supplementary table 1. composition of the experimental diets1 · supplementary data supplementary...

SUPPLEMENTARY DATA

Supplementary Table 1. Composition of the experimental diets1

DietSL SO

g/kg dietTotal milk protein and casein2 212.8 212.8Fat3 140.4 140.4Carbohydrate 510.7 510.7Indigestable dextrin 63.8 63.8Vitamin and mineral mixture4 72.3 72.3Fatty acid composition (%)

Palmitic acid 16:0 7.3 6.7Stearic acid 18:0 2.6 4.2Oleic acid 18:1 (n-9) 13.4 72.3Linoleic acid 18:2 (n-6) 76.4 10.7α-Linolenic acid 18:3 (n-3) 0.2 4.4Other fatty acid 0 1.7

Carbohydrate composition, (%)Sucrose 70 70Dextrin 30 30

Total energy, (kJ/g)5 17.8 17.81 All of the diets were identical except for the type of fat used: linoleic acid in the SL diet and oleic acidin the SO diet. 2 The percentage of total milk protein and casein was 60% and 40%, respectively. 3 The fat component of SL and SO was derived from safflower oil and high-oleic sunflower oil blendedwith perilla oil, respectively. The percentage of high-oleic sunflower oil and perilla oil was 90% and10%, respectively. 4 The composition of the vitamin and mineral mixture (per kg of diet) was as follows. Vitaminssupplied: 3.2 mg retinyl palmitate, 31.9 µg cholecalciferol, 340.4 mg α-tocopherol, 42.6 µg menadioneand phylloquinone, 255.3 mg thiamine HCl, 21.3 mg riboflavin, 89.4 mg nicotinamide, 12.8 mgpyridoxine HCl, 42.6 mg calcium pantothenate, 1.7 g L-ascorbic acid, 825.5 mg choline, 2.1 mg folicacid, 38.3 µg cyanocobalamin, and 12.3 µg biotin. Minerals supplied: 3.0 g sodium, 3.4 g calcium, 42.6mg iron, 3.4 g phosphorus, 1.1 g magnesium, 3.4 g potassium, 2.1 mg copper, 0.4 mg manganese, 42.6mg zinc, 2.6 g chloride, 148.9 µg selenium, 127.7 µg chromium, 119.1 µg iodine, and 123.4 µgmolybdenum. 5 The percentage of energy as fat, carbohydrate, and protein was 29.7%, 50.3%, and 20.0%, respectively.

©2011 American Diabetes Association. Published online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db10-1338/-/DC1

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Supplementary Table 2. Body weight (BW), liver weight (LW), epididymal fat weight (FW), andblood glucose level (glucose) after a 16-hour fast in WT and Gck+/- mice fed standard diet.

WT Gck+/-

week 25 Standard diet Standard dietBW (g) 39.21 ± 2.66 39.81 ± 3.12LW (g) 1.88 ± 0.29 1.85 ± 0.36FW (g) 1.04 ± 0.24a 1.13 ± 0.31a

LW / BW (%) 4.79 ± 0.31 4.64 ± 0.46FW / BW (%) 2.65 ± 0.38a 2.86 ± 0.35a

glucose (mg/dl) 64 ± 9b, c 114 ± 18d

a: p < 0.05 relative to Gck+/- SL in Table 1, b: p < 0.05 relative to Gck+/- standard diet, c: p < 0.05relative to WT SO, WT SL, Gck+/- SO and Gck+/- SL in Table 1, d: p < 0.05 relative to Gck+/- SO andGck+/- SL in Table 1.

Supplementary Figure 1. Biochemical parameters in WT and Gck+/- mice after 25 weeks on the SOdiet or SL dietPlasma alanine aminotransferase (ALT), free fatty acid (FFA), total cholesterol (TChol), triglyceride(TG) and low-density lipoprotein cholesterol (LDL-C) in the groups of mice indicated after 25 weeks onSO diet or SL diet (n = 7-8).

