Requirement for the L-type Ca²⁺ channel α_1D subunit in postnatal pancreatic β cell generation

Pancreatic β cells are the source of insulin, which directly lowers blood glucose levels in the body. Our analyses of α_1D gene-knockout (α_1D^–/–) mice show that the L-type calcium channel, α_1D, is required for proper β cell generation in the postnatal pancreas. Knockout mice were characteristically slightly smaller than their littermates and exhibited hypoinsulinemia and glucose intolerance. However, isolated α_1D^–/– islets persisted in glucose sensing and insulin secretion, with compensatory overexpression of another L-type channel gene, α_1C. Histologically, newborn α_1D^–/– mice had an equivalent number of islets to wild-type mice. In contrast, adult α_1D^–/– mice showed a decrease in the number and size of islets, compared with littermate wild-type mice due to a decrease in β cell generation. TUNEL staining showed that there was no increase in cell death in α_1D^–/– islets, and a 5-bromo-2′ deoxyuridine-labeling (BrdU-labeling) assay illustrated significant reduction in the proliferation rate of β cells in α_1D^–/– islets.

Voltage-dependent Ca²⁺ channels (VDCCs) provide a pathway for the entry of extracellular Ca²⁺ into the cytoplasm in a membrane voltage-dependent manner (1). VDCC is a heteromeric protein complex, composed of a pore-forming α₁ subunit and regularly associated β and disulfide-linked α₂δ subunits (2). The L-type Ca²⁺ channel is defined by its pharmacological sensitivity to dihydropyridines, activation by relatively strong depolarization, and slow inactivation (3). Genes encoding the L-type channels are α_1S, α_1C, α_1D, and α_1F (Ca_v1.1-1.4) (4). Besides excitation-contraction coupling in muscle cells (5), L-type channels play critical roles in hormone or neurotransmitter release (6, 7), synaptic plasticity (8), and regulation of gene expression (9) especially in the nervous and neuroendocrine systems. Two different genes, α_1C and α_1D, encode neuronal L-type channels. The α_1D gene is expressed in various organs including brain, pancreas, heart, and cochlea (10–13).

Pancreatic β cells express the two isotypes of the L-type channel, α_1C and α_1D (11, 14). In the β cell, glucose metabolism causes an increase in ATP or the ATP/ADP ratio, which in turn closes K_ATP channels. This leads to membrane depolarization, opening of VDCC, influx of Ca²⁺, and a rise in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i). The elevation of [Ca²⁺]_i directly triggers insulin exocytosis. L-type channels are known to play a physiological role in insulin secretion because L-type channel blockers inhibit the rise in intracellular calcium and insulin secretion in response to various insulin secretagogues (7). Analysis of mRNA encoding α_1C and α_1D demonstrated that the Ca²⁺-conducting subunits of the L-type channel in pancreatic β cells mainly consist of the α_1D subunit (11, 14). On the basis of these considerations, we tried to elucidate the physiological role of α_1D in the pancreas by using α_1D-knockout (α_1D^–/–) mice. Our results indicate that α_1D is required for the proper generation of β cells in the postnatal pancreas.

α_1D –/– mice are deaf and smaller than their littermates. The α_1D+/– heterozygotes showed no apparent abnormality and were fertile. Homozygotes (α_1D–/–) were obtained by mating heterozygotes (Figure 1b). Western blotting of cerebral membrane proteins (Figure 1c) and immunostaining of pancreas sections (see Figure 3b) confirmed the lack of α_1D protein in knockout mice. Homozygous α_1D–/– mice appeared healthy, but smaller (80% of normal body weight) than the littermate controls. The lack of motor reflex in α_1D–/– mice in response to an auditory stimulus (Preyer reflex) and the significantly elevated auditory brainstem response (ABR) threshold for a broadband click (data not shown) confirmed that α_1D–/– mice were deaf, consistent with the phenotype of α_1D knockout mice reported by Platzer et al. (13).

