Patent Application: US-25715502-A

Abstract:
a transgenic diabetes type ii model laboratory animal is disclosed which comprises β - cells expressing a dominant negative form of fgfr1c . also disclosed is the use of the ipf1 / pdx1 promoter for controlling expression of fgfr1c ; β - cells in which the expression of pc1 / 3 is down - regulated or absent or which are competent to express a dominant negative form of fgfr1c ; mature β - cells incompetent to express glut2 or in which the processing of proinsulin is substantially impaired ; a method of preventing or treating type ii diabetes .

Description:
analysis of fgfr1 expression in the adult mouse pancreas revealed that fgfr1 is predominantly expressed in the adult β - cell , with no expression observed in the glucagon - producing α - cells ( fig1 ). a lower level of fgfr1 expression was also observed in the exocrine cells of the pancreas ( data not shown ). fgfr2 is also selectively expressed in the adult β - cells but in contrast to fgfr1 , fgfr2 expression was not observed in the exocrine cells of the adult pancreas ( data not shown ). the expression of fgfr in adult β - cells suggests a role for fgf - signalling in terminal differentiation and / or maturation of these cells . the expression of fgfr1 in the pancreas led us to examine whether signalling via this receptor may be required for pancreas development , as has already been implied for fgfr2b . we also examined whether fgf receptor signalling was required for the specification and differentiation of adult β - cells . to begin to address this issue we generated transgenic mice expressing dominant negative fgfr1c construct in the pancreas using the ipf1 / pdx1 promoter . this consisted of the three loop extracellular domain of fgfr1c fused in frame with the rat igg fc region . in the resulting mice ipf1 - expressing cells would secrete the hybrid protein from the cell allowing competitive binding of fgf ligands such as fgf1 , fgf2 , fgf4 , fgf5 and fgf6 which are known to bind to the fgfr1c variant . ipf1 / dnfgfr1c express high levels of the transgene in β - cells with lower levels of the transgene being expressed in the exocrine part of the pancreas ( fig1 and data not shown ). the ipf1 / dnfgfr1c transgenic mice are viable and fertile with a grossly well developed pancreas , providing evidence that signalling via fgfr1c is not important for pancreas growth , morphogenesis and differentiation . the mice appeared healthy until approximately 15 weeks of age when elevated non - fasting urine glucose concentrations greater than 2 % were observed suggesting a diabetic phenotype . fasting blood glucose measurements on mice were & gt ; 20 mm , confirming that these animals were severely diabetic . close monitoring of urine and blood glucose levels revealed that already at 3 weeks of age ipf1 / dnfgfr1c mice showed elevated glucose levels , albeit still within the normal range ( table 1 ). at six weeks of age their fasting blood glucose levels had increased by 1 mm and they had detectable levels of glucose in their urine when compared to wild type age matched littermates ( table 1 ). weekly monitoring of the urine glucose revealed a steady increase in glucose and by the age of 9 – 12 weeks urine glucose levels were in excess of 2 %. non - fasting and fasting blood glucose measurements taken at this stage revealed 4 - fold higher glucose levels compared with age - matched wild type littermates ( table 1 ). these findings demonstrate that impaired fgfr1c - signaling in adult β - cells results in the development of diabetes and points to a crucial , hitherto unknown role for fgf - signalling in β - cell glucose homeostasis . legend to table 1 : blood glucose levels were measured in ipf1 / dnfgfr1c mice and wild type littermates at the time points shown . at 3 weeks of age ipf1 / dnfgfr1c mice showed non - fasted blood glucose levels within the normal range . six - week old ipf1 / dnfgfr1c mice had slightly elevated non - fasted blood glucose levels , still within the normal range albeit at the upper level . overt diabetes ( od ) develops in 9 – 12 weeks old mice in whom both non - fasted and fasted blood glucose levels were 4 - fold higher than wild type , age - matched littermates . the overtly normal development of the pancreas in ipf1 / dnfgfr1c mice homozygous mutants suggested that the growth and differentiation of the pancreas is independent of fgfr1c - signaling . to assess this further we analyzed the expression of the transcription factors isl1 , ipf1 / pdx1 , nkx6 . 