The overall aim of this thesis is to look into the ordinance of beta cell mass in grownup mice in vivo. We employ a & A ; acirc ; ˆ?switchable transgenic mouse theoretical account pIns-c-MycERTAM, in which controlled beta cell extirpation and regeneration can be initiated and followed accurately across clip. This strain has been crossed with different mice transporting cistron smashers and or other transgenes in order to carry on functional surveies of beta cell mass ordinance and regeneration in vivo. Specifically, to research the function of IGF-II in beta cell regeneration pIns-c-MycERTAM mice were crossed with IGF-II knock outs. To look into the function of PML in beta cell programmed cell death, reproduction and tumor formation, pIns-c-MycERTAM and pIns-c-MycERTAM/Bcl-xL dual transgenic mice were crossed with PML knock outs. Cardinal consequences from these surveies and restrictions are discussed in the undermentioned subdivisions.
6.1.1 Impact of IGF-II Loss in Regulating Beta Cell Mass
There are multiple mechanisms involved in beta cell reclamation. Many attempts have been devoted to place those growing factors which can potentially advance and back up beta cell regeneration. However, the cardinal signals triping beta cell reproduction and regeneration still stay to be discovered. The IGF household has drawn a batch of attending in the recent decennaries as has been discussed in item in the debut. IGF-II is widely expressed during murine embryologic development whereas the look degree beads dramatically postnatally ( D’Ercole, Applewhite et Al. 1980 ; Moses, Nissley et Al. 1980 ; Brown, Graham et Al. 1986 ; DeChiara, Robertson et Al. 1991 ) . In the grownup, Igf2 loss of forming ( LOI ) and reactivation have been reported in human malignant neoplastic disease ( Kaneda and Feinberg 2005 ; Cui 2007 ) , and in beta cell derived malignant neoplastic diseases in mice ( Christofori, Naik et Al. 1994 ) . Taken together one can adumbrate that IGF-II is of import in modulating pancreatic beta cell mass during growths, might be usefully deployed therapeutically in the grownup, and is functionally of import in beta cell neoplasia ( Christofori, Naik et Al. 1995 ) . However, whether endogenous IGF-II plays any function in regeneration of murine beta cells in the normal grownup is non known yet and forms the footing of this survey. Here for the first clip we exploit one of the new conditional beta cell extirpation theoretical accounts to turn to this inquiry in vivo.
In this survey we investigated IGF-II re-expression in pancreas. Numerous surveies in different theoretical account systems highlight the similarities between regeneration, neoplasia and growth. By triping c-Myc, beta cells are killed, raising the possibility that such hurt may ensue in the reactivation of a developmental plan which could in turn support regeneration later. We have shown, for the first clip that IGF-II is re-expressed after beta cell hurt in vivo. We chose to analyze this at an early clip point ( after 48 hours of Myc activation ) , as clear beta cell hurt and programmed cell death is present but islet mass is still reasonably maintained, maximising the opportunity of observing alterations in low copiousness messenger RNA for IGF-II. We detected IGF-II messenger RNA during 24hr and 48hr beta cell hurt in IGF-II wildtypes but non in IGF-II KO mice ( Figure 3.3.2d ) . Quantitative real-time PCR was performed to corroborate and quantify the IGF-II re-expression from RT-PCR informations. The consequences confirmed reexpression of IGF-II messenger RNA in IGF-II wildtype pancreas after 24hr c-Myc activation ( Figure 3.3.2d ) . In IGF-II KO mice no look was detected after either 24hr or 48hr beta cell hurt.
In order to place the beginning of freshly re-expressed IGF-II, we extracted RNA from islets isolated following Myc activation in both wildtype and IGF-II void mice following beta cell hurt. Again early clip points were needed as it has proven hard to insulate Myc-activated islets after 24 hours because of crumbliness through loss of cell adhesion molecule look. IGF-II look was non detected in islet messenger RNA. It is possible that the clip point chosen was excessively early to observe IGF-II re-expression. However, it is every bit possible that IGF-II re-expression takes topographic point in the exocrine pancreas or other site without the islets. Unfortunately, neither IHC nor Western blotting has proved successful, due to the deficiency of nice effectual antibodies against IGF-II. Future surveies could turn to this inquiry by agencies of in situ hybridisation.