Figure 3

Ca²⁺ channel activities in pancreatic islet cells. (a) The current density histogram of peak I_Ba in wild-type (filled bars, n = 22) and α_1D^–/– (open bars, n = 27) islets. Total refers to total current density; L represents L-current density; Non-L is the remaining current density. I_Ba was activated by depolarizations from –80 mV to 0 or 10 mV every 10 seconds. (b) Immunostaining of L-type calcium channel subtypes in pancreatic islets. Primary antibodies used are indicated on the left side of the panel. Four independent experiments were performed from three animals per genotype. Bar, 100 μm. (c) Representative current records of I_Ba by whole cell patch clamp in wild-type and α_1D–/– pancreatic islet cells. Depolarizations from –80 mV to levels ranging from –30 to 0 mV in 10-mV increments for 400 microseconds every 10 seconds. (d) Voltage dependence of Ca²⁺ channel currents in wild-type (filled circles, n = 16) and α_1D–/– (open circles, n = 17) β cells. *P < 0.001. The peak I_Ba current was normalized to cell capacitance. Average cell capacitance was 4.35 ± 0.32 pF for α_1D–/– islet cells and 4.16 ± 0.17 pF for wild-type islet cells. (e) Stimulation of insulin release from cultured islets by glucose stimulation. Islets of similar sizes were selected from the α_1D–/– (open bars) and the control mice (α_1D+/+ and α_1D+/–, filled bars). Five to seven animals were used per genotype. The number of measurements (n) for each concentration for each genotype ranged from 4 to 25. *P < 0.05.

α_1D–/– mice are hypoinsulinemic and glucose intolerant. Given that α_1D is the dominant L-type channel protein in pancreatic β cells (11, 14), and inhibitors of L-type channels block most of the glucose-stimulated and depolarization-evoked insulin release (7), we investigated blood glucose homeostasis in the mutants. In nonfasting states, the serum insulin levels in adult α_1D–/– mice were lower than those observed in littermate control mice (Figure 2a), although blood glucose levels did not differ significantly between the wild-type and mutant mice (154.1 ± 8.9 mg/dl, n = 7 males, wild-type; 159.1 ± 17.7 mg/dl, n = 8 males, α_1D–/–, P = 0.81). Glucose tolerance tests were performed to verify the pancreatic dysfunction in α_1D–/– mice. As shown in Figure 2b, at 12–14 weeks of age, α_1D–/– mice remained hyperglycemic 2 hours after intraperitoneal injection of glucose, whereas in the wild-type mice, blood glucose levels returned to the baseline. The level of serum insulin increased twofold in wild-type mice 30 minutes after glucose injection (after overnight fasting, i.e., before glucose injection, 0.24 ± 0.06 ng/ml, n = 6; 30 minutes after glucose injection, 0.49 ± 0.07 ng/ml, n = 6). We could not quantify the serum insulin in mutant mice, because for the majority of the mutants, the serum insulin levels after overnight fasting were less than 0.1 ng/ml (n = 7), which was the lowest limit of the insulin measuring kit used. Serum insulin levels in α_1D–/– mice, measured 30 minutes after glucose injection, were still below 0.1 ng/ml (n = 5). These data suggest that glucose intolerance in the α_1D–/– mice might be caused by the insulin deficiency. However, α_1D–/– mice exhibited normoglycemia in fed states compared with age-matched littermates and did not develop diabetes over the 1-year observation period. Because enhanced insulin sensitivity might account for these results in α_1D–/– mice, we injected insulin and examined changes in blood glucose levels. Indeed, the glucose-lowering effect of insulin was significantly enhanced in α_1D–/– mice, compared with wild-type mice (Figure 3c).

Figure 2

Serum insulin levels and glucose tolerance test. (a) Serum insulin concentrations in nonfasting 10-week-old α_1D^–/– mice (open bar, n = 4) and control littermates (filled bar, n = 5). *P < 0.005. (b) Glucose tolerance tests of 12- to 14-week-old female mice. Blood glucose was determined at indicated time points after intraperitoneal glucose injection (2 mg/g body weight). Open circles, α_1D^–/– mice (n = 6); filled circles, wild-type littermates (n = 9). (c) Insulin tolerance tests were performed on fed animals. Results are expressed as percentage of initial blood glucose concentration. Open circles, α_1D^–/– mice (n = 7); filled circles, control littermates (n = 11). *P < 0.05; **P < 0.01.