Supplementary Figure 2. Epididymal adipocyte size in WT and Gck+/- mice after 25 weeks on theSO diet or SL dietHistogram of adipocyte size WT or Gck+/- mice after 20 weeks on the standard diet or high-fat diet(HFD32, CREA Japan) (n = 4) (left panel). Average size of adipocyte from WT and Gck+/- mice after 25weeks on the SO diet or SL diet is shown in right panel. Results of average size of adipocyte from WT orGck+/- mice after 20 weeks on the standard diet or high-fat diet (HFD32) are also shown as references.


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Supplementary Figure 3. F4/80 staining of epididymal fat tissue in WT and Gck+/- mice after 25weeks on the standard diet Epididymal fat tissue was stained with anti-F4/80 antibody in WT and Gck+/- mice after 25 weeks on thestandard diet.

Supplementary Figure 4. CD4+ subset and CD8+ subset of CD3+ T cells among splenocytes in WTand Gck+/- mice after 25 weeks on the SO diet or SL dietFlow-cytometric analysis of the CD4+ subset and CD8+ subset of CD3+ T cells among splenocytes fromthe groups of mice indicated (n = 5).

Supplementary Figure 5. Biochemical parameters in WT and Gck+/- mice after 20 weeks on the SLdiet or SL plus DFS dietPlasma alanine aminotransferase (ALT), free fatty acid (FFA), total cholesterol (TChol), triglyceride(TG) and low-density lipoprotein cholesterol (LDL-C) in the groups of mice indicated after 20 weeks onthe SL or SL plus DFS diet (n = 6).


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Supplementary Figure 6. Epididymal adipocyte size in WT and Gck+/- mice after 20 weeks on theSL diet or SL plus DFS dietAverage size of adipocyte from WT and Gck+/- mice after 20 weeks on the SO diet or the SL plus DFSdiet. Results of average size of adipocyte from WT or Gck+/- mice after 20 weeks on the standard diet orhigh-fat diet (HFD32) are also shown as references.

Supplementary Figure 7. CD4+ subset and CD8+ subset of CD3+ T cells among splenocytes in WTand Gck+/- mice after 20 weeks on the SL diet or SL plus DFS diet Flow-cytometric analysis of the CD4+ subset and CD8+ subset of CD3+ T cells among splenocytes fromthe groups of mice indicated (n = 5).


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Supplementary Figure 8. Expression of mouse CD26 on CD4+ and CD8+ T cellsFlow-cytometric analysis of the CD4+ T cells and CD8+ T cells with anti-CD26 antibody before (day 0:red), 1 day (blue), or 2 day (green) after stimulation with plastic-coated anti-CD3 and anti-CD28 mAbs.


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Supplementary Figure 9. No impact of DFS on the production of MCP-1, TNF-α, IL-10, or IFN-γby antibody-stimulated CD4+ or CD8+ T cells.(A-D) Primary splenic CD4+ T cells (left panel) and CD8+ T cells (right panel) from male WT mice (bars1-5) or Gck+/- mice (bars 6-10) were stimulated with or not with plastic-coated anti-CD3 and anti-CD28mAbs for 3 days. IFN-γ (A), TNF-α (B), MCP-1 (C) and IL-10 (D) concentrations in culturesupernatants were analyzed by ELISA (n = 3). Samples were assayed as follows. Bars 1 and 6:unstimulated; Bars 2 and 7: stimulated with anti-CD3 and anti-CD28 mAbs alone; Bars 3 and 8:stimulated with anti-CD3 and anti-CD28 mAbs in the presence of 100 nM exendin-4 (Ex-4); Bars 4 and9: stimulated with anti-CD3 and anti-CD28 mAbs in the presence of 1 μM des-fluoro-sitagliptin (DFS);Bars 5 and 10: stimulated with anti-CD3 and anti-CD28 mAbs in the presence of 100 μM DFS. N.D.:not detected.


