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. 2009 Feb;58(2):433-41.
doi: 10.2337/db08-0749. Epub 2008 Nov 25.

A role for von Hippel-Lindau protein in pancreatic beta-cell function

Sapna Puri  1 , David A Cano, Matthias Hebrok
Affiliations

A role for von Hippel-Lindau protein in pancreatic beta-cell function

Sapna Puri et al. Diabetes. 2009 Feb.

Abstract

Objective: The Vhlh gene codes for the von Hippel-Lindau protein (VHL), a tumor suppressor that is a key player in the cellular response to oxygen sensing. In humans, a germline mutation in the VHL gene leads to the von Hippel-Lindau disease, a familial syndrome characterized by benign and malignant tumors of the kidney, central nervous system, and pancreas.

Research design and methods: We use Cre-lox recombination to eliminate Vhlh in adult mouse pancreatic beta-cells. Morphology of mutant islets is assessed by immunofluorescence analysis. To determine the functional state of Vhlh(-/-) islets, insulin secretion is measured in vivo and in vitro, and quantitative PCR is used to identify changes in gene expression.

Results: Loss of VHL in beta-cells leads to a severe glucose-intolerant phenotype in adult animals. Although VHL is not required for beta-cell specification and development, it is critical for beta-cell function. Insulin production is normal in beta-cells lacking VHL; however, insulin secretion in the presence of high concentrations of glucose is impaired. Furthermore, the loss of VHL leads to dysregulation of glycolytic enzymes, pointing to a perturbation of the intracellular energy homeostasis.

Conclusions: We show that loss of VHL in beta-cells leads to defects in glucose homeostasis, indicating an important and previously unappreciated role for VHL in beta-cell function. We believe that the beta-cell-specific Vhlh-deficient mice might be a useful tool as a "genetic hypoxia" model, to unravel the possible link between hypoxia signaling and impairment of beta-cell function.

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Figures

FIG. 1.
FIG. 1.
VHL inactivation in β-cells leads to glucose intolerance. A: In the absence of tamoxifen, control (black line, Pdx-1-CreER;VHL+/LoxP, n = 6) and Pdx-1-CreER;VHLLoxP/LoxP(gray line, n = 3) mice normalize blood glucose 120 min after challenge. B: At 120 min after glucose challenge, Pdx-1-CreER;VHLLoxP/LoxP mice (gray line) injected with tamoxifen have elevated glucose levels compared with control mice (black line). Eleven animals were analyzed per group from eight independent cohorts. *P < 0.001, ***P < 10−8. C: PCR on genomic DNA from transgenic islets heterozygous and homozygous for the VHLLoxP/LoxP allele demonstrates the extent of Cre-mediated excision. Both samples are positive for Pdx-1-CreER. Top panel: The genotype of the islets. The wild-type allele in the heterozygous sample (*) runs below the floxed allele. Both mice were injected with tamoxifen and display a robust signal for the excised allele (lower panel). D: Adult Ins-Cre;VHLLoxP/LoxP mice are glucose intolerant. Two hours after the glucose challenge, the 8- to 10-week-old Ins-Cre;VHLLoxP/LoxP mice (gray line, n = 14) have elevated glucose levels compared with the control littermates (black line, n = 11). Control and mutant animals from nine independent cohorts were used for the analysis. *P < 0.001, ***P < 10−8. Error bars represent SD in all cases.
FIG. 2.
FIG. 2.
Islet morphology upon VHL inactivation in adult β-cells. Hematoxylin and eosin (H&E) analysis of control (A) and Vhlh−/− (B) tissue shows aberrant islet architecture due to increased blood vessel density. Bar, 20 μm. Immunostaining for insulin (C and D) and PECAM-1 (E and F) reveals that islet morphology in tamoxifen-injected Pdx-1-CreER;VHLLoxP/LoxP mice (D, F, and H) displays increased vasculature compared with the control mice (C, E, and G). The overlay images are shown in G and H. Bar, 50 μm. I: Real-time PCR analysis on islets isolated from tamoxifen-injected Pdx-1-CreER; VHLLoxP/LoxP mice shows an increase in Vegf expression (error bars represent SD, n = 5. **P < 0.005). (Please see http://dx.doi.org/10.2337/db08-0749 for a high-quality digital representation of this image.)
FIG. 3.
FIG. 3.
VHL is not essential for islet formation and β-cell differentiation. Adult mice lacking VHL in β-cells maintain islet architecture. Insulin-expressing (green, A–H), glucagon-expressing (red, A and B), and somatostatin-expressing (red, C and D) cells are found in the control (A and C) and mutant tissue (B and D). Mature β-cell markers Nkx6.1 (red, E and F) and Pax6 (red, G and H) continue to be expressed in the absence of VHL. Bar, 50 μm. (Please see http://dx.doi.org/10.2337/db08-0749 for a high-quality digital representation of this image.)
FIG. 4.
FIG. 4.
VHL inactivation in adult β-cells leads to a block in insulin secretion. A: Serum insulin measurements 30 min after glucose challenge show an absence of circulating insulin in tamoxifen-injected Pdx-1-CreER;VHLLoxP/LoxP mutant mice compared with control littermates. Four animals per group were included in the analysis from four independent cohorts. *P < 0.05. B: In vitro insulin secretion assay carried out on isolated islets demonstrates a block in insulin secretion upon incubation with high (16.67 mmol/l) glucose in the absence of VHL. Basal secretion (low glucose, 1.67 mmol/l) was comparable between control and mutant islets. n = 3. ***P < 0.0005. C: Incubation of mutant islets with 40 mmol/l KCl in the presence of high (16.67 mmol/l) glucose induces insulin secretion in vitro. n = 3. **P < 0.001. Significance (A–C) was determined between the amount of insulin secreted under high glucose conditions from control and mutant islets using the Student's t test. D: Real-time PCR of insulin transcript levels shows a moderate decrease of insulin in mutant animals (gray bars) when compared with the control animals (black bars) (Ins1, P > 0.1; Ins2, P > 0.01). n = 3. E: Total insulin levels were calculated in islets isolated from control (black bar) or mutant animals (gray bar). P > 0.1, n = 3. Error bars represent SD in all cases.
FIG. 5.
FIG. 5.
Glucose metabolism genes dysregulated in islets upon VHL inactivation. A: Quantitative PCR analysis of relative transcript levels of glycolytic enzymes reveals an upregulation of several members of the metabolic machinery in the absence of VHL. The housekeeping gene cyclophilin A was used to normalize gene expression. Changes in glucose transporters were also observed. The canonical HIF target gene Glut-1 is upregulated, whereas Glut-2 is downregulated. The change in expression of genes in Vhlh−/− islets was normalized to expression in control animals that was set to one (represented by the dotted line). Between three and five mutant and control animals were used for isolation of islets for RNA. B: Immunostaining for GLUT2 confirms significant downregulation of the transporter in tamoxifen-injected Pdx-1-CreER;VHLLoxP/LoxP islets. C: Increased expression of genes regulating the formation and export of lactate in islets. Transcript levels of lactate dehydrogenase (Ldha), pyruvate dehydrogenase kinase (PDK), and the monocarboxylate transporter MCT4 are dramatically increased in Vhlh−/− islets, as detected by quantitative PCR. D: β-Cells lacking VHL secrete an increased amount of lactate into the surrounding medium (gray bar graph) compared with control islets (black bar graph). Islets isolated from three animals per group from three independent cohorts were included in the analysis. *P < 0.05, **P < 0.01, ***P < 0.005. Error bars represent SD in all cases. (Please see http://dx.doi.org/10.2337/db08-0749 for a high-quality digital representation of this image.)
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