Compensatory overexpression of α_1C in α_1D –/– pancreatic β cells. Given that calcium entry through L-type calcium channels is crucial for insulin secretion in β cells (7), we examined the effects of the loss of the α_1D channel on calcium currents and insulin secretion in response to glucose stimulation in α_1D–/– pancreatic β cells. Ca²⁺ currents, supported by 10 mM Ba²⁺ as a charge carrier, were activated by step depolarizations from a holding potential of –80 mV. Unexpectedly, mutant cells did not show a decrease in either total current density (Figure 3a) or L-type current density, as confirmed by current analyses in the presence of 10 μM nifedipine (Figure 3a). These results suggest the possibility of a compensatory increase in the other L-type channel, namely α_1C. Immunostaining with anti-α_1C antibody confirmed that α_1C was indeed overexpressed in mutant pancreatic islets, compared with wild-type mice (Figure 3b). Significantly, a closer examination of electrophysiological data revealed that a current-voltage plot of the mutant cells was shifted by about 10 mV toward more positive potentials at the lower voltage range (Figure 3d). Therefore, the current density at –10 mV and –20 mV were reduced by approximately 50% and approximately 70% in mutant β cells, respectively (Figure 3d). However, at potentials of 10 mV or above, the curves looked virtually identical between mutant and wild-type. These results effectively demonstrate the loss of α_1D channels and subsequent compensation by α_1C in the mutant, as α_1D channels have a lower activation threshold than α_1C (10, 13). Because β cell action potentials in response to glucose stimulation rarely exceed –10 mV (21, 22), the decreased channel activity below the membrane potential of –10 mV in mutant β cells may affect the secretion of insulin. To examine the consequences of the altered channel profile on the insulin secretory function of islet β cells, we measured insulin secretion from isolated islets in culture in response to ambient glucose, using the batch incubation method. Mutant islets secreted less insulin than control islets at 3 mM ambient glucose (0.019 ± 0.003 ng/30min/islet for control; 0.011 ± 0.002 ng/30min/islet for mutant; P = 0.03), although no statistically significant difference was observed at glucose concentrations of 6 mM or higher (Figure 3e). These results suggest that the glucose-sensing and subsequent insulin secretion machinery might be largely preserved in isolated α_1D -deficient β cells.

Reduction of β cell mass in α_1D –/– pancreas. Another possible mechanism underlying the hypoinsulinemia and glucose intolerance in α_1D–/– mice could be a reduction of total β cell mass. We examined islet morphology in the mutant pancreas. Pancreatic islets of α_1D–/– mice were well organized with the insulin-producing β cells forming a central core surrounded by a discontinuous mantle of non β cells (Figure 4a). A simple scanning of the histological slides under a microscope, however, revealed a marked decrease in islet numbers and a scarcity of larger islets in the mutant pancreas, compared with control (Figure 4a). At the day of birth (P0), mutant mice showed smaller body weight and a slightly smaller number of islets (Figure 4b), although the relative islet number (normalized as their body weight) was equivalent to that in littermate wild-type mice (88.1 ± 3.9 per body weight in wild-type mice, and 81.8 ± 3.1 per body weight in α_1D–/– mice at birth [n = 4]; P = 0.24). However, at postnatal day 14 (P14), the islet number was approximately 60% less than that of wild-type littermates, and by P30, we observed approximately 40% fewer islets than in wild-type littermates (Figure 4b). The relative islet number was also significantly reduced in α_1D–/– pancreas by approximately 40% at P14 (n = 4, P < 0.05) and approximately 30% at P30 (n = 5, P < 0.05). But the pancreas weight (measured as percent body weight) did not differ between the α_1D–/– mice and littermate controls (0.25 ± 0.01% for wild-type mice versus 0.28 ± 0.01% for α_1D–/– mice at P14, P = 0.15; 1.15 ± 0.05% versus 1.25 ± 0.03% at P30, P = 0.15d). Distribution of islet size (shown in Figure 4 by cell numbers in the cross-section), represented as a percentage of islets measured, revealed a significant reduction in the number of larger islets in the mutant pancreas (Figure 4c). The proportion of β cells in individual islets was significantly reduced, from approximately 72% in wild-type islets to approximately 48% in mutant islets (Figure 4d). Moreover, the percentage of α cells was relatively increased in mutant islets (data not shown), indicating that reduction of islet mass was mostly caused by a decrease in β cell mass. Somatostatin- and pancreatic polypeptide–positive cells were well preserved in the mutant islets (data not shown). Our data collectively demonstrate that postnatal β cell expansion is impaired in α_1D–/– mice. The reduction in β cell mass may, in turn, lead to hypoinsulinemia and glucose intolerance in α_1D–/– mice.

