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Molecular Endocrinology 16 (4): 884-895
Copyright © 2002 by The Endocrine Society

Disturbances in Glucose-Tolerance, Insulin-Release, and Stress-Induced Hyperglycemia upon Disruption of the Cav2.3 ({alpha}1E) Subunit of Voltage-Gated Ca2+ Channels

Alexey Pereverzev, Marina Mikhna, Rolf Vajna, Cornelia Gissel, Margit Henry, Marco Weiergräber, Jürgen Hescheler, Neil Smyth and Toni Schneider

Institute of Neurophysiology (A.P., M.M., R.V., C.G., M.H., M.W., J.H., T.S.) and Institute of Biochemistry (N.S.), University of Cologne, D-50931 Köln, Germany

Address all correspondence and requests for reprints to: Toni Schneider, University of Cologne, Institute of Neurophysiology, Robert-Koch-Strasse 39, D-50931 Köln, Germany. E-mail: Toni.Schneider{at}uni-koeln.de.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Multiple types of voltage-activated Ca2+ channels (T, L, N, P, Q, R type) coordinate Ca2+-dependent processes in neurons and neuroendocrine cells. Expressional and functional data have suggested a role for Cav2.3 Ca2+ channels in endocrine processes. To verify its role in vivo, Cav2.3(-/-) mutant mice were generated, thus deficient in {alpha}1E/R-type Ca2+ channel. Intraperitoneal injection of D-glucose showed that glucose tolerance was markedly reduced, and insulin release into plasma was impaired in Cav2.3-deficient mice. In isolated islets of Langerhans from these animals, no glucose-induced insulin release was detected. Further, in stressed Cav2.3-deficient mice, the rate of glucose release into the blood was only 29% of that observed for wild-type animals. Thus, the deletion of Cav2.3 causes deficits not only in insulin release but also in stress-induced hyperglycemia. The complex phenotype of Cav2.3-deficient mice has dual components related to endocrine and neurological defects. The present findings provide direct evidence of a functional role for the Cav2.3 subunit in hormone secretion and glucose homeostasis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE CaV2.3 ({alpha}1E) Ca2+ channel subunit is a member of the non-L-type high-voltage-activated Cav2 channel subfamily (1, 2, 3, 4, 5). Human Cav2.3-subunits were cloned as neuronal (6) and endocrine splice variants (7, 8), the latter being detected initially in the insulinoma cell line INS-1 (9). Channels containing Cav2.3 as an ion-conducting pore constitute part of the R-type current that "remains" after the block of other high-voltage-gated channels by specific antagonists. It was found recently to be selectively blocked by SNX-482, a toxin from the tarantula Hysterocrates gigas and to be involved in oxytocin release (10).

Recent reports about the ablation of Cav2.3 revealed only a subtle phenotype for Cav2.3-deficient mice when compared with the inactivation of other voltage-gated Ca2+ channels (11, 12, 13, 14, 15). In cultured cerebellar granule neurons and in a subpopulation of dorsal root ganglion neurons, only part of the R-type current was shown to be lost when Cav2.3 was deleted (16), confirming the assumption, that E-type Ca2+ channels produce only a fraction of the R-type current. It was also suggested that these animals showed different responses to pain (17). Basic excitatory synaptic transmission as well as long-term potentiation were intact in the hippocampal CA1 region of Cav2.3-deficient mice. Further, spatial memory, but not fear memory formation, differed when compared with control animals (18).

So far, endocrine functions were not investigated in Cav2.3-deficient mice. The influx of calcium ions through voltage-gated Ca2+ channels is a major trigger for the secretion of insulin from the islets of Langerhans (19). Both L-type and non-L-type Ca2+ channels are involved in Ca2+-induced insulin secretion (20, 21). Based on immunohistochemical and in vitro functional studies, Cav2.3 has been suggested as a candidate channel involved in the release of peptide hormones (22, 23, 24). To test this hypothesis in vivo, glucose homeostasis and insulin release, as well as stress-induced hyperglycemia, were analyzed in mice lacking the Cav2.3 Ca2+ channel subunit.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Gene Inactivation of cacna1e Using Deleter Mice
The cacna1e gene encoding Cav2.3 (Fig. 1Go) was disrupted in vivo by mating agouti-colored Cav2.3(fl/+) and deleter mice expressing cyclic DNA-producing recombinase (Cre-recombinase) constitutively under the control of the cytomegalovirus promoter. The offspring was genotyped by Southern blot analysis. In 20 of 83 pups (24%) exon 2 was deleted by Cre-mediated recombination. Heterozygous Cav2.3(+/-) mice were intercrossed to receive null mutants, and the genotype identified in the offspring from 12 different litters followed a Mendelian inheritance with 27 Cav2.3(-/-) mice of 106 newborn pups (29%). Thus, the general ablation of the Cav2.3-subunit is not lethal before birth. The litter size of Cav2.3(-/-) mice was indistinguishable from the litter size of Cav2.3(fl/fl) mice, which were generated by inbreeding of Cav2.3(fl/+) mice containing the mutated cacna1e gene. Here, the exon 2 was flanked by two loxP sites (recognition site for Cre) that did not impair the expression of Cav2.3 (not shown). Both, the Cav2.3(-/-) and Cav2.3(fl/fl) mice were fertile and exhibited no obvious anatomical abnormalities after a lifespan of 42 wk.



