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Impaired insulin secretory capacity in mice lacking a functional vitamin D receptor¹

UTE ZEITZ^*, KARIN WEBER^*, DESI W. SOEGIARTO^,2, ECKHARD WOLF, RUDI BALLING^,3 and REINHOLD G. ERBEN^*4

^* Institute of Animal Physiology, Ludwig Maximilians University, 80539 Munich, Germany;
Institute of Mammalian Genetics, GSF National Research Center for Environment and Health, 85758 Neuherberg, Germany; and
Institute of Molecular Animal Breeding, Ludwig Maximilians University, 81377 Munich, Germany

⁴Correspondence: Institute of Animal Physiology University of Munich, Veterinaerstrasse 13, D-80539, Munich, Germany. E-mail: R.Erben{at}lrz.uni-muenchen.de

SPECIFIC AIMS

Apart from its essential role in mineral metabolism, the vitamin D hormone 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] may have important functions in other organ systems such as the endocrine pancreas. By gene targeting, we have recently generated mice in which a lacZ reporter gene is driven by the endogenous vitamin D receptor (VDR) promoter. These mice express a functionally inactive mutant VDR. To explore further the functional role of vitamin D for the endocrine pancreas, we investigated glucose tolerance, insulin secretion, and pancreas islet morphology in these VDR mutant mice.

PRINCIPAL FINDINGS

1. Strong reporter gene expression in pancreatic islets
To visualize lacZ reporter gene expression, we performed pre-embedding X-gal staining of the pancreas from wild-type and homozygous VDR mutant mice. In agreement with earlier findings of VDR expression in insulin-producing ß cells, our VDR mutant mice showed strong lacZ reporter gene expression in pancreatic islets (Fig. 1 ). The surrounding exocrine pancreas cells did not exhibit lacZ expression.

View larger version (82K): [in this window] [in a new window]   Figure 1. Strong lacZ reporter gene expression in pancreas islets of VDR mutant mice. Paraffin sections of pancreas from 10-wk-old wild-type and homozygous VDR mutant mice after lacZ staining. A) Islet cells of the pancreas show intense lacZ expression in the homozygous VDR mutant mouse. Exocrine pancreas cells are negative. B) Staining is completely absent in the wild-type mouse. Five µm-thick paraffin sections; lacZ expression is visualized by X-Gal staining.

2. Glucose tolerance and insulin secretory capacity are impaired in VDR mutants
It is well known that calcium is an important factor in regulating insulin secretion in pancreatic ß cells. Therefore, the hypocalcemia present in VDR mutant mice on a standard mouse diet with 0.9% calcium and 0.7% phosphorus (means±SE: 0.94±0.02 mmol/L ionized blood calcium in homozygous mutants vs. 1.24±0.01 in wild-type controls, P<0.05) could be a major confounding factor in the current investigation. To correct calcium homeostasis in VDR mutant mice, we fed a so-called ‘rescue diet’ containing 2% calcium, 1.25% phosphorus, and 20% lactose, starting from 16 days of age. This rescue diet fully normalized body weight, total serum calcium, ionized calcium, serum phosphate, alkaline phosphatase, and serum parathyroid hormone in 10-wk-old homozygous VDR mutants.

We then performed oral glucose tolerance tests in homozygous VDR mutants and wild-type control mice on normal and rescue diets. After an overnight fasting period, blood glucose was unchanged in VDR mutant mice (Fig. 2 A, B). Baseline insulin levels were comparable in wild-type and VDR mutant mice on the normal diet, but mutant mice on the rescue diet showed lower serum insulin levels (Fig. 2C ). When the mice were challenged with a single oral dose of glucose, blood glucose reached significantly higher values in VDR mutants than wild-type controls regardless of diet (Fig. 2A, B ). When we measured maximum insulin serum levels 10 min after oral glucose administration, we found a 65% and 63% reduction in insulin levels relative to wild-type controls in VDR mutant mice on the normal and the rescue diet, respectively (Fig. 2C ). These findings indicate that oral glucose tolerance and insulin secretion are impaired in normocalcemic and normophosphatemic mice with a nonfunctioning VDR. To rule out that these changes were caused by endocrine signals originating from the gut in response to oral glucose loading, we performed subcutaneous (s.c.) glucose tolerance tests in wild-type and VDR mutant mice on the rescue diet (Fig. 2D ). The results showed that not only oral but also s.c. glucose tolerance was impaired in normocalcemic VDR mutant mice.

