Genetic Disruption of Adenosine Kinase in Mouse Pancreatic β-Cells Protects Against High-Fat Diet-Induced Glucose Intolerance

Abstract

Islet β-cells adapt to insulin resistance through increased insulin secretion and expansion. Type 2 diabetes typically occurs when prolonged insulin resistance exceeds the adaptive capacity of β-cells. Our prior screening efforts led to the discovery that adenosine kinase (ADK) inhibitors stimulate β-cell replication. Here, we evaluated whether ADK disruption in mouse β-cells affects β-cell mass and/or protects against high-fat diet (HFD)-induced glucose dysregulation. Mice targeted at the Adk locus were bred to Rip-Cre and Ins1-Cre/ERT^1Lphi mice to enable constitutive (βADKO) and conditional (iβADKO) disruption of ADK expression in β-cells, respectively. Weight gain, glucose tolerance, insulin sensitivity, and glucose-stimulated insulin secretion (GSIS) were longitudinally monitored in normal chow (NC)-fed and HFD-fed mice. In addition, β-cell mass and replication were measured by immunofluorescence-based islet morphometry. NC-fed adult βADKO and iβADKO mice displayed glucose tolerance, insulin tolerance and β-cell mass comparable to control animals. By contrast, HFD-fed βADKO and iβADKO animals had improved glucose tolerance and increased in vivo GSIS. Improved glucose handling was associated with increased β-cell replication and mass. We conclude that ADK expression negatively regulates the adaptive β-cell response to HFD challenge. Therefore, modulation of ADK activity is a potential strategy for enhancing the adaptive β-cell response.

Figure 1

Conditional disruption of ADK gene expression. A: Schematic representation of the Adk locus and targeting construct: mutagenic orientation (ADK¹), Flp recombinase–dependent nonmutagenic orientation (ADK²), and Cre recombinase–dependent mutagenic orientation (ADK³). Forward primer (Fwd), reverse primer (Rev), B32 primer, recombination sequences (Frt and LoxP), SA, and the β-galactosidase/βgeo are shown. B: Histochemical staining of pancreatic sections from Adk-targeted mice for β-galactosidase activity (blue); counterstaining with eosin (pink, top row) or insulin (brown, bottom row) are shown. C: ADK-directed Western blot of hepatic tissue lysates from Adk-targeted and control mice. A nonspecific upper band reflects sample loading (loading control [LC]). D: Liver and islet lysates probed for ADK and enolase (LC).

Figure 2

Constitutive deletion of ADK in β-cells enhanced glucose tolerance. A: Body weights of NC- and HFD-fed Rip-Cre and βADKO female mice (n = 8 mice/group). Statistical comparisons: Rip-Cre (NC) vs. βADKO (NC), P < 0.05 for weeks 8 and 13 only; Rip-Cre (HFD) vs. βADKO (HFD), P > 0.05 for all time points; Rip-Cre (NC) vs. Rip-Cre (HFD), P < 0.05 for weeks 15–20; βADKO (NC) vs. βADKO (HFD), P < 0.05 for weeks 15–20. B: Weight gain of Rip-Cre and βADKO mice on HFD (no statistical differences detected). C: IPGTT of 13-week NC-fed Rip-Cre and βADKO mice (n = 15 mice/group; P > 0.05 for all time points). D: IPGTT of 52-week NC-fed Rip-Cre and βADKO mice (n = 8 mice/group). *P < 0.05 at 120 min (P > 0.05 for all other time points). E: IPGTT of Rip-Cre and βADKO mice after 2 weeks of HFD (age 15 weeks; n = 7 mice/group). *P < 0.05 at 0 min (P > 0.05 for all other time points). F: IPGTT of female Rip-Cre and βADKO mice after 6 weeks of HFD (age 19 weeks; n = 7 mice/group). *P < 0.05 at 30, 60, and 90 min. G: IPGTT of female Rip-Cre and βADKO mice after 18 weeks of HFD (age 31 weeks; n = 7 mice/group). *P < 0.05 at 30 and 60 min. H: IPGTT of male Rip-Cre and βADKO mice after 6 weeks of HFD (age 19 weeks; n = 7 mice/group). *P < 0.05 at 30, 60, and 120 min. I: Fasting glucose values of female Rip-Cre and βADKO mice after 23 weeks of HFD (age 30 weeks; n = 8 mice/group). *P < 0.01. J: Random glucose values of female Rip-Cre and βADKO mice after 12 weeks of HFD (age 25 weeks; n = 8 mice/group). *P < 0.01.

