells formation are hitherto unclear. Accordingly, a high lethality was expected, especially in older hyperglycemic and weakened mice. However, while all control and double transgenic mice older than 10 weeks died, 41% of the younger animals survived during two months post STZ injection. Closer examination revealed a progressive reconstitution of the islets and normalization of the glycemia and indicated that the new insulin+ cells behaved as true β-cells. Also under these conditions, clusters of glucagon+ cells were detected at one pole of the islets, adjacent to duct structures, such cells subsequently adopting a β-cell phenotype. Furthermore, the endocrine tissue in animals that died during the course of these experiments showed significant β-cell neogenesis, but, most likely, not sufficient to allow survival.
The observation that only double transgenic animals younger than four weeks of age could survive STZ treatment was intriguing. Interestingly, this age corresponds to the period when such animals, initially hypoglycemic, progressively develop a hyperglycemic condition. While the low blood glucose levels observed after birth can be easily explained by the β-cell hyperplasia, the development of a diabetic condition was unexpected: the steady increase in insulin-producing cell content and the reduced number of glucagon-expressing cells are in contrast with the observed high glucose levels. In the light of the data reported in this study, it appears that the newly-formed insulin-expressing cells are functional for at least four weeks following birth and respond normally to a glucose challenge. During this time, they express all the β-cell markers we tested and are negative for α-, δ- and PP-cell marker genes. However, glycemia, glucose tolerance, insulin secretion and insulin sensitivity deteriorate in older animals despite a normal expression of a typical β-cell complement of transcription factors, suggesting that older β-cells fail to trigger an optimal response upon glucose challenge. Interestingly, these retain some capacity to secrete insulin when challenged with arginine or a long-acting GLP-1 analog. The reasons for the establishment of such a condition are unclear, but may result from progressive (1) β-cell exhaustion, (2) β-cell alterations, (3) desensitization of the insulin receptor/pathway as a consequence of increased insulin levels, and/or (4) unknown effects induced by the decreased glucagon/somatostatin/PP hormone contents. Defining the mechanisms involved would require further work in which the impact of β-cell hyperplasia, but also of the decrease in α-, δ- and PP-cells contents, could be modulated and analyzed independently. It is interesting to notice that younger double-transgenics subjected to streptozotocin treatment display an extended lifespan in comparison to untreated counterparts (data not shown). This indicates that the development of the diabetic condition in these mice is not age-related, but may rather depend on the islet hyperplasia state. Clearly, using different activation times of exposure to Pax4 would allow us to determine its long-term effect on islet function. Together, our results suggest that the sole ectopic expression of Pax4 in glucagon+ cells can, in younger animals, reverse the consequences of streptozotocin-mediated diabetes through the induction of α-cell neogenesis and their ensuing conversion into functional β-cells. Based on these findings, we suggest that drug-mediated modulation of Pax4 and/or its targets may open new avenues for treatment of diabetes and, in addition, may contribute to strategies aiming to differentiate β-cells from stem, progenitor or other cell types.
The strategy used to generate the POE mouse line is depicted is
To assess the effects of glucagon on islet size, mice were injected intraperitoneally twice daily (every 12h) with 5 μg of glucagon and sacrificed after 3 weeks of treatment. For STZ-mediated diabetes induction, a freshly prepared 50 mg/ml solution in 0.1 mol/l sodium citrate, pH 4.5 was injected intraperitoneally (200 mg/kg). Lentivirus production and injection were performed as described previously (Xu et al., 2008). Lastly, BrdU was injected intraperitoneally (200 μl of 100 μl/ml) and was detected by immunohistochemistry (Invitrogen).