As mentioned in Chapter 1.2.2, IGF-II can adhere to IGF-IR/IR to trip IRS/PI3K/Akt every bit good as Ras/Raf/MAPK tract to trip cell proliferation, growing and supply endurance signals ( Baserga et al. , 1997 ; Franke et al. , 1997 ; Kulik et al. , 1997 ; Parrizas et al. , 1997 ) . Given the re-expression of IGF-II after hurt in grownup pancreas, we hypothesized that IGF-II might be a critical factor in the successful recovery of beta cell mass in grownup mouse islets after hurt.
To look into the function of IGF-II in modulating beta cell mass, we activated c-Myc in IGF-II wildtypes and KO mice for 11 yearss. Leting the mice to so retrieve for 4 yearss or 3 months, we examined the beta cell mass and Numberss. Previous surveies have suggested that IGF-II KO mice have 40 % less organic structure weight than normal mice at birth ( DeChiara, Robertson et Al. 1991 ) . We can corroborate these findings here: IGF-II KO mice have fewer beta cells and a lower beta cell mass compared with IGF-II WT mice. Most of this difference can be accounted for by the little body/pancreas weight/size of the IGF-II KO mice, because, the cross sectional country entirely, ( which is non multiplied by pancreas/body weight ) are really similar at twenty-four hours 0 ( before c-Myc activation ) in both strains ( See Appendix 2 ) . As expected, c-Myc activation leads to a big grade of beta cell extirpation in both IGF-II wildtypes every bit good as in KO pancreas. However, beta cell mass quantification suggested IGF-II wildtypes may endure more beta cell extirpation as we observed similar beta cell mass after extirpation in both strains. To look into the inside informations of beta cell regeneration after 4 yearss or 3 months c-Myc inactivation in IGF-II WT and KO mice, beta cell mass/numbers at the extirpation clip point ( c-Myc activation for 11 yearss ) was set as a starting line for comparing. After 4 yearss recovery both beta cell figure and beta cell mass have increased significantly in the IGF-II wildtype mice, whereas this is non seen in IGF-II smasher mice. Furthermore, after 3 months following Myc inactivation, although both strains achieved recovery, IGF-II wildtypes systematically increased more beta cell mass/numbers than IGF-II KO mice. And in both strains such beta cell mass/numbers recovery was sufficient to command blood glucose even during an IPGTT.
These findings suggested the re-expression of IGF-II following c-Myc-induced beta cell hurt is non sufficient to forestall c-Myc induced programmed cell death. Both strains suffered big figure of beta cell loss after c-Myc activation. However, the re-expression of IGF-II may be of import for the early recovery of beta cell mass, as it has been clearly shown impaired beta cell regeneration at 4 yearss c-Myc inactivation in IGF-II KO mice. Upon long term c-Myc inactivation, informations suggested that re-expression of IGF-II may go on to impact beta cell regeneration, as we found IGF-II WT systematically increased more beta cell mass/numbers than KO mice. However such consequence may be less likely to be indispensable in beta cell regeneration in long term recovery as we besides observed some grade of beta cell mass recovery in IGF-II KO mice. The consequences suggested other factors/mechanisms may besides be activated transverse clip. This could besides be a response to beta cell loss or a compensation of loss of IGF-II.
In the grownup beta cell Numberss can be renewed by distinction of root cells ( Bock 2004 ; Hao, Tyrberg et Al. 2006 ) or by reproduction of bing differentiated cells, of the same ( Dor, Brown et al. 2004 ) or of a different line of descent ( Bouwens and Rooman 2005 ) . An intriguing survey showed that alpha cells can transdifferentiate to beta cells after utmost beta-cell loss ( Thorel, Nepote et Al. 2010 ) . Besides recent work by Dr. Abouna from our group has shown that beta cells may originate from a non-beta cell beginning, such as a root cell primogenitor, during gestation ( Abouna, Old et Al. 2010 ) . We have besides hence determined the rate of beta cell proliferation in IGF-II wildtypes and KO mice after 3 months recovery. However, no difference was detected between the two strains. Hence loss of IGF-II may non impact beta cell proliferation in long term recovery. However, given observed more beta cell addition in IGF-II WT mice, one possible account could be that IGF-II look promoted beta cell neogenesis by triping other beginnings, other than proliferation.