1 and nkx2 . 2 , the endocrine hormones insulin ( ins ), glucagon ( glu ), somatostatin ( som ), and the exocrine enzymes amylase and carboxypeptidase a . each of these markers were expressed in the pancreas of ipf1 / dnfgfr1c mice and the organization of endocrine cells into islet - like clusters and of exocrine cells into acinar - like structures appeared normal ( data not shown ). nevertheless , as revealed by double immunohistochemical analysis , there was a 35 % decrease in the total number of isl1 + cells paralleled by ˜ 30 % net decrease in the number of ins + - cells and a concomitant 20 % increase in the relative number of glu + - cells ( fig2 ). these results suggest that the genesis and / or survival of β - cells partly depends on fgfr1c - signalling . tunnel assays failed , however , to detect any increased β - cell apoptosis suggesting that the decreased number of β - cells are not caused by β - cell death ( data not shown ). the 30 % decrease in total number of insulin cells in the transgenic mice was reflected by a 28 % decrease in total pancreatic insulin content ( fig2 ). moreover , although islets form in the ipf1 / dnfgfr1c mice the typical structure of maturing islets with α - cells at the periphery surrounding a core of β - cells is perturbed ; instead , glu + cells are found scattered throughout the islets ( fig3 ). in combination these histological analyses support the idea that the development of the pancreas to generate both exocrine and endocrine cells is unaffected despite the expression of a dn form of fgfr1c during pancreas development and in the adult β - cell . in addition these results provide evidence of the genesis of pancreatic β - cells appearing to be partly dependent on , and that normal organization of islet - cells requires , fgfr1c - signaling . the decrease in total insulin production by 28 % appears unlikely to be sufficient to cause the diabetes observed in the transgenic mice and suggests that additional complications underlie the development of the diabetic phenotype observed in the ipf1 / dnfgfr1c mice . to determine whether other key characteristics of the adult β - cells were affected in the transgenic mice we next monitored the expression of factors crucially required for normal glucose homeostasis . glut2 is a key component in glucose sensing machinery within the β - cell . analyses revealed that the expression of glut2 was virtually lost in overt diabetic ipf1 / dnfgfr1c mice ( fig3 ). to exclude that the loss of glut2 expression was a consequence of the hyperglycemic state rather than a direct effect of the ipf1 / dnfgfr1c transgene expression , 5 - week old , prediabetic transgenic mice were analyzed . prediabetic , 5 week old ipf1 / dnfgfr1c mice exhibit a clearly reduced level of glut2 expression as compared to wild type ( not shown ). these results indicate that the reduction of glut2 expression observed in ipf1 / dnfgfr1c mice is a direct consequence of impaired fgfr1c signalling . although the loss of glut2 may be sufficient to cause their diabetic phenotype , the severity of the ensuing hyperglycemia in a short period of time suggested that there might be additional defects in the β - cell of ipf1 / dnfgfr1c mice . type 2 diabetes patients and animal models of the disease , often suffer from hyperproinsulinemia , reflecting an impaired processing of proinsulin to mature , active insulin which is believed to be a major contributing factor to their disease . to elucidate a potential processing defect in the ipf1 / fgfr1dn mice we performed an immunohistochemical analysis for investigating the expression of the proinsulin processing enzymes , pc1 / 3 and pc2 . pc1 / 3 expression was found to be severely down - regulated so as to be virtually absent in the ipf1 / dnfgfr1c mice , whereas only a minor decrease was observed with respect to pc2 expression in overt diabetic mice ( fig3 and data not shown ). next we wanted to determine whether this aberrant processing enzymes expression could be directly involved in the development of the diabetic phenotype in the ipf1 / dnfgfr1c