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In physiological situations such as pregnancy, beta-cell mass expansion is generally associated with beta-cell function enhancement to accommodate the new insulin requirement particular to the pregnant state (Sorenson and brelje 1997, horm metab res). Similarly during obesity, an augmentation of both beta-cell number and beta-cell responsiveness is also observed to compensate for the insulin resistance induced by this state, a key trait of this condition (Kargar and Ktorza, Diabetes, obesity and metabolism 2008). However, in specific models of pancreatic injury associated with hyperglycemia, such as 90% subtotal pancreatectomy (laybutt et al. diabetologia 2007) or selective beta-cell ablation induced by activation of caspase 8(Wang et al diabetes 2008, panic attac), the regenerative process characterised by an increase beta-cell mass was not correlated with an improvement of the beta-cell functionality. Similar features were observed in the Kir6.2G123S transgenic mice, a spontaneous model of beta-cell regeneration (Oyama et al. Diabetes 2206). In those models, the regenerated beta-cells are not fully functionally mature (Kargar and Ktorza, Diabetes, obesity and metabolism 2008). Consequently, in our system in which beta-cells do regenerate after massive ablation by c-Myc (Pelengaris et al., Cano et al.), it is also of interest to address the properties of new beta-cells and determine to which extent their functional capacity matches that of original beta-cells. Using single cell microfluorimetry, we examined the beta-cell function after diabetic recovery in the pIns-c-MycERTAM mice. We stress that the mice studied here are singly and not triply transgenic.
We analysed the beta-cell responsiveness in islets isolated from pIns-c-MycERTAM mice after diabetic recovery using calcium microfluorimetry. Single transgenic pIns-c-MycERTAM and wild-type mice about three months of age were daily injected with a dose regime of 1mg/ml of 4-hydroxytamoxifen (4-OHT) for six consecutive days, while control pIns-c-MycERTAM mice were injected with peanut oil, the control vehicle. At the end of the pulse, the pIns-c-MycERTAM mice upon 4-OHT withdrawal were allowed to recover. Nine months after 4-OHT withdrawal, the islets from mice after diabetic recovery, from wild-type and control pIns-c-MycERTAM mice were isolated for analysis. It is important to point out that pIns-c-MycERTAM mice, at around three months of age at the beginning of the experimentation, were injected contemporaneously with peanut oil with or without 4-OHT, and euthanized concurrently at the end of the experiment to isolate their islets. The wild-type mice, on the contrary, were injected with 4-OHT at different times, at three months of age, but their islets were still isolated at the same time as those of the single transgenic mice for consistency. Three to four replicates were used per group for the experiment and islets were isolated from each individual animal. Also, we isolated islets from three month old pIns-c-MycERTAM control and wild-type mice that did not received any treatment, for additional control tests of functionality. Islets were isolated from three mice per group. Fed blood glucose levels and glucose tolerance tests (see Material and methods for further details) were performed on the animals during the experiment before euthanizing the mice for islet isolation. The islets were isolated using the collagenase digestion procedure, then the purified islets were cultured on 3-aminopropyltriethoxysilane-coated glass coverslips in a standard RPMI medium and incubated at 37oC in the presence of 5% CO2 for two to three days until analysis ( details of these procedures are described in Materials and Methods). The calcium microfluorimetric measurements to study beta-cell functionality were performed by Dr Paul E. Squires according to the protocol described by Squires et al. (Diabetes 2000).
What is the mechanism that allows beta-cells to tightly regulate the release of insulin in mammals and how can the single-cell microfluorimetric method be used to evaluate the efficiency of this process?