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  		Figure 1. Targeting of the cacna1e Gene by Homologous Recombination

A, The murine cacna1e gene locus was targeted by homologous recombination close to exon 2 encoding the first transmembrane segment of domain I (IS1) and the adjacent loop to IS2. Exon 2 represents nt 269–375, and exon 3 represents nt 376–516 of the murine Cav2.3 sequence (GB L29346). Restriction sites: A, AseI; B, Bst 1107 I; E, EcoR V; H, HindIII; n, NsiI; N*, NsiI site was destroyed. B, Targeting vector containing a thymidine kinase cassette (tk) upstream of the EcoRV site as a negative selection marker for random incorporation into DNA, three loxP sites, and a neomycin cassette. The first loxP site is adjacent to HindIII, and the second and third sites are flanking the neomycin cassette used as a positive selection marker after incorporation into the cacna1e gene locus. C, Targeted gene locus after homologous recombination. The ES cell clone T{alpha}1E1E8 was used for the transient expression of Cre-recombinase in ES cells to delete the neomycin cassette. D, Recombined products of the cacna1e gene locus after transient expression of Cre-recombinase.

 
The deletion of exon 2 from the cacna1e gene was analyzed on the transcript level by RT-PCR (Fig. 2BGo). The transcript between exon 1 and 3 was amplified as cDNA, and the fragment from Cav2.3-deficient mice was larger than the expected 220-bp fragment in wild-type mice (Fig. 2BGo). Sequencing showed that exon 1 was spliced differently, producing a novel second exon of 204 bp, which also contained an in-frame stop codon. This results in chain termination within the new exon 2.



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  		Figure 2. Genotyping of ES Cells, and Expression Analysis of cacna1e by RT-PCR and Western Blot

A, After digestion of genomic DNA with HindIII and AseI, a 2.3-kb fragment was detected in all four ES cell types that is indicative for the wild-type allele of cacna1e. The neomycin cassette that was incorporated into the Cav2.3 gene between exon 2 and 3 caused a 1.8-kb shift in the genomic DNA from T{alpha}1E1E8 ES cells (1E8). The expected size was 4.12 kb. ES cell subclone T{alpha}1EPuNr6 (#6) did not contain a visible additional band, because the expected size of the HindIII–AseI fragment from the mutated allele is 2,316 bp and close to the size of the wild-type fragment of 2,282 bp. ES cell subclone T{alpha}1EPuNr9 (#9), which was analyzed because it should lack both exon 2 as well as Neo, showed a 1.3-kb fragment (expected size, 1,307 bp) which is indicative for the deletion of exon 2 and neomycin cassette. B, PCR amplification of Cav2.3 cDNA fragments from total brain-RNA of wild type (wt), heterozygous (+/-), and homozygous mice (-/-). In the heterozygous mouse, only the shorter cDNA fragment of the wild-type allele was amplified although the heterozygous genotype was clearly defined by genotyping. A 100-bp ladder was used as size marker. C, Microsomal membranes were isolated from brain of Cav2.3 null mutants (-/-), and wild-type (wt) or heterozygous (+/-) littermates. Microsomal membranes from untransfected (HEK-) and stably Cav2.3({alpha}1E) transfected HEK-293 cells (HEK-E: 2 and 5 µg) were used as negative and positive controls, respectively. Two different peptide-directed sera were used as the primary antibodies during Western blot analysis. Probing of membrane proteins by anti-{alpha}1E-spec serum, which was raised against an epitope, present only in longer Cav2.3 splice variants. D, Probing of membrane proteins by anti-{alpha}1E-com serum, which was raised against an common epitope in all Cav2.3 splice variants.

 
Brain microsomal membranes were isolated from wild-type, heterozygous, and homozygous mice. The transfer membranes were probed with two different sera directed against a specific (Fig. 2CGo) or a common epitope (Fig. 2DGo). No expression of any Cav2.3-subunit from brain was detected.