Tissues were fixed in 4% PFA overnight at 4° C., embedded in paraffin and 8-μm sections applied to slides. These sections were assayed as described previously (Collombat et al. 2003). In order to perform co-immunofluorescence, the GFP signal was bleached when necessary, as described (Collombat et al., 2007). The primary antibodies used were the following: mouse monoclonal anti-insulin, anti-glucagon ( 1/1000 —Sigma), anti-Ngn3 ( 1/2000—1F25A1B3, BCBC Antibody Core), anti-c-Myc ( 1/100 —Abcam); guinea pig anti-insulin, anti-glucagon ( 1/1000—Sigma); rabbit anti-somatostatin ( 1/600—Dako), anti-PP ( 1/200—Dako), anti-Nkx6.1 ( 1/3000), anti-Nkx2.2 ( 1/1000—kindly provided by T. Jessell), anti-Pax6 ( 1/500 Chemicon), anti-Pax4 ( 1/500), and anti-Arx ( 1/1000); chicken anti-β-galactosidase ( 1/500 Biozol); goat anti-Ngn3 ( 1/1000—kindly provided by M. Sander). The secondary antibodies ( 1/1000—Molecular Probes) used were: 594-alexa anti-mouse; 488-alexa anti-mouse; 594-alexa anti-rabbit; 488-alexa anti-rabbit; 594-alexa anti-guinea pig; 488-alexa anti-guinea pig. Pictures were processed using confocal microscopy. For quantification purpose, stained cells were counted manually on every tenth section and the count reported to the pancreatic area estimated in silico.
For glucose challenge tests, 6 mice per genotype were fasted for 24 h and injected intraperitoneally with glucose (2 g/kg) and blood glucose levels measured 0, 30, 60, 90, 120, 150, and 210 min afterwards. At each time point, one animal per genotype was sacrificed immediately after glycemia assessment for serum insulin level determination using RIA (Linco). Glucose levels (mg/dl) were determined with the One Touch Glucose monitoring kit (Johnson & Johnson).
All values are depicted as mean±standard error of the mean from at least three independent experiments and considered significant if p<0.05. All data were statistically analyzed by multivariate comparison (two-way ANOVA) with Bonferroni correction or one-way ANOVA with Newman-Keuls correction.
RNA isolation (RNAeasy, Qiagen) and cDNA synthesis (Supercript choice system, Invitrogen) were performed according to the manufacturer's instructions. cDNA synthesis for analyses aiming to detect Pax4 transcripts was performed using a mixture of eight Pax4-derived (and β-actin-derived) antisense oligonucleotides instead of the provided oligo-dT primers (
Quantitative RT-PCR were carried out using the QuantiTect SYBR Green RT-PCR Kit (Qiagen) and validated primers (Qiagen) according to the manufacturer's instructions. The PCR reactions and detection were performed on a Mastercycler® ep realplex cycler using GAPDH and HPRT1 as internal controls for normalization purposes.
A 5566-bp long fragment of the Ngn3 promoter spanning from position −5286 to +280 relative to transcription start site was amplified by PCR from a genomic lambda clone containing the mouse Ngn3 locus using the high fidelity Phusion DNA polymerase (FINNZYMES) with the following primers: pNgn3 Fw=5′ CACCAAGCTTTGTGTGGAAGGAAATGTC and pNgn3 Rv=CGCGCGCCCCTCATCCACCCTTTG. The resulting PCR product was cloned into the pENTR/D-Topo plasmid (Invitrogen) and the insert sequenced to rule out PCR-mediated mutations. The resulting plasmid was used to perform LR Clonase II (Invitrogen) in vitro recombination in order to insert the Ngn3 promoter fragment upstream of either DSRed2::c-Myc or eGFP reporters in the pTrip deltaU3 lentiviral backbones. The lentiviral vector stocks were produced as described before (Castaing 2005).
The lentiviral vector expressing eGFP under the control of the 5566-bp long fragment of the Ngn3 promoter was injected into the perivitelline space of mouse fertilized eggs as described previously (Lois et al. 2002). The injected eggs were next re-implanted into pseudo pregnant females and the resulting embryos isolated 14.5 days afterwards. They were genotyped with eGFP specific primers (Fw=5′ GACGTAAACGGCCACAAGTTC 3′ and Rev=5′ GTCGCCCTCGAACTTCACCTC 3′) and fixed in 4% PFA overnight, cryoprotected in 15% sucrose PBS and frozen. Cryo-sections were stained for eGFP (Castaing et al., 2005) and Ngn3 (Zahn et al., 2004), and counterstained with DAPI.