Beta-cells can be compared to a micro-factory that manufactures insulin that is stored in secretory vesicules or granules before its release into the medium by exocytosis. Insulin secretion is highly regulated and depends on intricate intracellular signalling in response to a variety of stimuli. The process involved in glucose-induced insulin release by beta-cells, is succinctly presenting in Figure 28 and it follows the following steps. First, an increase of glucose concentration in the extracellular medium leads to glucose uptake by the beta-cells, catalysed by the glucose transporter Glut2. Then, the metabolic breakdown of the sugar that ensues results in the increase of the intracellular ATP concentration at the expense of ADP (or the increase of the ATP/ADP ratio) which induces the closure of an ATP-dependent potassium channel (KATP-channel). The closing of this channel causes a progressive depolarisation of the plasma membrane and the initiation firing of action potentials. These in turn open voltage-sensitive, L-type Ca2+ channels and provoke the influx of Ca2+ in the beta-cells. This results in an increase of the intracellular free Ca2+ concentration ([Ca2+]i) that can be considered as the key stimulus that triggers insulin-containing granules to fuse with the plasma membrane and release insulin into the medium by exocytosis (Rutter, G.A., Diabetologia 2004; Rorsman and Renstrom, Diabetolgia 2003). Glucose-stimulated insulin release by beta-cells, it is in fact, ultimately due to the Ca2+ influx. Accordingly, one can quantify this specific part of the process to appraise the efficiency of the beta-cell functionality by measuring a change in [Ca2+]i in the cells after appropriate treatments known to induce insulin release. The single cell fluorimetric technique is based on the measurement of a change in [Ca2+]i by using a Ca2+-fluorophore fura-2A/M (Squires et al., Diabetes 2000). Upon binding calcium the maximum excitation wavelength of the fura-2A/M undergo a blue shift from 380nm (when it is calcium free) to 340nm (when it is saturated with calcium). By detecting the intensity of the emitted light (wavelength of 510nm) coming from the fluorophore after illumination alternatively with the excitation wavelength of 340nm and that of 380nm, the increase of [Ca2+]i due to the influx of calcium into the beta-cells will be characterized ( Palmer and Moore, 2000). Changes in [Ca2+]i is represented by the ratiometric values of the intensity of fluorescence emitted by fura-2A/M after excitation with the 340nm wavelength over the intensity of fluorescent emitted by fura-2A/M after excitation with the 380 wavelength. This emission ratio can be written as 340/380nm.
To trigger insulin release from the beta-cells in vitro, the cells maybe incubated with high concentrations of glucose about 20 to 30mM, but other components can be used to elicit the release of insulin. Potassium chloride (K+Cl-) at 20mM depolarises the membrane and forces the voltage-sensitive, L-type Ca2+ channel to open, which in turn leads to the influx of extracellular Ca2+ in the cells and insulin release by exocytosis. Tolbutamide (100µM), actives the sub-unit Sur1 of the KATP-channel, which also results in depolarisation of the membrane, which in turn induces the opening of the voltage-sensitive, L-type Ca2+ channel and so forth. Finally, ATP (100µM) which binds to P2-purinoreceptors, in turn leads to the formation of IP3 (Inositol 3phosphates) resulting in the release of Ca2+ from the endoplasmic reticulum to the cytoplasma of the cell. In this situation the Ca2+ is released from inside the cells. An important point regarding this technique is that the fluorescence measurement is carried out only in regions (called region of interest (RIO)) located at the periphery of the islets because only these areas are in focus under the binocular microscope, so that the fluorescence coming from fluorophore fura-2A/M can be recorded.
Fed blood glucose and glucose tolerance tests in pIns-c-MycERTAM and wild-type animals
Wild-type and transgenic pIns-c-MycERTAM mice were injected with 4-OHT or a control vehicle for six consecutive days to induce diabetes by ablation of beta-cells upon c-Myc activation. Figure 29A presents the fed blood glucose (a) in wild-type animals, (b) in pIns-c-MycERTAM mice injected with control vehicle (c-MycERTAM off), (c) in pIns-c-MycERTAM mice on the last day of c-Myc activation by 4-OHT (c-MycERTAM on), and (d) in pIns-c-MycERTAM mice in which c-Myc was activated and then deactivated upon 4-OHT removal after nine months (c-MycERTAM on-off). The values were, (a) 7.3±1mM, (b) 4±0.4mM, (c) 30±3mM and (d) 4.1±.5mM respectively. The difference between all the groups was statistically significant (One-way Analysis of Variance (ANOVA) P-value <0.05), except for the mean fed blood glucose of pIns-c-MycERTAM mice after diabetic recovery (4.1±.5mM, c-Myc on-off) and that of control pIns-c-MycERTAM (4±0.4mM, c-Myc off). These results indicate that upon activation of c-Myc by 4-OHT, the mice developed a diabetic phenotype (30±3mM) but following removal of 4-OHT, the transgenic mice recovered from diabetes (4.1±.5mM). The difference between the two groups is highly significant (One-way Analysis of Variance (ANOVA) P-value <0.0001). These results corroborate Pelengaris (Pelengaris et al.) and Cano (Cano et al.) reports. Also, the data show that the mean fed blood glucose is higher in wild-type mice in general (7,3±1mM) than in the pIns-cMycERTAM mice in which c-Myc is off (4±0.4mM), control transgenic animals (One-way Analysis of Variance (ANOVA) P-value <0.05).