Figure 4

Islet morphology. (a) Upper panel: insulin- and glucagon-positive cells, respectively in wild-type and mutant mice. Bars, 100 μm. Lower panel: lower magnification (×40) of pancreatic islets of wild-type and α_1D–/– mice. Immunostaining with anti-insulin antibody shows sparse distribution and lack of larger islets in the α_1D–/– pancreas. (b) Decreased number of islets in α_1D–/– mice, compared with that in littermate controls. Summed islet numbers obtained from examining every fifth section of 20-μm thickness from a whole pancreas. Filled bars, littermate control mice (wild-type for P0, P14, and P30 and heterozygote for 7 months); open bars, α_1D–/– mice. The number of animals examined is indicated at the bottom of each bar. *P < 0.05. **P < 0.001. (c) Morphometric analysis of islet area in pancreas from wild-type (filled bars, n = 5) and α_1D–/– (open bars, n = 5) female mice at P30. Distribution of islet areas less than 100 cells, 100–150 cells, or more than 150 cells is shown as a percentage of islets measured. The proportion of larger islets (more than 150 cells in a cross-section) was significantly reduced in the α_1D–/– pancreas. *P < 0.05. (d) Percentage of β cells in individual islets at P30. Filled bar, wild-type islets (n = 58 islets from five mice); open bar, α_1D^–/– islets (n = 79 islets from five mice). *P < 0.001.

Decreased β cell proliferation in the α_1D–/– mice. To determine whether the decrease in β cell mass was due to a defect in β cell genesis or in survival, we first examined the degree of cell death in islets at P14 and P30, using the TUNEL assay. Previously, it was reported that the frequency of apoptotic β cells is higher throughout the neonatal period than in the adult rat, peaking at 13–17 days after birth (23). We found no increase in the cell death in mutant islets either at P14 or P30. At P14, the percentage of apoptotic cells was 5.73 ± 0.01% in wild-type islets (n = 2 mice) and 5.42 ± 0.17% in mutant islets (n = 2 mice). At P30, there were only six apoptotic cells over 49 islets in the wild-type and four cells over 41 islets in the mutant pancreas, respectively. This indicates that the decrease in β cell mass in the mutant was not due to an increase in cell death but rather due to a decrease in the proliferation and/or differentiation of β cells. Pancreatic β cell growth can be mediated by neogenesis or differentiation from ductal precursor cells, and replication of differentiated β cells (24, 25). β Cell neogenesis and replication normally proceeds in mice up to around 3 weeks of age (26, 27). After weaning, a low level of replication is maintained (24). As shown in Figure 4b, during the first 2 weeks after birth, increase in islet number was severely impaired in the mutant pancreas. We examined β cell proliferation at P14, by incorporation and immunohistochemical detection of bromodeoxyuridine (BrdU) in the DNA of dividing cells. After 2-hour BrdU labeling, the number of BrdU and insulin double-positive cells per total insulin-positive cells significantly decreased in mutant islets to 53% of that in wild-type at P14, and the difference persisted at P30 (Figure 5b). In conclusion, the β cell proliferation rate is decreased in the mutant islets, and this may partly contribute to the decrease in β cell mass in α_1D–/– mice.

Figure 5

β Cell proliferation rate. (a) Anti-BrdU and anti-insulin double immunostaining of pancreatic islets. The β cells have dark violet cytosols, and BrdU-positive cells appear with red nuclei. Representative islets each from the wild-type (left) and the α_1D–/– mice (right) at P14 were shown. Bar, 50 μm. (b) Reduced β cell proliferation rate in the α_1D–/– islets. The percentage of BrdU-positive β cells among β cells was obtained from each mouse, and the average value was calculated for each genotype. Filled bars, wild-type (n = 5 mice for P14 and P30); open bars, α_1D–/– (n = 2 mice for P14 and n = 5 mice for P30). *P = 0.05; **P < 0.005.