6.1.2 Impact of PML Loss in Regulating Beta Cell Mass
The protein PML is a tumor suppresser foremost identified in acute promyelocytic leukaemia. PML and its atomic macromolecular construction PML-NB are mostly involved in cell programmed cell death. Research from pml void mice and cells indicated that PML can bring on programmed cell death in a p53 dependant or independent mode. Continual groundss suggests PML is of import in bring oning growing apprehension and tumour suppression ( Gottifredi and Prives 2001 ; Pearson and Pelicci 2001 ; Bernardi and Pandolfi 2003 ; Ito, Bernardi et Al. 2009 ) . However, comparative few surveies have been done to associate PML and c-Myc. The lone direct grounds was done by Saint lawrences group showed that Max, an of import c-Myc spouse, is concentrate within PML organic structures, beef uping the relationship between PML organic structures and Myc/Max ( Smith, Byron et Al. 2004 ) . Whether PML is playing a cardinal function in pancreatic beta cell has non been good described yet. Therefore, we aimed to find if PML is involved in c-Myc induced programmed cell death.
First, we investigated the function of PML in modulating beta cell mass in the pIns-c-MycERTAM/PML+/+ ( PML wildtype ) and pIns-c-MycERTAM/ PML-/- ( PML KO ) mice. Before c-Myc activation islet morphology in PML KO mice was identical from that of PML wildtype mice. No self-generated tumors were found in PML KO pancreatic islets at twenty-four hours 0 ( before c-Myc activation ) . As antecedently described c-Myc activation in pancreas can bring on early beta cell proliferation and afterwards big figure of beta cell extirpation ( Pelengaris, Khan et al. 2002 ; Radziszewska, Choi et Al. 2009 ) . After 4hr or 24hr c-Myc activation we found a similar addition of beta cell proliferation in both PML KO and WT mice. Following up to 3 hebdomads of Myc activation, we observed a similar extent of islet extirpation in both PML wildtypes and KO mice. These consequences indicated that with the loss of PML in beta cells and attendant c-Myc activation, the beta cells do undergo early proliferation. However, the loss of PML in beta cells is non able to overrule the prevailing apoptotic signal conferred by sustained c-Myc activation in beta cells.
Furthermore, unlike the Myc-induced malignant neoplastic disease formation that occurs, when Myc is activated in pIns-c-MycERTAM mice that have been crossed with those showing anti-apoptotic lesions such as Bcl-xL, or where p19ARF or p53 are knocked out, even after 3 hebdomads c-Myc activation no grounds of islet tumor development was observed in the crosses with PML void mice. The consequences indicated that the c-Myc induced apoptotic signal is non modified by loss of PML. Finally, we tested the map of PML in our tumour theoretical account RIP7-Bcl-xL/pIns-c-MycERTAM by traversing with PML KO strain. As antecedently mentioned c-Myc activation in RIP7-Bcl-xL/pIns-c-MycERTAM mice can bring on beta cell tumorigenesis ( Pelengaris, Khan et al. 2002 ) . Hence in this survey we investigated whether loss of PML accelerates or aggravates beta cell tumour tumorigenesis. After 2 hebdomads c-Myc activation in both RIP7-Bcl-xL/pIns-c-MycERTAM/PML+/+ ( RIP/PML wildtype ) and RIP7-Bcl-xL/pIns-c-MycERTAM/PML-/- ( RIP/PML KO ) mice, there was no seeable difference in the extent, nature or spread of islet tumors between mice with or without PML.
