Elevated Hedgehog/Gli signaling causes beta-cell dedifferentiation in mice - PubMed (original) (raw)
Elevated Hedgehog/Gli signaling causes beta-cell dedifferentiation in mice
Limor Landsman et al. Proc Natl Acad Sci U S A. 2011.
Abstract
Although Hedgehog (Hh) signaling regulates cell differentiation during pancreas organogenesis, the consequences of pathway up-regulation in adult β-cells in vivo have not been investigated. Here, we elevate Hh signaling in β-cells by expressing an active version of the GLI2 transcription factor, a mediator of the Hh pathway, in β-cells that are also devoid of primary cilia, a critical regulator of Hh activity. We show that increased Hh signaling leads to impaired β-cell function and insulin secretion, resulting in glucose intolerance in transgenic mice. This phenotype was accompanied by reduced expression of both genes critical for β-cell function and transcription factors associated with their mature phenotype. Increased Hh signaling further correlated with increased expression of the precursor cell markers Hes1 and Sox9, both direct Hh targets that are normally excluded from β-cells. Over time, the majority of β-cells down-regulated GLI2 levels, thereby regaining the full differentiation state and restoring normoglycemia in transgenic mice. However, sustained high Hh levels in some insulin-producing cells further eroded the β-cell identity and eventually led to the development of undifferentiated pancreatic tumors. Summarily, our results indicate that deregulation of the Hh pathway impairs β-cell function by interfering with the mature β-cell differentiation state.
Conflict of interest statement
The authors declare no conflict of interest.
Figures
Fig. 1.
Glucose intolerance correlates with increased Hh signaling in adult β-cells. _Pdx1_-CreER;CLEG2 (A_–_D, blue lines; n ≥ 4) and _Pdx1_-_CreER;CLEG2;Kif3a_f/f (D_–_G, red lines; n ≥ 7) transgenic (tg) and nontransgenic (non tg; black lines; n ≥ 8) males were i.p. injected with TAM (2 mg per mouse on 5 consecutive days at 8–10 wk of age). Glucose tolerance test were performed before TAM injection (A and E) and 1 wk (B and F) or 4 wk (C and G) after injection. (A_–_C and E_–_G) After overnight fasting, mice were i.p. injected with dextrose (2 mg/g body weight) and their blood glucose levels were measured at indicated times. (D) Islets from control and transgenic mice were isolated 3–5 wk after TAM injection, their RNA was extracted, and Ptch1 and Gli1 expression levels were analyzed by qPCR (n = 3). *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001, and *****P < 0.0005 (Student's t test). Data represent mean ± SD.
Fig. 2.
Impaired glucose-stimulated insulin secretion by transgenic β-cells. Pdx1-CreER;CLEG2;_Kif3_af/f mice were presented with defects in β-cell function 4 wk after TAM injection. (A) Reduced serum insulin levels in transgenic mice (black) compared with nontransgenic control (gray). Blood was collected from tail veins before and 30 min after glucose injection (3 mg/g body weight), and serum insulin levels were determined by ELISA (n = 3). (B) Transgenic islets have impaired glucose-stimulated insulin secretion. Isolated islets from TAM-treated transgenic (black) and nontransgenic (gray) mice were incubated with either low (30 mg/dL) or high (300 mg/dL) glucose, and supernatant was collected and analyzed by ELISA (n = 3). (C) Protein extract of isolated islets treated as described in B was analyzed for insulin levels by ELISA. Transgenic islets show significantly reduced insulin levels (n = 3). (D) Pancreatic tissues from TAM-injected transgenic (Right) and control (Left) mice were immunostained for Insulin (green) and Glut2 (red). (E) RNA was extracted from transgenic islets (black) and nontransgenic (gray) mice 3–5 wk after TAM injection. Islets from untreated transgenic mice served as additional controls (white bars). The expression of Insulin1, Insulin2, and Glucagon genes was analyzed by qPCR (n = 4). (F and G) Reduced expression of mature β-cell genes in transgenic islets. RNA was extracted from isolated islets (see above in E), and expression of indicated genes was analyzed by qPCR (n = 4). (H and I) Reduced Glut2 protein levels in transgenic islets. Western blot analysis for Glut2 was performed on isolated islets 4 wk after TAM. A representative blot is shown (H), and quantification of data from four mice in each group is provided (I). Protein levels were normalized to GAPDH, and average levels in nontransgenic controls was set to “1”. For statistical analysis, unpaired two-tailed Student's t test was performed. All data are in comparison with TAM-treated nontransgenic controls. P values: *P < 0.05, **P < 0.01, ***P < 0.005, NS, nonsignificant. Data represent the mean ± SD.
Fig. 3.