View larger version (29K): [in this window] [in a new window]   Figure 2. Mice with a nonfunctioning VDR have impaired oral glucose tolerance and reduced maximum insulin secretory capacity. A, B) Oral glucose tolerance (n=9-11 each) in 10-wk-old male wild-type (wt/wt) and homozygous VDR mutant mice (/) fed a standard (A) or a rescue diet (B). Glucose (1.5 mg/kg body weight) was administered at time 0 by gavage after an overnight fast. C) Serum insulin levels at baseline and 10 min after an oral glucose challenge (1.5 mg/kg bw) in wild-type and VDR mutant mice on the normal (ND) and rescue (RD) diet (n=7-16 each). D) Glucose (s.c.) tolerance (1.5 mg/kg bw) in wild-type and VDR mutant mice on the rescue diet (n=6 each). E) Representative Northern analysis of total RNA (10 µg per lane) isolated from the whole pancreas of nonfasting, 10-wk-old male wild-type and homozygous VDR mutant mice on the normal or rescue diet. Blots were hybridized with a 0.4 kb probe specific for both mouse insulin genes, using a 0.9 kb ß-actin-specific probe as loading control. Data represent mean ± SE. *P < 0.05 vs. wild-type by t test or U test.

3. Pancreas from VDR mutants shows reduced insulin mRNA content
To examine changes in the expression of insulin at the mRNA level, we performed Northern analyses of total RNA isolated from the whole pancreas of nonfasting mice. Relative to wild-type controls, VDR mutant mice showed a distinct decrease in insulin mRNA levels (Fig. 2E ). The rescue diet failed to restore insulin mRNA levels in VDR mutants.

4. VDR mutant mice have pancreas islets of normal size and number
It is evident that all the observed changes in VDR mutant mice could be explained by a reduced pancreatic ß cell mass. Therefore, we analyzed the number and size of pancreas islets by histomorphometry and cell distribution within the islets by immunohistochemistry in 10-wk-old mice. Because the impairment in endocrine pancreas function was similar in mutants on the normal and the rescue diet, we performed these measurements only in mice on the normal diet. We found that both the size (means±SE: 7320±160 µm² in VDR mutants vs. 7739±635 µm² in wild-type controls) and the number of islets (means±SE: 0.51±0.06 per mm² pancreas tissue in VDR mutants vs. 0.53±0.05 in wild-type controls) were unchanged in the pancreas of VDR mutants relative to wild-type mice. It has become increasingly clear that ß cell mass is dynamic and that ß cells in the pancreas are subject to a lifelong renewal process, at least in rodents. Therefore, we measured the number of small clusters of ß cells as an index of islet neogenesis. However, mutant mice had unchanged numbers of small ß cell clusters (means±SE: 0.48±0.10 per mm² pancreas tissue in VDR mutants vs. 0.49±0.06 in wild-type controls), indicating normal islet neogenesis in the absence of VDR signaling. Similarly, immunohistochemical analysis showed that the distribution of cells producing insulin, glucagon, somatostatin, and pancreatic polypeptide within the islets of Langerhans was indistinguishable between wild-type and mutant mice. These results indicate that the impaired glucose tolerance and insulin secretion in VDR mutant mice cannot be explained by alterations in pancreatic ß cell mass or microanatomical changes within pancreas islets.

CONCLUSIONS

This study has demonstrated that disruption of the VDR signaling pathway is associated with a pronounced impairment in oral glucose tolerance and insulin secretory capacity, together with a reduction in pancreatic insulin mRNA levels in normally fed mice. These changes were independent of alterations in body weight or mineral homeostasis. Thus, the data provided by the current experiments corroborate earlier findings of impaired insulin secretion in vitamin D-deficient rats and clearly establish a molecular role of the VDR in the endocrine function of the pancreas in vivo.