In the present study, we show a marked reduction in total islet mass in α_1D–/– mice, due to impairment of postnatal β cell generation. The actual β cell mass in α_1D–/– mice at P30 is approximately 24–30% of that of wild-type mice, as the total islet number in mutant is approximately 60% of that in wild-type (Figure 4b), and the number of β cells in individual islets is approximately 40–50% of that in wild-type (Figure 4d).

We observed an overexpression of α_1C in islets of α_1D–/– mice both in adult (Figure 3b) and at P14 (data not shown). The maximal current density was restored in the mutant β cells, although the calcium current density at –10 mV to –30 mV was reduced by 50–70% in mutant β cells (Figure 3d). In spite of the compensatory overexpression of α_1C in mutant β cells and the normal maximal current density, proliferation rate was reduced. These results may suggest two possible roles of α_1D in β cell proliferation. First, calcium entry through α_1D channels at the membrane potentials of below –10 mV may play a role in β cell proliferation. Second, α_1D protein itself may function as one of the components in the mitogenic signaling pathway. These two possibilities do not necessary exclude each other. For the first time, to our knowledge, this study demonstrates that the L-type channel α_1D is one of the major requirements for proper β cell generation. In addition, these results clearly demonstrate distinct functions of the L-type channel isotypes in the pancreas.

The β cell mass reduction in α_1D–/– mice is an unexpected and novel finding, as there have been no reports to show that L-type channels contribute to postnatal β cell generation. With hindsight, however, earlier observations are consistent with the possible involvement of L-type channels in the production of β cells. In pancreatic β cells, glucose uptake leads to an increase in intracellular ATP/ADP ratio and a closure of K_ATP channels. This results in membrane depolarization, opening of VDCC, calcium influx, and finally activation of the insulin secretory machinery. It is known that glucose itself induces β cell mitosis in vivo or in vitro (28). Glibenclamide, a hypoglycemic drug that inhibits K_ATP channels, affects the replication rate and mass of β cells in young mice (29). Moreover, glucagon-like peptide-1 (GLP-1), a regulator of postnatal β cell growth and differentiation, increases levels of β cell cAMP and glucose-stimulated Ca²⁺ influx (30). Recent studies have shown that islet size and function may be determined by the extent of activin receptor-mediated TGF-β signaling (31). Pancreatic expression of a dominant negative type II activin receptor (dn-ActR) in transgenic mice results in islet hypoplasia (32, 33), and ActRIIA^+/–B^+/– mice show hypoplastic pancreatic islets, hypoinsulinemia, and impaired glucose tolerance (34). Interestingly, activin causes Ca²⁺ influx through membrane depolarization by inhibiting the activity of the K_ATP channel and directly modulates VDCCs in β cells (35). However, until now, there have been no reports that L-type channels are involved in the β cell growth-promoting activity of GLP-1 and activin.

Recent studies have demonstrated that a number of transcription factors are necessary in the proper development of the pancreas (31). Genetic studies have outlined the hierarchy of transcription factors involved in β cell development. For example, the homeodomain factor, IPF1/PDX1, has been proposed to regulate the expression of a variety of different pancreatic endocrine genes including insulin, somatostatin, glucokinase, and glucose transporter 2 (Glut2). Recently, Hart et al. reported that attenuation of FGF signaling in mouse β cells leads to impairment of postnatal β cell expansion (36) and the expression of glucose transporter 2 (Glut2) in these mice. Moreover, the expression of FGF receptors was impaired in IPF1/PDX1 inactivated β cells, and it was claimed that the IPF1/PDX1 acts upstream of FGF receptor (FGFR) signaling. It would therefore be worthwhile to examine the levels of α_1D gene expression in these mice and the transcriptional regulation of α_1D gene in β cell development.