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Supplementary Figure 10. No impact of DFS on the intracellular production of MCP-1, TNF-α,IL-10, or IFN-γ by antibody-stimulated CD4+ or CD8+ T cells(A-D) Primary splenic CD4+ T cells (left panel) and CD8+ T cells (right panel) from male WT mice (bars1-5) or Gck+/- mice (bars 6-10) were stimulated with or not with plastic-coated anti-CD3 and anti-CD28mAbs for 3 days. Intracellular IFN-γ (E), TNF-α (F) and MCP-1 (G) syntheses were analyzed in thesecells by flow cytometry. The samples were assayed as follows. Bars 1 and 6: unstimulated; Bars 2 and 7:stimulated with anti-CD3 and anti-CD28 mAbs alone; Bars 3 and 8: stimulated with anti-CD3 and anti-CD28 mAbs in the presence of 100 nM exendin-4 (Ex-4); Bars 4 and 9: stimulated with anti-CD3 andanti-CD28 mAbs in the presence of 1 μM des-fluoro-sitagliptin (DFS); Bars 5 and 10: stimulated withanti-CD3 and anti-CD28 mAbs in the presence of 100 μM DFS.


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Supplementary Figure 11. No impact of DFS and rsCD26 on the production of MCP-1, TNF-α,IL-10, or IFN-γ by specific antigen-stimulated CD4+ or CD8+ T cells.(A-D) Primary splenic CD4+ T cells from female OT-II mice (upper panel) and CD8+ T cells fromfemale OT-I mice (lower panel) were stimulated with CD11c+ splenic APCs from female WT mice inthe presence or absence of ova peptide for 3 days. IFN-γ (A), TNF-α (B), MCP-1 (C) and IL-10 (D)concentrations in culture supernatants were analyzed by ELISA (n = 3). The samples were assayed asfollows. Bar 1: stimulated without ova; Bar 2: stimulated without ova in the presence of 100 nM Ex-4;Bar 3: stimulated without ova in the presence of 100 μM DFS; Bar 4: stimulated with ova; Bar 5:stimulated with ova in the presence of 100 nM Ex-4; Bar 6: stimulated with ova in the presence of 100


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μM DFS; Bar 7: stimulated with ova plus 0.5 mg/ml soluble recombinant CD26 (rsCD26); Bar 8:stimulated with ova plus rsCD26 in the presence of 100 nM Ex-4; Bar 9: stimulated with ova plusrsCD26 in the presence of 100 μM DFS.

Supplementary Figure 12. SO and SL diet-induced hepatic steatosis in WT and Gck+/- mice(A) Masson-Goldner staining of liver sections from the groups of mice indicated. (B) Grade of hepaticsteatosis (grade 0 - 3) (n = 7) as described in Supplementary Fig. 14. (C) Concentration of livertriglyceride in the groups of mice indicated (mg/g tissue) (n = 6). (D) Hepatic gene expression ofSREBP-1c, SCD-1, PPARα, SREBP-2, FAS, and TNF-α in the groups of mice indicated normalized tothe β-actin mRNA level (n = 6). (E) Hepatic gene expression of PEPCK and G6Pase in the groups ofmice indicated normalized to the β-actin mRNA level (n = 5). Experiments were performed in WT andGck+/- mice after 25 weeks on the SO diet or SL diet.


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Supplementary Figure 13. Steatosis were not occur in WT and Gck+/- mice fed standard diet or POdiet, and DFS improved steatosis in mice fed SO(A) Masson-Goldner staining of liver sections from WT and Gck+/- mice after 25 weeks on the standarddiet. (B) Masson-Goldner staining of liver sections from WT and Gck+/- mice after 25 weeks on the PO(palatinose and oleic acid) diet. (C) Masson-Goldner staining of liver sections from WT and Gck+/- miceafter 25 weeks on the SO plus DFS diet.


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Supplementary Figure 14. Grading system for hepatic steatosis.(A) Grade 0: Normal liver with no lipid deposits; (B) Grade 1: Liver tissue exhibiting mild steatosis; (C)Grade 2: liver tissue exhibiting a degree of steatosis between mild (grade 1) and severe (grade 3); (D)Grade 3: Liver tissue exhibiting severe steatosis with almost all cells affected and many containing largefat vacuoles.


supplementary table 1. composition of the experimental diets1 · supplementary data supplementary...

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