Basal Glucose and Insulin Levels in Fasting Mice Are Unaffected upon Deletion of Cav2.3
Cav2.3 is expressed as a specific splice variant in the insulinoma cell line INS-1 (7, 9) and is functionally involved in glucose-stimulated insulin release, which can be reduced by the peptide-toxin SNX-482 (24). Therefore, blood glucose and serum insulin were compared between C57Bl/6 and Cav2.3-deficient mice. After the animals were starved for 14 h, both parameters are not significantly different between the two genotypes of 10-, 21-, and 40-wk-old mice.

Impaired Glucose Tolerance in Cav2.3(-/-) Mice
To examine the glucose homeostasis in Cav2.3-deficient mice, glucose tolerance was compared after ip injection. For 10-wk-old male mice (Fig. 3AGo), the time course of glucose disposal was significantly slower in Cav2.3-deficient mice, yielding a 1.2-, 1.5-, and 1.7-fold higher blood glucose after 30, 60, and 120 min, respectively. Similar results were obtained for females of the same age (Fig. 3BGo) although the differences were not so marked compared with males. In conclusion, Cav2.3-deficient mice at the age of 10 wk are less tolerant against a glucose injection than age-matched C57Bl/6 mice.



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  		Figure 3. Intraperitoneal Glucose Tolerance Test

Ten-week-old mice were fasted for 14 h. Before and after injection of 2 g D-glucose/kg body wt, glucose was determined in the tail vein. A, Blood glucose from 21 C57Bl/6 ({bullet}) and from 16 Cav2.3-deficient male mice ({circ}). B, Blood glucose from 6 C57Bl/6 ({bullet}) and from 7 Cav2.3-deficient female mice ({circ}).

 
Decreased Acute Insulin Release in Cav2.3(-/-) Mice
A reduced glucose tolerance could be mediated by a reduced release of insulin from the ß-cells or by a slower transport of glucose into liver and skeletal muscle as the major glucose-consuming tissues. To determine the insulin release during glucose challenge by ip injection, the serum insulin content as well as blood glucose were monitored (Fig. 4Go). In wild-type mice, the insulin content increased 1.6-fold after injection of glucose within 2 min of glucose administration, and preceded the rise and subsequent reduction of glucose concentration in blood. Compared with basal insulin levels in wild-type mice, insulin is still elevated 30 min after the glucose injection. However, in Cav2.3-deficient mice, the serum-insulin content is unchanged. Thus, the insulin secretion after ip administration of glucose is grossly impaired in Cav2.3-deficient mice. This result supports our assumption, arising from functional studies in the insulinoma cell line INS-1, that Cav2.3 is involved in Ca2+-stimluated insulin release in wild-type mice and is important for the fast response to elevated blood glucose levels.



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  		Figure 4. Blood Glucose and Serum Insulin Concentrations of Cav2.3(-/-) and C57Bl/6 Wild-Type Mice That Underwent a Glucose Tolerance Test

D-Glucose (2 g/kg body wt) was injected ip in 10-wk-old male mice. A, Determination of glucose for 41 wild-type ({bullet}) and 36 Cav2.3-deficient mice ({circ}). B, Determination of insulin for 19–20 wild-type ({blacksquare}) and 17–20 Cav2.3-deficient mice ({square}).

 
Histological Analysis of the Pancreas
In elder KATP channel-deficient mice, histological abnormalities of pancreatic islets were observed (25, 33). To check whether the islets of Langerhans from Cav2.3-deficient mice were anatomically and numerically normal, the pancreas from both genotypes were analyzed histologically. Within randomly selected slices of pancreas, the number of islets per slice (Fig. 5AGo) as well as the distribution of glucagon-positive {alpha}- and insulin-positive ß-cells (Fig. 6Go) was indistinguishable between wild-type and Cav2.3-deficient mice. No histological changes were observed within the islets, relating to the peripheral distribution of {alpha}-cells (Fig. 6Go, E and F) vs. the more central arrangement of ß-cells (Fig. 6Go, C and D).



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  		Figure 5. Islets of Langerhans in C57Bl/6 and Cav2.3-Deficient Mice

A, Number of islets detected by histochemical methods. Randomly selected slices from wild-type and Cav2.3-deficient pancreas were counted under the microscope for islets visualized by histochemical staining. B, Comparison of total insulin extracted from isolated islets. Insulin was quantified by RIA after isolation of islets and extraction of insulin by acidic ethanol (n = 5 mice). C, Glucose-induced insulin-release from isolated islets. Basal insulin release was determined at 2 mM glucose. Release is plotted after normalization as percentage of remaining insulin per assay (n = 5 mice).