Change in transcription factors expression in transgenic β-cells. (A_–_C) Reduced expression of β-cell transcription factors in transgenic islets 3–5 wk after TAM. (A) RNA was extracted from Pdx1-CreER;CLEG2;_Kif3_af/f (black) and nontransgenic (non tg, gray) isolated islets and analyzed by qPCR for mRNA expression levels of indicated genes (n = 4). (B and C) Western blot analysis shows reduced levels of Pdx1 protein in transgenic islets (black bars) 4 wk after TAM (compared with nontransgenic control; gray bars). (C) Quantification for Western blot. Data represent protein levels normalized to GAPDH. Mean levels in nontransgenic control was set to “1” (n = 5) (D_–_H) Sox9 expression in TAM-treated transgenic β-cells. (G) RNA was isolated from control (gray) and transgenic (black) islets, and qPCR analysis was performed (n = 4). (E and F) Western blot analysis of Sox9 protein expression. A representative blot is shown (E), and quantification of data from four mice in each group is provided (F). Data represent protein levels normalized to GAPDH. Mean levels in nontransgenic control was set to “1”. (G and H) Pancreatic tissues from transgenic (H) and control (G) mice were immunostained for Sox9 (red) and Insulin (green). Arrows point to Insulin and Sox9 double-positive cells. (I_–_K) Hes1 is expressed in TAM-treated transgenic islets. (I) qPCR analysis for Hes1 expression was performed on islets isolated from transgenic islets (black bars) and nontransgenic controls 4 wk after TAM (n = 4). (J and K) Immunohistochemistry for Hes1 (brown) and Hematoxilin (blue) of transgenic (K) and control (J) tissues. *P < 0.05, **P < 0.01, ***P < 0.005 (Student's t test). Bar diagrams represent mean ± SD.
Fig. 4.
Restoration of glucose sensitivity and mature β-cell gene expression in aged Pdx1-CreER;CLEG2;_Kif3_af/f mice. (A and B) Blood glucose levels of transgenic (black bars) and control (gray bars) mice after overnight fast (A) or upon normal fed conditions (B) were analyzed at different time points after TAM injection (n = 5). (C) Normal glucose response of transgenic mice 10 wk after TAM injection (red line) compared with control (black line). Mice were treated as described in Fig. 1 (n = 4). (D and E) Restoration of Insulin and Glut2 expression in transgenic islets 10 wk after TAM injection. qPCR analysis (D) shows normal expression levels (n = 4) and immunofluorescence (E) confirms normal expression pattern of Insulin (green) and Glut2 (red) in the majority of islet cells. Asterisks mark cells negative for both markers. (F) Reduced number of Myc/GLI2-expressing β-cells 10 wk after TAM injection. Transgenic tissues 4 and 10 wk after TAM injection were stained with antibodies against the Myc-tag (which detects the Myc/GLI2 transgenic fusion protein) and Insulin, and the percentages of Myc/GLI2+ (identified as Myc+Insulin+ double-positive cells) out of total β-cell population (identified as Insulin+ cells) were counted. Images were exposed for extended period to ensure detection of all insulin-expressing cells. (n = 3). *P < 0.05 (Student's t test). Data represent the mean ± SD.
Fig. 5.
Prolonged Hh activation in β-cells promotes tumor formation. (A and B) Pancreatic tumors express the Myc/GLI2 transgene, are proliferative, and lack insulin expression. Pancreatic tissue from TAM-treated _Pdx1_-_CreER;CLEG2;Kif3a_f/f mouse (A) and an 8-mo-old _Ins1_-_Cre;CLEG2;Kif3a_f/f mouse (B) were stained with antibodies against insulin (blue), the proliferation marker KI67 (green), and Myc-tag (red, identifying the Myc/GLI2 fusion protein). (C) Tumors display increased Hh signaling levels. RNA was extracted from islets isolated from nontransgenic mice (gray bars) and _Ins1_-_Cre;CLEG2;Kif3a_f/f tumors (black bars). Expression of the Hh signaling target genes Ptch1 and Gli1 was analyzed by qPCR (n = 4). (D) Tumors lack Insulin1 transcript expression. RNA was extracted as described in C, and Insulin1 expression levels were analyzed by qPCR (n = 4). (E) Tumors express precursor cell genes. RNA was extracted from islets as described in C, and Sox9 and Hes1 expression levels were analyzed by qPCR (n = 4). *P < 0.05, **P < 0.01, ***P < 0.005 (Student's t test). Data represent the mean ± SD.
Fig. 6.
A model for the effect of increased Hh signaling on β-cell differentiation state. Mature β-cells express low levels of Hh signaling (Left) (5). Increased Hh signaling leads to dedifferentiation of β-cells (Center), accompanied by reduced expression of β-cell marker genes, and increased precursor genes expression. β-cells dedifferentiation is reversible, and cells can reacquire their differentiation state upon down-regulation of Hh signaling. However, when high Hh signaling persists, cells further lose their β-cells identity (Right) and no longer express insulin. Precursor genes expression increases in those β-cell–derived undifferentiated cells.
References
- Grant SF, et al. Variant of transcription factor 7-like 2 (TCF7L2) gene confers risk of type 2 diabetes. Nat Genet. 2006;38:320–323. - PubMed
- Hanley SC, et al. beta-Cell mass dynamics and islet cell plasticity in human type 2 diabetes. Endocrinology. 2010;151:1462–1472. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Molecular Biology Databases
Research Materials