For RNA in situ hybridization, pancreata were collected, fixed, incubated overnight in 30% sucrose, embedded in cryomatrix, and 18-μm sections were applied to Probe-on Plus slides (Fisher Scientific). Defrosted sections were hybridized overnight with DIG-labeled probes in a medium containing 50% formamide, 10% dextran sulphate, 1 mg/mL yeast tRNA, 0.02% BSA, 0.02% Ficoll, and 0.02% PVP. Tissues were washed successively in 50% formamide, 1×SSC, 0.1% Tween-20 at 70° C. for 70 min, and MABT at pH 7.5 (100 mM maleic acid; 150 mM NaCl; 0.1% Tween-20) at room temperature for 1 h and blocked in PBS containing 20% inactivated fetal calf serum for 90 min. Anti-DIGantibody (1:2500) was applied overnight in the same solution at room temperature. Tissues were washed thoroughly in MABT for 2 h, rinsed with NTMT (100 mM NaCl; 100 mM Tris-HCl at pH 9.5; 50 mM MgCl2; 1% Tween-20) for 1 h, stained in a solution containing 350 μg/mL NBT, 175 μg/mL BCIP, rinsed in PBS, and fixed in 4% paraformaldehyde.
Animals of the indicated genotypes were injected subcutaneously with Actrapid insulin (0.75 mU/g—Novo Nordisk—Denmark), intravenously with arginine (5 mg—Sigma) or subcutaneously with liraglutide (200 micrograms per kg—Novo Nordisk—Denmark). Following insulin challenge, blood glucose levels were measured as described in the main manuscript. Next to arginine challenge, at each time point, one animal per genotype was sacrificed immediately for serum insulin level determination using RIA (Linco). Lastly, in the case of liraglutide, the latter was injected 30 min prior to glucose challenge. At each time point, three animals per genotype were sacrificed immediately for serum glucose and insulin level determination.
A quantitative endocrine marker analysis was performed on pancreata of the indicated genotypes and ages. Using immunohistochemical detection, hormone-expressing cells were counted on every tenth section of pancreas and reported to the pancreatic area estimated in silico. The values presented correspond to the averages of hormone-producing cells per square centimeter (also reported in
A quantitative endocrine marker analysis was performed on sections of pancreata of the indicated genotypes and ages using immunohistochemical detection, hormone-expressing cells were counted on every tenth section of pancreas and reported to the pancreatic area estimated in silico. The values presented correspond to the averages of positive cells per square centimetre of pancreas (n>3, P<0.05, except when labeled with U, Unchanged). Note the loss of α-cells and the concomitant increase in the number of β-galactosidase+ insulin-producing cells and associated markers (Nkx6.1, Pdx1, Glut2, Isl1, HB9) in double transgenic animals, concurrently with an increase in the total endocrine cell number.
The expression of the indicated genes was assayed in pancreatic tissues of POE::Glucre, POE::Glucre supplemented with glucagon, and glucagon-receptor mutant (GluR-KO) mice using age-matched tissues of WT and POE pancreata as reference (values set at 100%). POE::Glucre, but also GluR-KO pancreata display increased contents of Ngn3 and Reg3b transcripts. Additionally, an augmentation in β-cell-specific transcripts is outlined in POE::Glucre, whereas the expression of the α-cell marker genes is reduced Importantly, concomitantly with a significant decrease in Ngn3 and Reg3b transcript contents, a reduction in insulin- and glucagon-producing cell numbers (and associated marker genes) is evidenced in POE::Glucre animals supplemented with glucagon. All values are statistically significant (P<0.05, n>2) versus POE/WT.