Both c-Myc and PML seem to hold a strong relationship with p53 in their apoptotic maps. Both of them can stabilise p53 via ordinance of MDM2, either through the formation of trimeric complex or advancing p19ARF ( Zindy, Eischen et al. 1998 ; Kurki, Latonen et Al. 2003 ; Louria-Hayon, Grossman et al. 2003 ; Bernardi, Scaglioni et Al. 2004 ; de Stanchina, Querido et Al. 2004 ; Qi, Gregory et Al. 2004 ; Alsheich-Bartok, Haupt et Al. 2008 ) . Hence the omission of PML is expected to cut down the stableness of p53 and prevent cell from undergoing programmed cell death. However, in our survey loss of PML didnt protect beta cells from c-Myc induced programmed cell death. Besides loss of PML didnt accelerate c-Myc induced tumorigenesis, which suggests PML may non be involved in the c-Myc related programmed cell death tract, at least in beta cells in vivo. A fresh PML/PTEN/Akt/mTOR/FoxO signaling web was described late ( Ito, Bernardi et Al. 2009 ) , proposing an interesting relationship between PML and PTEN. The PTEN ( phosphatase with tensin homology ) is an of import tumour suppresser. PTEN loss or mutation is often found in malignant neoplastic disease patterned advance ( Li, Yen et Al. 1997 ; Liaw, Marsh et Al. 1997 ; Zhou, Loukola et Al. 2002 ) . PTEN could dephosphorylate PIP3 to PIP2 and acts as a powerful negative regulator of the PI3K/Akt signaling pathway. By using the same transgenic theoretical account pIns-MycERTAM the effects of the omission of PTEN has been studied in modulating beta cell mass ( Radziszewska, Choi et Al. 2009 ) . Research found PTEN omission does non take to tumor formation in beta cells. Furthermore, surprisingly, they found PTEN loss did non protect beta cells from the overpowering programmed cell death and development of diabetes induced by c-Myc activation. And the decision was drawn that there was no grounds of tumor development with sustained c-Myc activation despite of the combined consequence of loss of a tumor suppresser and activation of an transforming gene in beta cells. Evidences to day of the month suggest that loss of either PML or PTEN does non strong plenty to suppress c-Myc induced programmed cell death or lead to tumour development.
6.1.3 Quantification of Beta Cell Mass and Numbers
Quantification of beta cell mass/number is of great importance in diabetes research. However, most decisions were drawn establishing on the measurings of cross sectional country or a comparative volume ( beta cell mass ) . Although the methods for beta cell mass quantification differ, to find a per centum or proportion of the tissue in this survey we foremost obtained the insulin cross sectional country ( insulin positive country divided by pancreas country ) . And in this thesis to gauge the beta cell mass pancreatic weight was multiplied by insulin cross sectional country consequences. To avoid any prejudice caused by overestimated pancreatic weight, the parametric quantity of organic structure weight was considered during standardization. Furthermore, beta cell numeration was performed both by homo and package which provided the comparative sum beta cell figure for each animate being. Thus the information of beta cell mass/number was achieved to look into the function of IGF-II in beta cell regeneration.
Bioimage information is now a fresh subdivision of the quickly coming field. Due to developments in the field of bioimaging, an increasing sum of informations demands to be analyzed. Data volume additions in different dimensions: higher declarations, faster readying and imagination, clip oversight imagination, multispectral imagination. As a consequence, manual rating is no longer executable without proper package support. The beta cell figure quantification by human manus labeling, as mentioned above, is proven to be really clip consuming and it can restrict the analysis of informations volume. In this survey more than 2000 islet images were manus labeled for beta cell figure numeration and besides a machine larning based system was trained and employed accomplishing the same consequences ( Herold, Zhou et Al. 2009 ) . As described in Chapter 5 such system was validated by human labeling consequences. The comparing informations suggested the package is dependable and numbering beta cell figure indifferent. We can anticipate that the packages, TIScover and Pankreas Analyzer, will mostly salvage clip and offer research workers the chance to accomplish even more accurate information in diabetes survey.
6.1.4 Restrictions of Thesis and Directions for Future Work
To day of the month we have non been able to find the exact cellular beginning of IGF-II in the pancreas. Our informations demonstrated that messenger RNA for IGF-II is found within the pancreas, but we have non been able to successfully or faithfully place peptide look to any given cell type utilizing immunohistochemistry, farther compounded by the inaccessibility of an antibody that works with Western smudges either, which has antecedently besides been noted by other groups. It is hoped that in situ hybridisation may assist decide this issue and we are prosecuting this affair farther at the present clip.
To quantify beta cell mass/numbers experiments were set up with n=3 at each clip point from IGF-II KO and WT strains. And we did noticed carnal fluctuation during the informations analysis which likely can be corrected by presenting more replicates. However, limited by carnal care and clip of analysis, n=3 for each clip point was the maximal. Hence it is recommended to utilize more replicates in future beta cell mass quantification survey.