Contrary to our results, Platzer et al. (13) reported recently that α_1D-deficient mice showed no growth retardation or glucose intolerance. There are a number of possible explanations for this discrepancy. First, the difference in the genetic background of the embryonic stem (ES) cell line used for generating knockout mouse might have caused different phenotypes: AB2.2 originating from the 129/SvEv substrain was used in their work, whereas J1 originating from the 129/SvJae substrain was used in our work (37). The genetic heterogeneity among 129 substrains is well documented (37, 38). The phenotypes resulting from the disruption of genes in insulin production and insulin signaling pathways are critically dependent on the genetic background of the mouse used (39–41). Second, we used a different targeting strategy to the other group. For example, we deleted the locus spanning the 3′ end of the first exon to the 5′ part of the second exon that included an intron in between, whereas they inserted neocassette into the second exon. Further molecular analysis of each mutant with regard to the status of the α_1D locus, such as aberrantly spliced products, may provide an answer.

Despite the reduction of β cell proportion in individual α_1D–/– islets (Figure 4d) and the decrease in Ca²⁺ channel activity at the lower range of membrane potentials in mutant β cells (Figure 3d), there is no decrease in insulin secretion in response to glucose stimulation except at the basal glucose concentration in α_1D–/– islets (Figure 3e). This discrepancy implies that insulin secretion per β cell may be enhanced in mutant islets. In fact, analysis of insulin content in individual islets revealed that mutant islets contained more insulin than did similar size of wild-type islets (41.0 ± 2.4 ng/islet in mutant islets, n = 63 islets from three mice; 27.8 ± 2.2 ng/islet in wild-type islets, n = 66 islets from three wild type mice, P < 0.001). Considering the decreased proportion of β cells in mutant islets (Figure 4d), insulin content per β cell increased twice in mutant islets. However, it is unlikely that there is an increase in individual β cell volume (hypertrophy) in α_1D–/– islets, because no significant difference was observed in cell capacitance (pF) between wild-type and α_1D–/– cells (Figure 3b, legend). This increase in insulin content per islet accounts for the increase in insulin secretion from mutant islets. The average total insulin content in the pancreas (μg/g pancreas) in 6- to 7-week-old α_1D–/– mice was approximately 39% that of control mice, which indicates that total β cell number might be approximately 20%, compared with wild-type mice.

It will be interesting to see whether there is any difference in the change of membrane potential in response to glucose between the wild-type and the mutant β cells. Glucose-induced membrane depolarization is the intervening step from glucose-sensing to insulin secretion (42). In addition, inward Ca²⁺ currents through VDCCs participate in the generation of action potentials in β cells (42). There is, however, a great deal of heterogeneity among β cells (22, 43), and the electrical activity of β cells induced by glucose is dependent on the cell configuration, such as in single cell, a cluster, or an intact islet (21, 22, 43). Therefore, this issue could be addressed by population studies using perforated patch-clamp recordings of β cells in intact pancreatic islets of wild-type and mutant mice (21).

The α_1D-deficient mice showed hypoinsulinemia and severely impaired glucose tolerance, which were caused mainly by the reduction of total β cell mass. Increases in insulin content in individual β cells and enhanced insulin sensitivity of peripheral tissues may offer explanations for the lack of overt diabetes in α_1D–/– mice. Similar phenotypes such as impaired glucose tolerance and enhanced insulin sensitivity are observed in the offspring of insulin-sensitive type 2 diabetes patients (44, 45).

Human type 2 diabetes is strongly associated with genetic and familial backgrounds (46, 47). Although insulin resistance is an important pathological sign in the early stages of type 2 diabetes, a failure in adequate β cell mass increase leads to progression to a diabetic state (48). For example, humans with type 2 diabetes have reduced β cell mass, compared with weight-matched nondiabetic subjects (48). Although mice lacking α_1D did not develop overt diabetes in the 12-month observation period, they might be more prone to diabetogenic conditions such as high-fat diet, stress, or mild insulin resistance, owing to reduced β cell mass. The possibility of using α_1D–/– mice as a model for type 2 diabetes remains to be examined.

The authors thank M.P. Kong for animal care, K. Jun for primary library screening, J. Jeong for technical help, and J.W. Yoon for critical evaluation of the manuscript. This work was supported in part by a Creative Research Initiative program grant from the Ministry of Science and Technology, Korea, and a medical science grant from the Ministry of Health and Welfare, Korea.

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