 


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  		Figure 6. Immunohistochemical Detection of {alpha}- and ß-Cells in C57Bl/6 and Cav2.3-Deficient Mice

Anti-glucagon-positive {alpha}-cells and anti-insulin-positive ß-cells are detected in the islets of Langerhans using the biotin-streptavidin-horseradish peroxidase technique with DAB as chromogen and hematoxylin as counterstain. Pancreas was isolated from C57Bl/6 (A, C, and E) and Cav2.3-deficient mice (B, D, and F). A and C, Immunocytochemical detection of ß-cells in C57Bl/6 mice. B and D, Immunocytochemical detection of ß-cells in Cav2.3-deficient mice. E, Immunocytochemical detection of {alpha}-cells in C57Bl/6 mice. F, Immunocytochemical detection of {alpha}-cells in Cav2.3-deficient mice.

 
Decreased Insulin Release in Isolated Islets of Langerhans from Cav2.3(-/-) Mice
In five independent experiments, islets of Langerhans were isolated from 10- to 11-wk-old C57Bl/6 and Cav2.3-deficient female mice. Total insulin per islet was indistinguishable between both genotypes (Fig. 5BGo).

Below the threshold level of glucose, basal release of insulin was not significantly increased at 2 mM glucose in Cav2.3-deficient mice. At low levels of glucose (7 mM), a slow increase in insulin secretion occurred in wild-type islets that was not seen in the Cav2.3-deficient islets. However, this was not statistically significant. At higher levels (20 mM), stimulated insulin release from wild-type islets rose 1.9 ± 0.1-fold, whereas for islets from Cav2.3-deficient mice, the insulin secretion was not significantly changed at 1.1 ± 0.1-fold (Fig. 5CGo). These results suggest that the glucose-induced insulin secretion from wild-type islets of Langerhans is dependent on the expression of Cav2.3.

The Glucose Tolerance Alters During Aging in Cav2.3-Deficient Mice
Mice lacking the ß-cell insulin receptor acquire a reduced glucose tolerance with aging (26). However, in Cav2.3-deficient mice, glucose tolerance altered differently with age. In 21- (Fig. 7AGo) and 40-wk-old animals (Fig. 7BGo) blood-glucose of Cav2.3-deficient mice is disposed differently than in age-matched wild-type mice. In 10-wk-old Cav2.3-deficient mice, the fast insulin release was grossly impaired after ip administration of glucose. But at the age of 42 wk, the Cav2.3-deficient mice still lacked the acute fast response to glucose challenge, and they showed a tendency for a slight increase of serum insulin 30 min after ip injection of glucose (Fig. 8AGo). Comparing the insulin release between the groups of 10- vs. 42-wk-old animals shows that the wild-type mice respond identically at both ages, whereas the Cav2.3-deficient mice have altered both their glucose tolerance (Fig. 7Go) as well as their kinetics of insulin release (Fig. 8BGo), suggesting that with aging the Cav2.3-deficient mice compensate partially their reduced glucose-induced insulin release by an unknown mechanism.



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  		Figure 7. Intraperitoneal Glucose Tolerance Test of 21- and 40-wk-Old Mice

Mice were treated as described in Fig. 3Go. A, Blood glucose from eight C57Bl/6 ({bullet}) and six Cav2.3-deficient male mice ({circ}; 21 wk old). B, Blood glucose from 15 C57Bl/6 ({bullet}) and 10 Cav2.3-deficient male mice ({circ}; 40 wk old).

 


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  		Figure 8. Blood Glucose and Serum Insulin Concentrations of Cav2.3(-/-) and C57Bl/6 Wild-Type Mice During Glucose Tolerance Testing

D-Glucose (2 g/kg body wt) was injected ip and glucose and insulin were determined as described in Fig. 4Go. A, Blood glucose and serum insulin from 42-wk-old male mice. At 15 and 30 min after glucose load, the blood glucose is significantly different between wild-type ({bullet}) and Cav2.3-deficient mice ({circ}). After 30 min, the increase of insulin is not significant in Cav2.3-deficient mice ({square}) compared with wild-type animals ({blacksquare}). The serum insulin differs significantly between genotypes 5 min after glucose load. B, Comparison of insulin release from 10 (squares) and 42-wk-old mice (circles). Closed symbols are from wild-type and open symbols are from Cav2.3-deficient animals. Number of mice: 17–20 of 10-wk-old mice, and 6–8 of 41-wk-old mice.

 
Stress-Induced Hyperglycemia
Stress is known to activate the hypothalamus-pituitary-adrenal axis and elicits the activation of sympathetic tone, which leads to hypertension and cardiovascular effects. Moreover, stress elicits hyperglycemic responses, and stress might occur during glucose tolerance testing. To determine whether the deletion of Cav2.3 influences the hyperglycemic response, 10- and 42-wk-old mice were stressed by being restrained in individual wire cages (27). Both mouse lines show a hyperglycemic response to the immobilization stress at both ages (Fig. 9Go). However, the response in the mutant animals, which is particularly marked with younger animals, is greatly attenuated and suggests that in the Cav2.3(-/-) mice during aging compensatory mechanisms also apply for this hormone cascade.



