Beta-cell-specific ablation of the hepatocyte growth factor receptor results in reduced islet size, impaired insulin secretion, and glucose intolerance

Abstract

Hepatocyte growth factor (HGF) and its c-met receptor consist of a paired signaling system that has been implicated in the regulation of pancreatic beta-cell survival, proliferation, and function. To define the role of HGF/c-met signaling in beta-cell biology in vivo, we have generated conditional knockout mice in which the c-met receptor gene was specifically inactivated in pancreatic beta cells by the Cre-loxP system. Mice with beta-cell-specific deletion of the c-met receptor (betamet-/-) displayed slight growth retardation, mild hyperglycemia, and decreased serum insulin levels at 6 months of age when compared with their control littermates. Deficiency of the c-met receptor in beta cells resulted in a complete loss of acute-phase insulin secretion in response to glucose and an impaired glucose tolerance. Glucose transporter-2 expression was down-regulated in the beta cells of betamet-/- mice. Compared to controls, betamet-/- mice exhibited reduced islet size and decreased insulin content in the pancreas, but displayed normal islet morphology. Therefore, HGF/c-met signaling plays an imperative role in controlling islet growth, in regulating beta-cell function, and in maintaining glucose homeostasis.

Figure 1

Tissue-specific ablation of the HGF receptor (c-met) in pancreatic β cells. A: Diagram illustrates the strategy of generating β-cell-specific c-met knockout mice. Shown is the deletion of exon 16 in the event of recombination of the c-met gene. B: Representative picture shows PCR analysis of the genomic DNA from tail clippings. The PCR bands of wild-type (300 bp), floxed (380 bp), and Cre (411 bp) are indicated. Genotypes of representative litters are indicated. fl, c-met floxed. C: Cre-mediated excision of the floxed c-met allele in a tissue-specific manner. DNA was isolated from various tissues of the control (met^lox/lox) and βmet^−/− (met^lox/lox, Cre) mice and analyzed by PCR with appropriate primers. A band after Cre excision was only observed in the isolated islets of βmet^−/− mice (lane 2). D to G: Immunofluorescence staining demonstrates c-met and Cre expression in pancreatic islets of the control and βmet^−/− mice. D and E, Cre (green); F and G, c-met (red); D and F, controls; E and G, βmet^−/− mice. The location of islets in pancreatic sections was confirmed by staining cell nuclei with PI (data not shown). H: Western blot analysis of c-met protein expression in the islets isolated from the control and βmet^−/− mice. Islet lysates were immunoblotted with antibodies against c-met and actin, respectively. The lower band (below 145 kd) is nonspecific (n. s.). I: Western blot analysis shows the activation of Erk-1/2 and Akt in the isolated islets in response to HGF stimulation. Pancreatic islets were isolated from the control and βmet^−/− mice, pooled from five different animals for each group and incubated with 20 ng/ml of HGF for various periods of time as indicated. Islet lysates were immunoblotted with antibodies against p-Erk-1/2, total Erk-1/2, p-Akt, and total Akt, respectively. Relative abundances (fold induction over 0 time point) of p-Erk1/2 and p-Akt after normalization are presented at the bottom of Western blot pictures.

Figure 2

βmet^−/− mice display impaired acute-phase insulin secretion in response to glucose but not to l-arginine. Glucose and l-arginine were injected intraperitoneally, respectively, in the male (A and C) and female (B and D) control and βmet^−/− mice at 6 months of age after a 24-hour fast. A loss of acute-phase insulin secretion was observed in both male and female βmet^−/− mice in response to glucose (A, B), but not to arginine (C, D). *P < 0.05, n = 4.

Figure 3

βmet^−/− mice show impaired glucose tolerance but normal insulin sensitivity. Glucose tolerance tests were performed in the male (A) and female (B) control and βmet^−/− mice at 6 months of age in a fasted state. Glucose intolerance was observed in both male and female βmet^−/− mice. *P < 0.05, n = 6 to 8. Insulin-sensitive tests were performed in the male (C) and female (D) control and βmet^−/− mice at 6 months of age under normal diet conditions. n = 3.

Figure 4

Specific deletion of c-met in mice decreased Glut-2 expression in pancreatic β cells. A, B: Semiquantitative RT-PCR demonstrates decreased Glut-2 mRNA levels in the pancreatic islets isolated from βmet^−/− mice, compared with the controls. Graphical presentation shows the relative Glut-2 mRNA abundance in the control and βmet^−/− islets after normalization to actin. *P < 0.05, n = 3. C, D: Representative Western blot and graphical presentation show decreased Glut-2 protein levels in the pancreatic islets isolated from βmet^−/− mice, compared with the controls. *P < 0.05, n = 4. E, F: Representative micrographs demonstrate localization and abundance of Glut-2 protein in the islets of control (E) and βmet^−/− mice (F).

Figure 5

β-Cell-specific ablation of c-met results in reduced islet size but maintains normal islet structure at 6 months of age. A: Reduced islet area was observed in βmet^−/− mice at 6 months of age, compared to the controls. The percentage of islet/pancreas areas was measured in pancreatic sections stained with insulin and data were presented as mean ± SE. *P < 0.05, n = 8. B: Pancreatic insulin content was decreased in βmet^−/− mice at 6 months of age, compared to the controls. Insulin levels were expressed as ng/mg pancreas. *P < 0.05, n = 4. C: No difference in relative insulin content in the islets of the control and βmet^−/− mice was found. Islet insulin content was measured in acid-ethanol extracts and expressed as ng/μg protein (n = 4). D: No difference in relative abundance of insulin mRNA levels was observed in the islets of the control and βmet^−/− mice (n = 3). E to J: Representative micrographs show islet structure and morphology in the control (E to G) and βmet^−/− mice (H to J) after staining with various antibodies. E and H, insulin; F and I, glucagons; G and J, somatostatin.