We carried out glucose tolerance tests by injecting intra-peritoneal glucose (2g/kg ) in animals after overnight fasting. The blood glucose level was recorded prior to and at 10, 30, 60 and 120minutes after glucose administration. Figure 29B presents the intra-peritoneal glucose tolerance test (IPGTT) results in non-treated wild-type (n=4) and pIns-c-MycERTAM (n=3) mice aged three months, and also pIns-c-MycERTAM mice (n=4) treated with control vehicle, aged twelve months. Following the IPGTT, one can see that 120min after the glucose challenge, the blood glucose level of animals of each group returns to normal. Only at the time point prior to and 120min after glucose administration, the wild-type animals show a glucose level different from the two other groups. The difference was significant (One-way Analysis of Variance (ANOVA) P-value <0.05). Also the calculation of the area under the curve (AUC), (prism 4, University of Warwick) of the glucose tolerance test curve for each individual mouse in each group, showed that the difference of the mean AUC between non-treated wild-type (1541±276), pIns-c-MycERTAM (1359±285) mice, both aged of three months and pIns-c-MycERTAM mice aged of twelve months (1015±326) was not statistically significant (One-way Analysis of Variance (ANOVA) P-value >0.05). First, these results indicate that the age of the transgenic pIns-c-MycERTAM mice, does not affect the capacity of their beta-cells to respond properly to a glucose challenge. Second, the mice of each group were glucose tolerant, suggesting that their beta-cells were functional. Third, it is of interest to note that the fed blood glucose of wild-type animals is higher than that of our transgenic animals.
Glucose tolerance tests in pIns-c-MycERTAM mice after diabetic recovery
Three pIns-c-MycERTAM mice about three months of age, were treated with 4-OHT for six consecutive days, and then allowed to recover from diabetes upon 4-OHT removal. IPGTT was carried out on the mice during the course of the experiment, that is before 4-OHT treatment, shortly after the end of the treatment (that is when the mice were overtly diabetic) and nine months after the last 4-OHT treatment (that is when the mice recovered from diabetes). The results are recorded in Figure 29C. The analysis of the IPGTT in the animals shortly after 4-OHT treatment(c- MycERTAM on) indicates clearly that the mice were glucose intolerant and 120min after the glucose challenge they were still not able to normalise their blood glucose level. The IPGTT values of animals shortly after treatment compared with those of both the mice before treatment and after diabetic recovery, were significantly different at all the time points (One-way Analysis of Variance (ANOVA) P-value <0.05). In stark contrast, IPGTT curves of animals before treatment (c- MycERTAM off) and after diabetic recovery (c- MycERTAM on-off) were not significantly dissimilar from each other. Thus, analysis of IPGGT curves in these two groups indicates that the animals were able to normalise their blood glucose level 120min after the glucose challenge. Also, the calculation of the AUC of the glucose tolerance test curve of each individual mouse, shows that the difference of the mean AUC of animal before treatment (1197± 273) and after diabetic recovery (1010±466) was not statistically significant (One-way Analysis of Variance (ANOVA) P-value >0.05).
By contrast, comparison of the mean AUC of the animals during treatment (2661±359) with that of animals before treatment or after diabetic recovery, indicates that the difference between the two groups was statistically significant (One-way Analysis of Variance (ANOVA) P-value <0.001). It is not noteworthy that the same animal was glucose tolerance tested prior to, at the end of and after the 4-OHT treatment, in this experiment. Collectively, these results indicate that upon 4-OHTtreatment, pIns-c-MycERTAM mice develop a diabetic phenotype (become glucose intolerant), but after 4-OHT removal, and nine months recovery, the animals recover from diabetes, as evidenced by normalisation of their fed blood glucose and by becoming glucose tolerant again. In addition, the fact that the animals after diabetic recovery were glucose tolerant again, suggested that their regenerated beta-cells are functional.