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  		Figure 9. Stress-Induced Glucose Release in 10- and 42-wk-Old Females

Blood glucose was determined for 10-wk ({bullet}) and 42-wk-old C57Bl/6 mice ({blacksquare}), and for 10-wk ({circ}) and 42-wk-old Cav2.3-deficient mice ({square}) before and after 15, 30, 60, and 120 min of restraint stress. Ten to 12 mice for each curve.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The inactivation of the cacna1e gene in mice was reported to generate a nonlethal phenotype with an altered pain response (17) and an impairment of spatial memory (18). Cav2.3-deficient mice were also helpful for defining more precisely the identity of the residual R-type voltage-gated calcium currents (16).

In the present study results are reported for a general knockout of Cav2.3, which was produced by mating Cav2.3(fl/+) with deleter mice expressing Cre-recombinase constitutively. The inbreeding of the heterozygous offspring led to homozygous Cav2.3(-/-) animals that are viable and fertile.

Glucose Tolerance and Insulin Release Are Altered in Cav2.3-Deficient Mice
Based on our recent results concerning the functional role of Cav2.3 in the insulinoma cell line INS-1 (24), the effect of the disruption of the cacna1e gene on the insulin secretion and on glucose homeostasis was investigated in Cav2.3-deficient mice. For Cav2.3-deficient males and females, the glucose tolerance at the age of 10 wk is significantly reduced compared with C57Bl/6 mice, although it is rather moderate when compared with the defect in insulin release. Presumably, the Cav2.3-mice have developed a higher insulin sensitivity by this age (but see Note Added in Proof).

A similar pattern of both a reduced glucose tolerance and a markedly impaired early insulin response after administration of glucose was observed for glucokinase-deficient (28, 29) and for KATP channel-deficient mice (25, 33). In elder KATP channel-deficient mice, histological alterations of pancreatic islets were observed (25, 33). However, neither histological abnormalities of pancreatic islets nor a reduced insulin content of islets was detected in Cav2.3-deficient animals.

Not only the ablation of proteins that are directly involved in the metabolic sensing of glucose and the subsequent excitation-secretion coupling cause a glucose intolerance and an impairment of glucose-stimulated insulin release, but also the disruption of proteins that are related to the glucose homeostasis through the regulation of insulin biosynthesis via insulin-stimulated insulin gene transcription, show the same two phenomena. The insulin receptor-deficient mice (26), as well as the gene inactivation of insulin receptor substrate 2 (30, 31) and S6 kinase 1 (32), cause phenotypes that show characteristics of diabetic organisms. In KATP channel-deficient mice (25) and after the tissue-specific gene inactivation of the insulin receptor (26), the glucose-induced insulin release is impaired. However, the latter animals demonstrate a progressively impaired glucose tolerance at 8, 16, and 24 wk (26). This is not observed for the Cav2.3-deficient mice. It is possible that Cav2.3-deficient mice have altered their impairment of glucose-stimulated insulin release by a higher insulin sensitivity (see Ref. 33).

Initially, another voltage-gated Ca2+channel, {alpha}1D (Cav1.3), was thought to be the major candidate for the Ca2+ entry and Ca2+-triggered hormone release in endocrine systems (34), especially as it was cloned from ß-cells (35, 36). However, the disruption of the gene encoding Cav1.3 produced no major disturbances in glucose metabolism (14). In Cav1.3-deficient mice, the fasting blood glucose levels were slightly, but not significantly, lower than in control mice, and no difference was observed after glucose injections. Also, no differences were detected for the insulin content after fasting or after a glucose challenge. Therefore, in mice the insulin secretion is not strictly dependent on the expression of Cav1.3 ({alpha}1D) in the ß-cells of pancreas (14). Hence, it was assumed that other L-type voltage-gated Ca2+ channels (Cav1.2) compensate for the ablation of the Cav1.3-subunit.