mice . to this effect pancreas from five - week old prediabetic and wild type littermates were analyzed for pc1 / 3 and pc2 expression . the analyses showed that already at 5 - weeks of age expression levels of the prohormone convertases was reduced in the ipf1 / dnfgfr1c mice as compared to controls ( not shown ). together , these results suggest that fgfr1c signaling is required for high level expression of pc1 / 3 , and to a lesser extent for pc2 expression . to address whether the impaired expression of the processing enzymes might affect insulin processing in the ipf1 / dnfgfr1c mice we examined the type of insulin made and stored in the β - cells . an antibody directed against intact human proinsulin , which do not cross - react with active insulin ( madsen et al ., 1983 ; madsen et al ., 1984 ; furuta et al ., 1998 ), and antibodies directed against mouse c - peptide 1 and 2 ( blume et al ., 1992 ) were used to evaluate the forms of insulin present in the β - cells of ipf1 / dnfgfr1c mice . there was a marked reduction in the levels of both c - peptide 1 and c - peptide 2 in the β - cells of the overt diabetic transgenic mice as compared to β - cells of wild type mice ( fig4 ). in contrast , high levels of proinsulin was observed in the β - cells of the transgenic mice while no or very little proinsulin was observed in the wild type β - cells ( fig4 ). notably increased proinsulin levels were manifest already at the prediabetic 5 - week stage ( data not shown ). these results indicate that the down regulation of the processing enzyme , pc1 / 3 in the ipf1 / dnfgfr1c mice results in an impaired processing of proinsulin to its active mature form . this processing defect is likely to contribute to the development of severe diabetes in the ipf1 / dnfgfr1c mice . thus impaired signaling via fgfr1c in the adult β - cell leads to both aberrant glucose sensing and impaired insulin processing that together ultimately progress to diabetes development . the disorganisation of islets and the down - regulation of glut2 observed in the ipf1 / dnfgfr1c mice resembles the islet phenotype associated with a β - cell specific activation of the transcription factor ipf1 / pdx1 demonstrated in the rip1 δ / ipf1 mice previously established in our laboratory ( ahlgren et al ., 1998 ). these mice develop diabetes at 10 – 15 weeks of age due to a cre - mediated excision exon 2 of the ipf1 / pdx1 gene . analysis of pancreas derived from rip1 δ / ipf1 mice showed that the expression of fgfr1 and fgfr2 was down - regulated in the β - cells ( fig5 and data not shown ). some residual expression could still be observed which probably reflects a residual ipf1 expression in some of the β - cells , since these mice develop diabetes at a stage when approximately 20 % of the β - cells still express ipf1 ( ahlgren et al ., 1998 ). to investigate whether the decrease in fgfr1c expression in the rip1 δ / ipf1 mice might lead to a perturbed insulin processing we performed an analysis of the expression of the prohormone convertases in the rip1 δ / ipf1 mice . pc1 / 3 was severely down - regulated in these mice as well ( fig5 ). the rip1 δ / ipf1 mice also displayed an increased proinsulin content in their β - cells coupled with a decreased levels of c - peptide 1 and 2 ( fig5 , and data not shown ). in combination these results provide evidence that ipf1 is required for the expression of fgfr1 in the adult β - cells , thereby ensuring a high level of glut2 and pc1 / 3 expression . thus , both perturbed ipf1 expression and impaired signalling via fgfr1c leads to diabetes due to impaired glucose sensing and insulin processing in the adult β - cells with diabetes manifestation as a consequence . these results suggest that signalling via fgfr1c is critical for the maintenance of a mature , functional β - cell phenotype , and that ipf1 / pdx1 by virtue of its key role in controlling , directly or indirectly , many aspects of the β - cell glucose homeostasis machinery is pivotal for the β - cell &# 39 ; s capacity to preserve normoglycemia . double immunohistochemical analyses of pancreas derived form non - diabetic humans show that both fgfr1 and fgfr2 are selectively expressed also in human insulin - producing β - cells and not glucagon producing α - cells ( fig6 a and 6 b ). similar to