It is well known that oral glucose induces the release of gastrointestinal hormones that stimulate pancreatic insulin secretion such as glucose-dependent insulinotropic polypeptide or glucagon-like peptide 1, resulting in augmented insulin secretion in response to oral glucose loading compared with systemic glucose administration. Therefore, it is conceivable that the effect of vitamin D on insulin secretion involves this enteroinsular axis. However, our finding of a similar impairment in both oral and s.c. glucose tolerance in VDR mutants suggests that alterations in the enteroinsular axis are not involved in the diminished insulin secretory response in mice with a nonfunctioning VDR.

VDR mutant mice demonstrated normal pancreatic ß cell mass, normal architecture of pancreas islets, and a normal number of small clusters of insulin-producing cells. These findings suggest that fetal and postnatal islet development as well as the intensity of islet neogenesis during postnatal life is unchanged in VDR mutant mice. Our study shows that the reduced insulin secretory capacity in VDR mutants is not based on a developmental or structural defect, but rather is caused by functional alterations within insulin-producing cells (Fig. 3 ). Because mice with a nonfunctioning VDR had normal fasting blood glucose and insulin levels, the defect in pancreatic insulin secretion is latent and is seen only when the mice are challenged with glucose.

View larger version (41K): [in this window] [in a new window]   Figure 3. Schematic diagram of the effects of the vitamin D hormone (1,25(OH)2D3) on the endocrine pancreas. Consistent with the strong expression of the lacZ reporter gene in ß cells, 1,25(OH)2D3 influences endocrine pancreas function through a direct effect on insulin-producing ß cells in the islets of Langerhans. Acting through the intracellular vitamin D receptor (VDR), the vitamin D hormone directly stimulates insulin synthesis in ß cells and may also influence the cellular mechanisms involved in insulin secretion. Disruption of the vitamin D signaling pathway results in reduced insulin secretion after glucose loading. However, the VDR has no role in fetal or postnatal islet development or postnatal neogenesis of pancreas islets (arrow marks small cluster of insulin-producing ß cells in murine pancreas immunolabeled with an anti-insulin antibody).

It is unclear whether 1,25(OH)₂D₃ regulates only the biosynthesis of insulin or whether 1,25(OH)₂D₃ can also influence the mechanisms of insulin secretion (Fig. 3) . In our study, the 60% reduction in maximum insulin secretion observed in response to an oral glucose challenge was associated with a distinct decrease in pancreatic insulin mRNA content in nonfasting VDR mutant mice. Thus, the impaired insulin secretion in VDR mutants may be caused by a reduction in the amount of insulin stored in ß cells. The stimulatory effect of 1,25(OH)₂D₃ on islet cell insulin synthesis may involve increased transcriptional activity of the insulin gene or increased insulin mRNA stability. So far, no vitamin D response elements were reported in the human or mouse insulin gene promoters.

In conclusion, the present data have demonstrated a molecular role of the VDR in pancreatic endocrine function and may provide a functional basis for the association between diabetes and VDR in epidemiological studies. Although our study has shown that the VDR is not involved in islet morphogenesis or postnatal regulation of ß cell mass in the mouse, disturbances in the vitamin D signaling pathway may compromise the ß cell’s ability to functionally respond to situations of an increased insulin demand such as in type 2 diabetes or in the prediabetic phase of type 1 diabetes. More extensive experimentation is required to define the molecular pathways by which 1,25(OH)₂D₃ regulates pancreatic insulin synthesis.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0424fje; to cite this article, use FASEB J. (January 22, 2003) 10.1096/fj.02-0424fje

² Present address: MBT Munich Biotechnology GmbH, Fraunhoferstrasse 10, 82152 Martinsried, Germany.

³ Present address: GBF Gesellschaft für Biotechnologische Forschung, Mascheroder Weg 1, 38124 Braunschweig, Germany.

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