L- and P/Q-type voltage-gated Ca2+ channels have also been reported to trigger the insulin release in islets of Langerhans (21, 37, 38, 39), and transcripts of multiple ion-conducting subunits of voltage-gated Ca2+ channels have been detected in INS-1 cells and islets of Langerhans (9, 22, 24). We assume that at least two, maybe even three, voltage-gated Ca2+ channels with different thresholds cooperate during Ca2+-triggered insulin release (Fig. 10Go). The KATP channel is directly linked to the metabolic activity of the ß-cell. Glucose metabolism in pancreatic ß-cells is tightly coupled to the mitochondria, and signals derived from mitochondrial metabolism include ATP and possibly glutamate (40). A high ATP/ADP ratio leads to closure of KATP channels, which depolarizes the ß-cell and may activate low-voltage-gated T-type Ca2+ channels at least in rat ß-cells from where a splice variant of Cav3.1 ({alpha}1G) has been cloned (41). Also Cav3.2 ({alpha}1H) is a candidate for T-type Ca2+ channels in endocrine cells as it was detected in the adrenal cortex (42). Furthermore, we assume that the Cav2.3-subunit forms a voltage-gated Ca2+ channel that is activated at more negative potentials than the L- and P/Q-type Ca2+ channels. If Cav2.3 would act in parallel to L- and P/Q-type channels, the insulin release should only be partially reduced in islets from Cav2.3-deficient mice. The assumed function of Cav2.3 as a mid voltage-gated Ca2+ channel is supported by electrophysiological measurements in rat supraoptic neurons (43) and in recombinant systems (44) as well as by the current-voltage relation of the resistant current component observed in INS-1 cells (24). Subsequent activation of the high-voltage-gated L- and P-/Q-type Ca2+ channels would then lead to the Ca2+-triggered release of insulin (Fig. 10Go). The disruption of Cav2.3 would interrupt the cascade of a successive activation and impair the insulin release. Thus, Cav2.3 would be an important modulator that determines the excitability of the ß-cell, and as hormonal regulation of Cav2.3 through G protein-coupled receptors is well known in vitro (45), it could modulate the insulin release in a positive and negative manner in vivo.



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  		Figure 10. Excitation Secretion Coupling in the ß-Cell of Islets of Langerhans

The secretion and the biosynthesis of insulin are involved in short- and long-term modulation of glucose homeostasis. The model describes our hypothesis concerning the successive activation of KATP channel, and low-, middle- and high-voltage-gated Ca2+ channels, which led to the final Ca2+-triggered release of insulin. Glucose is imported through the glucose transporter (Glut2) into the ß-cell. The rate of glucose phosphorylation, catalyzed via glucokinase (GK), determines the metabolic flux through the glycolysis and finally to the mitochondrial end oxidation (Mito). The cascade of membrane-spanning ion channels is activated by an increased ATP/ADP ratio dependent on the metabolic activity. The Cav2.3-subunit is activated as a midvoltage-activated Ca2+ channel that modulates the excitability of the ß-cell or other hormone-releasing cells. Successive activation leads to an increased depolarization of the membrane potential ({Delta}{Psi}), which finally triggers the Ca2+-induced release of insulin.

 
For the future, the model of successive activation needs further refinements to adopt to the more complex rhythmical electrical activity of the ß-cell and to the proposed spatial colocalization of Ca2+ channels with the exocytotic machinery of the ß-cell (46, 47).

Stress-Induced Hyperglycemia
The performance of the glucose tolerance test by itself causes stress and might influence the blood glucose content. Therefore, stress-induced effects on glucose homeostasis were investigated in the present study separately. Female mice were stressed by immobilization only after starvation. In rats and mice, stress-induced hyperglycemia is mediated mainly through epinephrine after starvation, whereas under fed conditions, glucagon and corticosterone interact with epinephrine-mediated increases of blood glucose (27, 48).

The reduced hyperglycemic response in Cav2.3-deficient mice demonstrates that the stress related to the whole procedure of the glucose tolerance testing probably does not interfere with the differences observed in glucose tolerance after ip injection of glucose. However, the results from the stress-induced hyperglycemia could be rather the consequence of an insufficient release of catecholamines from adrenal medulla. The detection of transcripts by RT-PCR in adrenal glands (9) and immunohistochemical data provide evidence for an expression of Cav2.3 in adrenal medulla but not in adrenal cortex (Weiergräber, M., and T. Schneider, unpublished results). Furthermore, the conclusion of an insufficient release of catecholamines is supported by the recent investigation of R-type Ca2+ channels in mouse adrenal chromaffin cells where this channel contributes to more than half of the rapid secretory response (49). Therefore, the drop in glucose release after ablation of Cav2.3 may be mediated through an impaired catecholamine release from adrenal gland, which would suggest that the deletion of Cav2.3 leads not only to an impaired insulin release but also influences other hormone release-triggered systems in the organism. Thus, it is assumed that Cav2.3 mediates and modulates the consecutive depolarizations during insulin secretion in islets of Langerhans as well as during the secretion of hormones from the adrenal medulla according to a model that includes the successive activation of low-, middle-, and high-voltage-gated Ca2+ channels (Fig. 10Go). Within both the stress-induced hyperglycemia and during insulin release, an alteration of the physiological response was observed during aging that might be interpreted as an adaptation of the Cav2.3-deficient organism by still unknown mechanisms to the impaired hormone release.