mice with diabetes due to impaired fgfr1 - signalling [ 5 , 6 ], type 2 diabetics display disorganized islets with glucagon - producing α - cells mixed with the insulin - producing β - cells ( fig6 d ), whereas non - diabetics have normal islets with glucagon - producing cells surrounding the core of insulin - producing β - cells ( fig6 c ). moreover , the expressions of both fgfr1 and fgfr2 are drastically reduced in the insulin - producing β - cells of type 2 diabetics as compared to that of control individuals ( fig7 ). consequently , as has been shown for mice with impaired fgfr1 signalling , type 2 diabetics show reduced expression of pc1 / 3 in their β - cells ( fig8 b , 8 e ) whereas the expression of pc2 appear less affected ( fig8 a , 8 d ). the reduction of pc1 / 3 expression is paralleled by an increase in β - cell proinsulin content in the type 2 diabetics as compared to the control individuals ( fig8 c , 8 f ). these experiments demonstrate that β - cells of type 2 diabetic patients have disorganized islets , reduced expression of fgfr1 , fgfr2 and pc1 / 3 as well as an increased proinsulin content in their β - cells . these findings when combined provide evidence that , similar to mice with diabetes due to an impaired fgfr1 - signalling in adult β - cells , attenuation of fgfr1 - signalling pathway in human β - cells is coupled to diabetes . we suggest that fgf - signalling in the adult pancreas ensures a functional β - cell identity and glucose homeostasis . thus an impaired expression , or activity , of components within the fgf - signalling pathway is coupled to diabetes in both mice and humans . in both mice and humans impaired fgf - expression and signalling is coupled to a decrease in pc1 / 3 expression . the decrease in pc1 / 3 in turn leads to a perturbed processing of insulin with elevated pro insulin levels as a result . consequently the fgf - signalling pathway , i . e . including components upstream and downstream of the fgfr - ligand interaction , is a suitable target for the development of new therapies to cure diabetes . we have also recent data from analyses of expression of id - proteins [ norton , 2000 ] that id2 and id3 are targets downstream of fgfr1 - signalling . as is shown in fig9 , both id2 and id3 are normally expressed in pancreatic endocrine cells ( fig9 a , 9 d ) . however , in mice with diabetes due to attenuation of fgf - signalling in β - cells , i . e . the frid1 and rip1 / ipf1 δ mice , the expression of both id2 ( fig9 b , 9 c ) and id3 ( fig9 e , 9 f ) are severely down - regulated . these results provide evidence that id2 and id3 expression in β - cells are dependent of fgf - signalling and hence represent downstream components of the fgf - signalling pathway in adult β - cells . id2 and id3 thus represent candidate targets for the development of new therapies to cure diabetes . fig1 : ( a – f ) analysis of fgfr1 expression in adult pancreas showing that fgfr1 - expression ( b , d ) coincides with insulin ( c ) but not glucagon ( a ) expression . ( e , f ) in ipf1 / dnfgfr1c transgenic mice the dnfgfr1c protein ( f ) is highly expressed in ipf1 + cells ( e ) as detected by antibodies against the rat igg fc region that is coupled to the dnfgfr1c domain . fig2 : ( a ) analysis of number ins + - cells over number of isl1 + - cells showing a 30 % decrease in the number of ins + - cells in ipf 1 / dnfgfr 1 c ( tg ) mice as compared to wild - type ( wt ) mice . data from at least 4 independent mice , n = 7435 cells . ( b ) measurement of total pancreatic insulin content ( μg insulin / mg pancreas protein ) from non - fasted animals ( n = 6 wt and n = 8 tg ) show that the 30 % decrease in number of ins + - cells ( a ) is accompanied by a 28 % decrease in total pancreatic insulin content in the ipf1 / dnfgfr1c mice as compared with their age - matched wild type littermates . ( c ) the 30 % decrease in number of insulin cells results in a relative 20 % increase in number of glu + - cells in ipf 1 / dnfgfr 1 c mice . data from at least 4 independent mice , n = 7525 cells . fig3 : ( a – d ) confocal microscopy analyses of insulin ( c , d ) and glucagon ( a , b ) expression in wild - type ( a , c ) and ipf 1 / dnfgfr1c transgenic ( b , d ) mice show that the islet organisation in the transgenic mice is abnormal