Objetivo Específico Nro 03

In several animal diabetes models, a deficiency of -cell functions was hypothesised to result from -cell dedifferentiation (Jonas et al., 1999; Laybutt et al., 2002), or from apoptosis (Pelengaris et al., 2002b; Pick et al., 1998). NOD

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(non-obese diabetic) mouse (Atkinson and Leiter, 1999; Makino et al., 1980) or streptozotocin (STZ) induced -cell deletion mouse model (Kolb, 1987; Like and Rossini, 1976) have been used to study T1DM. Apart from pancreatectomy or obese-induced diabetes mice, gene knockout mice, such as insulin receptor knockout mice, have also been widely applied to study the role of insulin resistance in T2DM (LeRoith and Gavrilova, 2006; Pattaranit et al., 2008).

A chimeric protein, c-MycERTAM, in which human c-MYC has been fused to a mutated form of the estrogen receptor (ER; ERTAM), can induce the activation of c-MYC by the injection of 4-hydroxytamoxifen (4-OHT; Littlewood et al., 1995; Pelengaris et al., 1999), and it has been set up as a transgenic animal model to turn MYC on or off. Pelengaris et al. (2002b) used plns-c-MycERTAM transgenic mice, in which the c-MycERTAM is regulated by the insulin promoter (pIns) so that this fusion transcriptional factor is specifically targeted to insulin producing -cells, to explore the role of c-MYC in apoptosis and proliferation in -cells. Activation of c-MYC induces proliferation in a short transition but this proliferation is subsequently overwhelmed by apoptosis in -cells. As a result, the transgenic mice become hyperglycaemic and unable to control the glucose homeostasis. However, deactivation of c-MYC by withdrawing 4-OHT leads to the regeneration of -cells in this c-MYC activated transgenic mouse. Thus this animal model can be applied to the study of -cells regeneration. Pelengaris et al. (2002b) also presented the doubly transgenic plns-c-MycERTAM/RIP-BclxL mice by giving c-MYC activation with

co-expression of Bcl-xL (which encodes an anti-apoptotic BCL-2 family protein).

This triggers carcinogenesis without apoptosis but shows hyperglycaemia in the early stage (6-9 days). They also found indications that the in vivo effects of c-MYC

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are reversible, i.e. deactivation of c-MycERTAM elicits -cell and islet regeneration and deactivation of c-MycERTAM in c-MycERTAM/Bcl-xL doubly transgenic model

triggers regression of cancerous cells to normal cells with normal cell behaviour. Furthermore, using the plns-c-MycERTAM transgenic mice, Cano et al. (2008) confirmed -cell regeneration after withdrawing tamoxifen in the transgenic animal system and suggested that retention of -cells results from replication of surviving -cells. However, Cano et al. (2008) also found early hypoglycaemia in this transgenic mouse and suggested that the low glucose level might be due to passive release of insulin because of on-going -cell apoptosis. However, this appears not to be the case since hyperinsulinaemia precedes any significant decrease in -cell mass in plns-c-MycERTAM mice. This view is supported by further functional studies in our group showing that hypoglycaemia is not prevented when MYC-induced apoptosis is fully prevented by co-expression of the anti-apoptotic protein BCLxL in plns-c-MycERTAM/RIP-BclxL doubly transgenic mice (see Figure 1.4.1; Dr. Linda

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Figure 1.4.1. Glucose level in plns-c-MycERTAM/RIP-BclxL doubly transgenic mice. (Dr. Linda

Cheung, unpublished data). Solid lines refer to the objects with treatment (4-OHT), whereas dash

lines refer to the control group (WT littermates treated with peanut oil). 4-OHT, 4-hydroxytamoxifen;

P.oil, a vehicle in which 4-OHT is made and acts as a control; ♂, male; ♀, female; ↑, the glucose

level is higher than the measurement range, which is 0.6-33.3 mmol/l.

Figure 1.4.1 shows the glucose level in the plns-c-MycERTAM/RIP-BclxL doubly

transgenic mice after treating with 4-OHT (treatment, n=4) or the same amount of vehicle (control, n=2). The glucose level is different (p<0.05, paired t-test) between control and treatment mice in day 1. The difference (p<0.01, paired t-test) between day 0 and day 1 in the treatment group indicates a decrease of glucose level on day1, whereas there is no significant difference (p=0.9126, paired t-test) in the glucose level in the control group. Apart from hyperglycaemia, which is regarded as a

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characteristic of diabetes, sudden hypoglycaemia was observed within 24 hours after c-MYC activation. This indicates that c-MYC-activated hypoglycaemia might not be directly linked to insulin released from apoptopic -cells because apoptosis was blocked by the expression of BCLxL.

MYC-activated diabetes animal models suggest that the activation of c-MYC reduces the expression of insulin, pancreatic duodenal homeobox factor-1 (PDX1), NKX6.1, GLUT2, and prepoinsulin I+II (PPI; Kaneto et al., 2002; Laybutt et al., 2002; Pascal et al., 2008; Pelengaris et al., 2002b). PDX1, also known as STF1, IDX1, IPF1, IUF1, which binds to A-box (Ohlsson et al., 1993), is an important transcriptional factor in pancreatic development and insulin transcription (Ahlgren et al., 1996; Ahlgren et al., 1998; Chakrabarti et al., 2002; Ohlsson et al., 1993). In PDX1-deficient animal models, impaired glucose tolerance and GSIS were found (Brissova et al., 2002; Johnson et al., 2003; Leibowitz et al., 2001; Seufert et al., 1998). Deficiency of PDX1 is further related to GLUT2, glucokinase (GK), MAFA, NKX6.1, and insulin, whose products are key factors in GSIS (reviewed in Babu et al., 2007). The plns-c-MycERTAM transgenic mouse can therefore be adopted for the study of -cell regeneration or diabetes treatment without other confounding factors, such as obesity. There are many studies related to -cell regeneration but a robust animal model is still necessary to demonstrate the process of -cell regulations in vivo.

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