It is nevertheless surely that these surveies reveal the public-service corporation of conditional beta cell extirpation theoretical accounts in the functional surveies of beta cell regeneration. It is anticipated that many more such will follow so that a more complete image can be drawn of those factors essential for beta cell regeneration in vivo. Such factors, are of obvious curative involvement as they may be exploitable as new curative agents in the intervention or even bar of diabetes.
Accrued groundss point out that PML is involved in many apoptotic tracts every bit good as suppression of survival signals. The omission of PML, a important tumour suppresser, was found to non impact c-Myc induced programmed cell death or supply protection of diabetes development. However, Here in this survey non merely does loss of PML in beta cells non take to tumorigenesis, but even with a attendant initiation of an transforming gene, a state of affairs which should make an environment that is favourable for tumour formation, we observed no tumorigenesis. Hence PML may non be a good campaigner for curative usage in bring arounding diabetes.
To quantify beta cell mass/numbers is a hard and clip devouring work. Here with the aid of package numbering system we are able to accomplish a more accurate cell figure informations in a faster manner. However, the appraisal of entire beta cell mass/numbers is still restricted by analyzing limited pancreas degrees. Recently ApErio and others have developed automatic slide scanning system, ScanScope, which with suited imaging package can be used to mensurate islet and beta cell country in an machine-controlled manner, therefore dramatically cut downing clip needed for such surveies. It is sensible to believe that more and more fresh instruments will be developed to offer a much rapid, more precise and dependable information on beta cell mass/number and would be a utile tool for all diabetes research workers.
In this thesis we demonstrated for the first clip that IGF-II is reactivated after beta cell hurt. We detected IGF-II messenger RNA during 24hr and 48hr beta cell hurt in IGF-II wildtype mice but as we expected non in IGF-II KO mice. However the re-expression of IGF-II didnt prevent IGF-II wildtype mice from c-Myc induced beta cell extirpation. After 4 yearss recovery beta cell mass and Numberss in IGF-II wildtype mice were significantly increased, whereas in IGF-II KO mice at that place was no recovery at all. After 3 months recovery both strains achieved some grade of beta cell mass and Numberss which was sufficient to command blood glucose even during an IPGTT. However, consequences suggested systematically more beta cell regeneration in IGF-II wildtypes cross to 3 moths recovery. Datas to day of the month back up the position that IGF-II is of import for beta cell regeneration.
In a related survey we investigated the function of PML in c-Myc induced both beta cell programmed cell death and proliferation. We demonstrated that PML loss does non forestall c-Myc induced diabetes. After 3 hebdomads c-Myc activation both PML wildtypes and PML KO mice developed hyperglycemia in 4 yearss and stayed high blood glucose in the whole process. Consequences from histological analysis confirmed islets from both strains were mostly ablated after c-Myc activation, which matches the old observation of hyperglycemia.
The effects of PML loss in c-Myc induced proliferation was examined by triping c-Myc for 4hr or 24hr. The consequences indicated that with the loss of PML in beta cells and attendant c-Myc activation, the beta cells do undergo early proliferation. However, the loss of PML in beta cells is non able to overrule the prevailing apoptotic signal conferred by sustained c-Myc activation in beta cells. Besides after 2 hebdomads c-Myc activation we didnt observe any terrible tumorigenesis status when PML is absent in our tumor mouse theoretical account RIP7-Bcl-XL/p-Ins-c-MycERTAM.
Finally in this survey we introduced a fresh machine based numeration system for beta cell figure designation and quantification. By analysing more than 4000 images from 30 pancreata the proof of package was successfully achieved with the support from human numbering informations. Furthermore consequences indicated when trying islets with little or average size ( incorporating less than 250 beta cells ) the package is numbering really similar consequences as worlds. But when trying big/huge islets ( incorporating more than 250 beta cells ) the package is systematically numbering more beta cells than human. The proof suggested package numeration is dependable in either numbering little, medium or large islets as it is sensible to believe that when confronting excessively many beta cells research workers can easy acquire lost or distracted. Such utile package based numeration would supply a much quicker, more accurate and efficient research tool in diabetes study in the hereafter.