but that there is no co - expression between insulin and glucagon within the islet cells . ( e – h ) expression of glucose sensing and insulin processing enzymes is impaired in ipf1 / dnfgfr1c mice . images show that glut2 - expression ( e , f ) in adult β - cells is lost as a result of ipf1 / dnfgfr1c expression and that only a very low level of pc3 expression ( g , h ) remains in ipf1 / dnfgfr1c mice , and preferentially in α - cells . fig4 : analyses of insulin variants in pancreas from wild - type ( a , c , e , g ) and ipf1 / dnfgfr1c ( b , d , f , h ) mice using c - peptide 1 ( a and b ), c - peptide 2 ( c and d ), insulin ( e and f ) and proinsulin ( g and h ) anti - sera . ( a , c , e , g ) in wild - type pancreas insulin is present predominantly in its fully processed form ( a , c , e ) with virtually no detectable unprocessed pro - insulin ( g ). note that the fluorescence in c represents background , non - β - cell reactivity due to the use of mouse monoclonal anti - sera . ( b , d , f , h ) ipf1 / dnfgfr1c mice display a perturbed proinsulin processing resulting in reduction of fully processed insulin ( b , d , f ) while a substantial , readily detectable fraction remains in the form of proinsulin ( h ). fig5 : ( a – f ) loss of ipf1 / pdx1 activity in β - cells ( ahlgren et al . 1998 ) results in a drastically reduced fgfr1 ( a , b ) and pc3 ( c , d ) expression . this loss of pc3 expression results in perturbed proinsulin processing , with a reduction in detectable levels of fully processed insulin ( not shown ) accompanied by increased levels of detectable proinsulin ( e , f ) within the β - cells of the rip1 / ipf1 δ mice ( b , d , f ) as compared to wild - type littermates ( a , c , e ). fig6 : expression of fgfr1 and fgfr2 in adult human islets . confocal microscopy analyses of fgf receptor and glucagon expression in adult human pancreas showing that the receptors fgfr1 ( a ) and fgfr2 ( b ) are expressed in □- cells and not in β - cells . the islets of human type 2 diabetic patients ( n = 3 ) ( d ) are disorganized with glucagon producing cells found scattered throughout the islets , as compared with non - diabetic pancreatic tissue where the glucagon cells are found at the periphery of the islet ( c ). fig7 : impaired expression of fgfr1 and fgfr2 in human type 2 diabetic patients . immunohistochemical analyses demonstrate that both fgfr1 ( a and c ) and fgfr2 ( b and d ) are expressed at clearly reduced levels in β - cells of patients ( n = 3 ) suffering from type 2 diabetes . non - diabetic pancreatic tissue ( a and b ), diabetic pancreatic tissue ( c and d ). fig8 : impaired expression of pc1 / 3 in human type 2 diabetic patients . immunohistochemical analyses show that pc2 ( a and d ) expression is unaffected in human type 2 diabetic patients ( n = 3 ), whereas expression of pc1 / 3 ( b and e ) is drastically reduced . the down - regulation of pc1 / 3 expression leads to functional impairment of insulin processing as revealed by the increase in intracellular proinsulin content ( c and f ). non - diabetic pancreatic tissue ( a – c ), diabetic pancreatic tissue ( d – f ). fig9 : id2 and id3 are downstream of fgfr1c - signalling . id2 and id3 down - regulated in β - cells of overt diabetic frid1 and rip1 / ipf1 δ mice . both id2 and id3 are normally expressed in mouse adult islets cells ( a , d ). the expression of id2 ( a – c ) is greatly reduced in β - cells of diabetic frid1 ( b , e ) and rip1 / ipf1 δ ( e , f ) mice as compared to that of wild type littermates and the expression of id3 ( d – f ) virtually absent ( e , f ) in these diabetic mouse models . the remaining id2 and id3 expression still observed in both the frid1 and rip1 / ipf1 δ islets represent expression in scattered glucagon cells . ahlgren u , jonsson j , jonsson l , simu k , edlund h ( 1998 ). β - cell - specific inactivation of the mouse ipf1 / pdx1 gene results in loss of the beta - cell phenotype and maturity onset diabetes . genes dev 12 , 1763 – 8 . apelqvist a , ahlgren u , edlund h ( 1997 ). sonic hedgehog directs specialised mesoderm differentiation in the intestine and pancreas . curr biol 7 : 801 – 4 . blume n , petersen j s , andersen l c , kofod h , dyrberg t , michelsen b k , serup p , madsen o d . 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