Figure 3

HFD-fed βADKO mice have enhanced insulin tolerance and GSIS in vivo. A and B: Normalized and raw glucose values from 20-week HFD-fed female Rip-Cre and βADKO mice subjected to an IPITT (n = 7 mice/group). C: In vivo GSIS of 24-week NC- or HFD-fed female Rip-Cre and βADKO mice (n = 5–8 mice/group). Significant differences are for comparisons made between Rip-Cre and βADKO mice on the same diet indicated. Insulin levels are significantly higher at all time-points in HFD-fed mice (P < 0.05). D: The total insulin secretion (AUC) is calculated from C. *P < 0.05.

Figure 4

HFD-fed βADKO mice demonstrate increased β-cell but not α-cell mass. A: Relative number of islets isolated from 28-week (21 weeks of HFD) female HFD-fed Rip-Cre and βADKO mice (n = 8). The average number of islets isolated per mouse was 107.5 ± 10.1 and 141.7 ± 17.0 from HFD-fed Rip-Cre and βADKO mice, respectively. B: Representative images of 24-week female HFD-fed Rip-Cre– and βADKO-derived pancreatic sections stained for DAPI (blue) and insulin (red). C: Size distribution of insulin-positive area obtained from Rip-Cre and βADKO pancreatic sections (minimum of six per mouse) stained as in B (n = 5 mice/group). D and E: Average and total insulin-positive cluster area (n = 5 mice/group). F: Total pancreas area measured on the basis of DAPI staining. Data are mean ± SEM. G: Percentage of pancreatic area (DAPI) that is insulin-positive (n = 5). H: Average pancreatic weight (n = 5). I: Calculated β-cell mass by using data obtained from G and H (n = 5). J: Number of insulin-positive clusters per pancreatic section (no statistical difference detected). K: Percentage of total pancreatic area (DAPI) that costains for glucagon. L: β-Cell replication in HFD-fed Rip-Cre and βADKO mice measured as BrdU-positive cells per square micrometer of insulin staining (n = 5 mice/group, 10 sections per mouse; P = 0.06). *P < 0.05.

Figure 5

Conditional deletion of ADK in the β-cells of HFD-fed mice enhances glucose tolerance and β-cell replication. A: The temporal relationship of experiments performed on iβADKO mice (red text indicates male mice only). B: Representative β-galactosidase histochemistry in pancreatic sections obtained from vehicle- and tamoxifen-injected animals 1 week postinjection. C: IPGTT of NC-fed female mice treated with vehicle or tamoxifen (n = 8 mice/group; no significant differences observed). D: Fed glucose values obtained from 22-week NC- and HFD-fed (4 weeks) mice (n = 8 mice/group). E and F: IPGTT of 21-week (3 weeks of HFD) and 29-week (11 weeks of HFD) female mice (n = 8 mice/group), respectively. G: IPITT of HFD-fed female mice treated with vehicle or tamoxifen (n = 8 mice/group; no significant differences observed). H: In vivo GSIS measurements of HFD-fed female mice treated with vehicle or tamoxifen (n = 8 mice/group). I: β-Cell replication index of male NC- and HFD-fed iβADKO mice that received vehicle or tamoxifen treatment (n = 8 mice/group). J: β-Cell replication index of vehicle- and tamoxifen-treated HFD-fed male iβADKO mice (n = 8 mice/group). K: Representative images of pancreatic sections used for β-cell replication analysis. Images were obtained from vehicle- and tamoxifen-treated iβADKO mice stained for DAPI (blue), insulin (red), and ki67 (green) (top panels) or DAPI (blue), PDX-1 (red), and BrdU (green) (bottom panels). L: In vitro β-cell replication index of DMSO-treated and 5-IT–treated (2 μmol/L) islet cultures obtained from vehicle-injected (ADK-expressing) and tamoxifen-injected (ADK-deficient) mice (n = 3–4). M: Western blot of islet lysates from vehicle- and tamoxifen-injected mice for ADK and β-actin. *P < 0.05.