Differences in the pain perception in Cav2.3-deficient mice (17) and the expression of R-type Ca2+ channels in sympathetic nerves (12) may also explain the different stress response and lead to reduced hyperglycemia after immobilization. Future experiments will have to address these details.

Cav2.3-deficient mice represent an animal model useful to study fundamental mechanisms involved in glucose-induced insulin release and stress-induced hyperglycemia. As under physiological conditions the Ca2+-dependent insulin release is the primary mode of glucose-dependent secretion, the present report provides evidence, for the first time, that Cav2.3 may be an important mediator of insulin release, maybe even more in general a mediator of hormone release in the endocrine regions in which Cav2.3 is expressed. The mouse lines should be helpful in understanding the factors that influence hormone secretion itself and the mechanisms of adaptation to chronic impairment of hormone release.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Animals
Mice were housed at a constant temperature (22–23 C), with light from 0700–1900 h and ad libitum access to food and water. Animals were fed a breeding diet containing 22.5% crude protein, 5% crude fat, and 4.5% crude fiber (no. 1310, Altromin, Lage, Germany). All animal experimentation described in the text was conducted in accordance with accepted standards of humane animal care. The studies were approved by the institutional committee on animal care.

Gene targeting of Cav2.3 by Homologous Recombination and Disruption of cacna1e in Vivo Using Deleter Mice
Genomic clones containing exons 2 and 3 of the cacna1e gene were obtained from the genomic library {lambda} fix-II (Stratagene, La Jolla, CA) of 129/Svj mice. For the generation of the targeting vector, a 14-kb genomic fragment was subcloned into SalI-digested pBlueskript-SK vector. The loxP-flanked neomycin cassette was inserted into the NsiI site of intron 2 (Fig. 1Go). The third loxP site was inserted downstream of the HindIII site by a PCR-based strategy amplifying a 421-bp long DNA fragment between the HindIII site and the PflmI site. The proper amplification was confirmed by sequencing of the DNA fragment, after which the fragment containing the loxP site was introduced into the targeting vector (Fig. 1BGo).

Homologous recombination was performed in E14.1 embryonic stem cells (ES cells), and correctly targeted clones were selected by Southern blot analysis with internal and external probes (Fig. 2AGo). They were transiently transfected with the pCre-pac vector, and the efficiency of the transient expression of Cre-recombinase in ES cells was improved by using a 2-d treatment with puromycin as a selection marker (50). Surviving clones were analyzed for the loss of the neomycin resistance gene cassette (Neo) and the presence of a loxP-flanked exon 2 corresponding to a type II deletion (Fig. 1Go). ES cell subclones lacking only the Neo cassette were injected into C57Bl/6 blastocysts, and resulting male chimeras were bred to C57Bl/6 females. The Cav2.3(fl/+) genotype of agouti-colored offspring was determined by Southern blot analysis using as probe the Bst 1107 I–AseI fragment (Fig. 1AGo).

The cacna1e gene encoding Cav2.3 was disrupted in vivo by deleting a region containing exon 2 on mating Cav2.3(fl/+) and deleter mice (51), which express Cre-recombinase constitutively under the control of the cytomegalovirus promoter.

RT-PCR to Detect cacna1e Expression
Transcripts of Cav2.3 were detected by RT-PCR in whole-brain RNA as described recently (9). The oligonucleotide 290 [GenBank L27745, nucleotides (nt) 217–257] was used as forward primer, 5'-GGA GAA GAT AAC ATT GTC AGG AAA TAT GCC AAG AAG CTC AT-3', and the oligonucleotide 2,294 (nt 434–418) as reverse primer, 5'-GCC ACA ATT TTG ATC CC-3'. The expected size of the cDNA fragment is 218 bp for wild-type Cav2.3.

Isolation of Microsomal Membranes and Immunoblotting
Brain microsomes were isolated according to standard procedures (7, 52). One half of a brain was used to isolate microsomes without freezing the tissue. Aliquots of microsomal membranes were stored at -80 C. After immunoblotting two different sera were used as primary antibodies, anti-{alpha}1E-com and anti-{alpha}1E-spec (22), which are designed to recognize either an epitope between the extracellular loop IS5 and the pore region that is common to all cloned Cav2.3 splice variants, or an epitope within exon 44 (insert 3) of the carboxy terminus that is specific for the longer Cav2.3 splice variant detected in cerebellar Purkinje neurons (22) and endocrine cells (9).