Analysis of beta-cell functionality using single-cell microfluorimetry
Using single-cell microfluorimetry we studied islet beta-cell functionality of pIns-c-MycERTAM mice before and after diabetic recovery. The study was performed in collaboration with P. Squires. For this experiment, wild-type animals were treated with 4-OHT prior to islet isolation. pIns-c-MycERTAM mice were treated with 4-OHT or control vehicle and islets were isolated nine months after 4-OHT or control vehicle administration. In each group, islets were isolated from three different animals. Figures 30A and C and 31C represent graphs of the islet beta-cell functionality of islets from wild-type animals Each figure represents the result of the beta-cell functionality test of an islet from an individual animal. The islets were incubated, first with glucose (20mM) then with different components not in a particular order, tolbutamide (100µm), ATP (100µm) or K+Cl- (20mM). The y-axis, corresponds to the changes in [Ca2+]i symbolised by an increase of the ratio 340/380nm, whilst the x-axis to the time in seconds. Series or RIO (region of interest) represents a region in the islet that was examined. At least eight different regions of interest were concurrently monitored per islet, for stimulated changes in [Ca2+]i. Each oscillatory curve characterizes changes in[Ca2+]i for each RIO or series tested. The three graphs show unequivocally an increase of the ratio 340/380nm, indicating an augmentation of the [Ca2+]i, due either to an influx of Ca2+ release into the cells or the cytoplasma. After the islets were stimulated with nutrient and non-nutrient. These results indicate clearly that the islet beta-cells from wild-type animals were completely functional. By contrast, the islet beta-cells from pIns-c-MycERTAM mice after diabetic recovery (Figures 30C and D) show very little increase of the ratio 340/380nm or change in [Ca2+]i after stimulation by glucose or other components. This indicates a marked impairment of the beta-cell functionality of mice after diabetic recovery. To verify that the impaired beta-cell functionality was a feature of the beta-cells after diabetic recovery, we checked the islet beta-cells functionality in pIns-c-MycERTAM mice treated with control vehicle (Figures 31A and B). Surprisingly, after stimulation of the islets with glucose, tolbutamide or K+Cl-, little increase in [Ca2+]i was observed, similar to islets of animals after diabetic recovery. To rule out the fact that the age of the -c-MycERTAM mice animals might affect their beta-cell responsiveness, we examined the islet beta-cell functionality of-c-MycERTAM (Figure 32A and B) and wild-type (Figures 32C and D) animals aged three months. The graphs of the islets from wild-type animals, once again, show a higher amplitude of the increase of the [Ca2+]i compared to those from pIns-c-MycERTAM mice.
Collectively, the profound attenuation of the beta-cell responsiveness in pIns-c-MycERTAM mice in untreated controls or after diabetic recovery , shows explicitly that beta-cell functionality is abnormal in this transgenic line. Also, the finding that the fura-2M/A uptake was similar in wild-type and pIns-c-MycERTAM islets, characterized by comparable dye base line, indicates that the beta-cells were viable at the start of the experiment. The graphs of islets from pIns-c-MycERTAM mice untreated controls and after diabetic recovery, show no evidence that the beta-cell functionality after diabetic recovery are different from those of untreated control pIns-c-MycERTAM mice.
In conclusion, based on the results of the glucose tolerance test, we concluded that the transgenic pIns-c-MycERTAM mice prior to c-Myc activation were glucose tolerant. This indicates that their beta-cells are functional in maintaining glucose homeostasis, and the age of the transgenic mice did not affect this property. By contrast, upon 4-OHTtreatment, pIns-c-MycERTAM mice developed a diabetic phenotype, becoming glucose intolerant, but upon 4-OHT removal, the animals recovered eventually from diabetes and became glucose tolerant again, suggesting that regenerated beta-cells are at least partially functional after diabetic recovery. To explore whether beta-cell function was normal after diabetic recovery, we tested their functionality using single-cell microfluorimetry. Based on the results of calcium microfluorimetry, we concluded that the islet beta-cells from control wild-type animals were completely functional. In stark contrast, the profound attenuation of the beta-cell responsiveness in pIns-c-MycERTAM mice untreated controls and after diabetic recovery, demonstrated that the mechanism of the beta-cell functionality in this line was altered. In addition, the comparison of the profile in the change of intracellular Ca2+concentration between islets from pIns-c-MycERTAM mice untreated controls and after diabetic recovery, showed no evidence that beta-cell functionality after diabetic recovery was different from the (albeit abnormal) functionality of untreated control pIns-c-MycERTAM mice. The discrepancy observed between the physiological responsiveness of transgenic animals and the beta-cell responsiveness in vitro in the pIns-c-MycERTAM line is quite intriguing and will be discussed in the next chapter.