Impact of Perturbed Pancreatic β-Cell Cholesterol Homeostasis on Adipose Tissue and Skeletal Muscle Metabolism
Article Figures & Tables
Figures
- Figure 1
Conditional deletion of ABCA1 and ABCG1 in β-DKO mice. A: Tissue homogenates from β-DKO and Abca1fl/flAbcg1fl/fl mice were subjected to SDS-PAGE and immunoblotted for ABCA1, ABCG1, and β-actin. B: Quantification of blots from A. Results represent the mean ± SD of three independent experiments.
- Figure 2
Glucose tolerance and insulin secretion are impaired in β-DKO mice. A: Body weight of 6- to 16-week-old Abca1fl/flAbcg1fl/fl mice, Ins2Cre mice, and β-DKO mice (n = 6/group). B: Daily food intake of 16-week-old Abca1fl/flAbcg1fl/fl mice, Ins2Cre mice, and β-DKO mice (n = 6/group). Fed and 5-h fasted blood glucose (C) and insulin (D) levels in 6-, 12-, and 16-week-old Abca1fl/flAbcg1fl/fl mice, Ins2Cre mice, and β-DKO mice (n = 8–10/group). Blood glucose levels (E) and incremental area under curve (AUC) data (F) during an intraperitoneal glucose tolerance test (glucose: 2 g/kg) in 5-h fasted, 16-week-old Abca1fl/flAbcg1fl/fl mice, Ins2Cre mice, and β-DKO mice (n = 6–8/group). G: Blood glucose levels during an intraperitoneal pyruvate tolerance test (sodium pyruvate: 2 g/kg) in 18-h fasted, 16-week-old Abca1fl/flAbcg1fl/fl mice and β-DKO mice (n = 6/group). H: Blood glucose levels during an intraperitoneal insulin tolerance test (insulin: 1 unit/kg; n = 7/group) in 16-week-old Abca1fl/flAbcg1fl/fl mice, Ins2Cre mice, and β-DKO mice. Plasma insulin levels during a glucose (I) and arginine (J) challenge in Abca1fl/flAbcg1fl/fl mice and β-DKO mice. The mice were fasted for 5 h before intraperitoneal administration of 3 g/kg glucose or 1 g/kg arginine. Plasma insulin levels were measured by ELISA (n = 7/group). K: Representative pancreas sections from Abca1fl/flAbcg1fl/fl mice and β-DKO mice immunostained for insulin. Scale bars = 1 mm. L: Quantification of β-cell mass in Abca1fl/flAbcg1fl/fl mice and β-DKO mice. Values represent mean ± SD. In the box-and-whisker plots, line in box represents median quartile; whiskers represent minimum-maximum values; box borders represent lower and upper quartiles. ****P < 0.001.
- Figure 3
Adipose tissue mass is increased and skeletal muscle mass is decreased in β-DKO mice. A: Representative images of visceral fat in 16-week-old Abca1fl/flAbcg1fl/fl mice and β-DKO mice. B: Representative transverse section of MRI displays the differences in visceral (red arrow) and subcutaneous (yellow arrow) adipose tissue and hind limb muscle (blue arrow) size in 16-week-old Abca1fl/flAbcg1fl/fl mice and β-DKO mice. C: Body fat percentage by EchoMRI in 6-, 12-, and 16-week-old Abca1fl/flAbcg1fl/fl mice and β-DKO mice (n = 6/group). D: Representative image of skeletal muscle in dissected hind limb in 16-week-old β-DKO mice and Abca1fl/flAbcg1fl/fl mice. E: Weight of isolated gastrocnemius muscle in 16-week-old Abca1fl/flAbcg1fl/fl mice and β-DKO mice (n = 8/group). F: Quantification of adipocyte size in 16-week-old Abca1fl/flAbcg1fl/fl mice and β-DKO mice (n = 8/group, 10 sections/animal). Values represent mean ± SD. ***P < 0.005; ****P < 0.001.
- Figure 4
β-DKO mice have increased adipose tissue macrophage content and systemic inflammation. A: Representative histology of F4/80+ staining in adipose tissue from Abca1fl/flAbcg1fl/fl mice and β-DKO mice. Scale bars = 200 µm. B: Quantification of F4/80 staining from A (n = 8/group, 10 sections/animal). Plasma IL-6 (C) and MCP-1 (D) levels in 16-week-old Abca1fl/flAbcg1fl/fl mice and β-DKO mice (n = 6/group). Values represent mean ± SD. **P < 0.01; ****P < 0.001.
- Figure 5
Impaired insulin secretion shifts glucose disposal from skeletal muscle to adipose tissue in β-DKO mice. Uptake of [3H]-2-deoxyglucose in muscle (A) and epididymal adipose tissue (B) in Abca1fl/flAbcg1fl/fl mice and β-DKO mice (n = 6/group). Fatty acid synthase (FAS) activity in isolated liver (C) and epididymal adipose tissue (D) in Abca1fl/flAbcg1fl/fl mice, Ins2Cre mice, and β-DKO mice (n = 6/group). FAS expression in Abca1fl/flAbcg1fl/fl and β-DKO mouse epididymal adipose tissue assessed by Western blotting (E) and quantification of FAS expression (F). G: Isolated adipocyte levels of malonyl-CoA levels were determined by ELISA. Mlycd (H) and Acaca (I) mRNA levels were determined by real-time PCR (n = 5/group). RQV, relative quantification value. Values represent mean ± SD. *P < 0.05; **P < 0.01.
- Figure 6
Insulin supplementation inhibits adipose tissue expansion but does not reduce inflammation in β-DKO mice. A: Body weight of β-DKO mice immediately before pump implantation at 12 weeks of age and at 16 weeks of age, after 4 weeks of PBS or insulin supplementation. Fed plasma insulin levels (B) and blood glucose levels (C) in PBS- and insulin-supplemented β-DKO mice and Abca1fl/flAbcg1fl/fl mice. D: Percentage of body fat in PBS- and insulin-supplemented β-DKO mice. E: Gastrocnemius muscle weight in 16-week-old PBS- and insulin-supplemented β-DKO mice and Abca1fl/flAbcg1fl/fl mice. Adipocyte size (F), β-cell mass (G), and adipose tissue macrophage content (H) in PBS- and insulin-supplemented β-DKO mice and Abca1fl/flAbcg1fl/fl mice. Plasma IL-6 (I) and MCP-1 (J) levels in PBS- and insulin-supplemented β-DKO mice. Values represent mean ± SD (n = 6/group for all). *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001.
Tables
- Table 1
Altered metabolic pathways in isolated gastrocnemius muscle from β-DKO mice
Pathway Upregulated Downregulated P value Glycolysis BPGM, FBP2, PGAM2, PGK1, PKM GAPDH, GPI 2.97E-13 Gluconeogenesis BPGM, FBP2, PGAM2, PGK1 GAPDH, GPI 3.50E-11 Rapoport-Luebering glycolytic shunt BPGM, PGAM2 4.61E-05 Glutaryl-CoA degradation ACoAAT, HSD17B10 5.00E-04 Isoleucine degradation ACoAAT, HSD17B10 7.91E-04 Gastrocnemius muscle was obtained from 16-week-old β-DKO mice and Abca1fl/fl/Abcg1fl/fl mice at the time of sacrifice. The samples were homogenized, and proteins were precipitated with acetone and digested with trypsin, as described in research design and methods. The resulting peptides were purified and analyzed by MS and Ingenuity Pathway Analysis.
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