The counts in cells positive for the indicated genes are reported per square centimetre of pancreatic tissues of POE::Glucre, POE::Glucre supplemented with glucagon, and glucagon-receptor mutant (GluR-KO) mice using age-matched tissues of WT/POE pancreata as reference. It is worth noticing that, in animals ectopically expressing Pax4 in glucagon expression domains or in GluR-KO mice, Ngn3-producing cells reemerge (note for the latter that due to the bias introduced by the high background, this count should be considered as indicative). However, their content becomes significantly reduced in POE::Glucre mice supplemented with glucagon. In the latter case, a concomitant decrease in BrdU-labelled proliferating cell numbers is highlighted (BrdU provided at 5 weeks of age, examination a week later). (n>2, **P<0.01, *P<0.05 using the POE/WT genotype as reference; ##P<0.01, #P<0.05 using the POE::Glucre genotype as reference).
+457%
+339%
In order to verify a putative relationship between phenotypic alterations and Pax4 expression levels, we crossed the five different POE founder lines with Glucre animals. A similar outcome was observed in all cases, resulting in an increase in the insulin-expressing cell content at the expense of the glucagon-producing cell number. Using a combination of GFP intensity examination and real-time PCR analysis, different levels of Pax4 expression were highlighted in these lines (see thereafter), most probably due to distinct chromosomal integration loci of the transgene. In 3-week-old wild-type mice, Pax4 is normally produced in β-cells representing 68.21% of endocrine cells. In age-matched double-transgenics, a quantitative analysis demonstrated an expression of Pax4 in 93.35% of islet cells. Hence, we postulated that, if Pax4 was to be expressed at the same physiological dosage in double transgenic pancreata as in wild-type, the global Pax4 pancreatic dosage found in controls should then be relatively increased 1.37-fold (93.35/68.21) in double-transgenic pancreata. However, one also needs to factor in the total endocrine cell number increase upon expression of Pax4 in α-cells. A careful quantitative analysis demonstrated a 3.67 augmentation of the islet cell count between controls and double-transgenic animals at that age. Thus, the expected increase in Pax4 dosage, assuming a similar cellular Pax4 concentration across genotypes, should be of 5.02 (1.37×3.67). Interestingly, in two of our transgenic lines (lines 1 and 4, in bold-face and underlined), the global pancreatic increase of Pax4 expression was found lower than this threshold with values of 4.57 and 3.39, respectively. However, even in these animals, severe alterations in islet cell content were evidenced, indicating that, not only the overexpression of Pax4, but also its ectopic expression in glucagon-producing cells, is able to induce a loss of the α-cell phenotype at the profit of β-cell features. All reported values are statistically significant (P<0.05, n=3) and were further confirmed by immunohistochemistry (data not shown).
Animals of POE::Glucre genotype were infected with lentiviruses containing a shRNA either targeting Ngn3 production or a scrambled version. A clear decrease in Ngn3 and Reg3b transcript contents and Ngn3+ cell counts was observed two weeks post-infection (note for the latter that due to the bias introduced by the high background, this count should be considered as indicative). Concurrently, insulin- or glucagon-expressing cells as well as insulin or glucagon transcript contents were found significantly reduced.
Islets of 4- and 12-week-old POE::Glucre mice were isolated by handpicking, following collagenase digestion (0.3 mg/ml) of the pancreata, to study their secretory capacity in a glucose perifusion assay as described by Stangé et al. (2003). Unfortunately, we failed to detect Pax4 ectopically expressing oversized islets, suggesting that these were overdigested, presumably because of differences in cell-cell adhesion, known to be crucial for islet structure and function, between control and double transgenic mice (Rogers G J et al., Cell Physiol Biochem, 2007; Caton D et al, Diabetes Metab, 2002; Pipeleers, Experientia, 1984). Obviously, a decrease in digestion time/collagenase concentrations would not have permitted proper islet isolation. We anyway perifused the islets and observed no difference in islet function of younger animals for all the genotypes analyzed. However, in older counterparts, we did detect a decrease in the glucose-stimulated insulin release, albeit not significant. It should be noticed that such a lack of significance might be attributed to the loss of oversized islets induced by experimental conditions.
| Number | Date | Country | Kind |
|---|---|---|---|
| 09 166 951.5 | Jul 2009 | EP | regional |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13387018 | May 2012 | US |
| Child | 14570874 | US |