Immunohistochemistry
Tissue sections of pancreas were fixed in 10% buffered formalin and embedded in paraffin. Immunohistochemistry was performed on 4- to 5-µm deparaffinized tissue sections that were mounted on silane-coated slides and dried at 50 C before staining. Sections underwent microwave-based, epitope-retrieval treatment (22). Sections were immunostained using polyclonal antiinsulin and antiglucagon sera (DAKO Corp., Carpinteria, CA) at a final protein concentration of 0.17 mg/ml. Visualization was through the streptavidin-biotin horseradish peroxidase technique using 3,3'-diaminobenzidine (DAB) as the chromogen.

Glucose Tolerance Test
Ten-, 21-, and 40-wk-old mice were used. Before each experiment, they were starved for 14 h but allowed free access to water. The glucose tolerance was tested by the ip injection of 2 mg D-glucose/g body wt (Delta-Pharma GmbH, Pfullingen, Germany). The blood glucose was determined in blood taken from the cut tail tip, before and 15, 30, 60, and 120 min after the administration of glucose. The glucose concentration was determined using the Glucometer Elite (Bayer Corp. Diagnostics GmbH, Leverkusen, Germany).

Measurement of Serum Insulin Levels
After ip injections, serum insulin was measured by the ELISA kit from Crystal Chem Inc. (Chicago, IL) using mouse insulin as a reference. Mice were fasted under the same regimen as described for the glucose tolerance test. Blood samples were taken from the cut tail before and 2, 5, 15, and 30 min after the administration of glucose. Insulin was determined in the serum according to the protocol of the manufacturer.

Isolation of Islets of Langerhans
Islets of Langerhans were obtained after ductal injection of 3–5 ml collagenase (no. 17449, Serva, Heidelberg, Germany) at a concentration of 0.3 mg/ml in Hanks buffer, pH 7.4, containing in addition 2 mM glucose and 5.8 mM CaCl2. The pancreas was removed and incubated in 6 ml Hanks buffer for 10 min at 37 C. The digestion was stopped by adding 30 ml of ice-cold Hanks buffer including 3% BSA and 2 mM glucose. The digested tissue fragments were diluted with Hanks buffer and transferred on ice. After mechanical disruption at 4 C, islets were collected under a dissection microscope (53) and maintained in a modified Hanks buffer supplemented with 2.0 mM glucose. The composition of Hanks buffer was (in mM): 137 NaCl, 5 KCl, 2.7 CaCl2, 0.8 MgSO4, 4.2 Na2HPO4, 4.4 KH2PO4, 4.2 NaHCO3, 1 HEPES.

For studying insulin release, five islets were maintained in Krebs-Ringer-bicarbonate solution at 2 mM glucose in a total volume of 0.5 ml. The basal and glucose-induced insulin release from the islets was analyzed by a RIA (24).

Immobilization Stress
Female mice were selected for the experiments to avoid the stress that is routinely observed for fighting male mice. Stress was induced by restraint in individual (7.5 cm x 4.0 cm x 2.5 cm) wire cages (27). Blood was taken from the tail as performed for the glucose tolerance test at the times indicated.

Data Analysis
Data are calculated and plotted throughout in the figures as the mean ± SEM. Significance was estimated by t test. Levels of P < 0.05 were considered statistically significant (*), and levels of P < 0.001 were considered as statistically highly significant (**).

Note Added in Proof
Endocrine disturbances in Cav2.3-deficient mice were also reported by Matsuda et al. (54). Furthermore, Cav1.3 ({alpha}1D) is required for proper ß-cell generation in the postnatal pancreas (55).


   ACKNOWLEDGMENTS
 
We thank Professor Dr. M. Taniguchi for the pCre-pac vector. We are grateful to Dr. Wolfgang Nastainczyk (Universität des Saarlandes, Homburg, Germany) for the synthesis of peptides and antibodies and to Professor Dr. Veit Flockerzi for the help during the screening of the genomic library. We are grateful to Prof. Dr. Klaus Rajewsky and Drs. Werner Müller and Ralph Kühn for their helpful gifts of reagents and advice. We thank Dr. Winfried Oswald, Frau Christiane Raab, Herr van de Burgwal, and the animal keepers of the central facility, as well as Frau Renate Clemens, Frau Ursula Tampier, and Frau Susanne Schulze for their technical assistance.


   FOOTNOTES
 
This work was supported by the Center of Molecular Medicine Cologne/Zentrum für Molekularbiologische Medizin Köln (Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie 01 KS 9502 to T.S. and J.H.).

Abbreviations: Cav2.3, Ion-conducting {alpha}1-subunit of E-type voltage-gated Ca2+ channels; Cre-recombinase, cyclic DNA-producing recombinase; E14.1, cell line of murine ES cells; ES cells, embryonic stem cells; loxP, recognition site for Cre; Neo, neomycin resistance gene cassette; nt, nucleotide.

Received for publication September 12, 2001. Accepted for publication December 3, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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