Supplementary Information
Product # / D12052701 / D12052702 / D12052703 / D12052705 / D12052706Control / Mediterranean / Japanese / Western / Ketogenic
Ingredient / gm / gm / gm / gm / gm
Casein / 200 / 44 / 0 / 38.5 / 160
Soy Protein Isolate / 0 / 0 / 16 / 0 / 0
Fish Protein Isolate / 0 / 27 / 27 / 8.5 / 0
Egg White / 0 / 9 / 6 / 55 / 0
Beef, Cooked / 0 / 61.9 / 7 / 76.9 / 0
L-Cystine / 3 / 3 / 3 / 3 / 3
Corn Starch / 641 / 0 / 0 / 30 / 0
Wheat Starch / 0 / 365 / 0 / 195 / 0
Rice Starch / 0 / 0 / 763.5 / 0 / 0
Potato Starch / 0 / 0 / 0 / 30 / 0
Sucrose / 0 / 61 / 70 / 205 / 0
Fructose / 0 / 19 / 6 / 22 / 0
Cellulose, BW200 / 75 / 40.7 / 10 / 18.2 / 37.5
Inulin / 25 / 13.5 / 38 / 6 / 12.5
Soybean Oil / 70 / 0 / 10.5 / 0 / 0
Corn Oil / 0 / 0 / 0 / 34.4 / 8
Menhaden Oil / 0 / 13.2 / 5 / 1 / 8
Trisun Sunflower Oil / 0 / 0 / 0 / 0 / 0
Palm Kernel Oil / 0 / 9.9 / 0 / 0 / 0
Butter, Anhydrous / 0 / 7.4 / 4.2 / 54.1 / 161
Lard / 0 / 0 / 0 / 0 / 161
Safflower Oil / 0 / 0 / 0 / 0 / 0
Flaxseed Oil / 0 / 6.1 / 0 / 1 / 0
Olive Oil / 0 / 117.7 / 11 / 27.5 / 0
Mineral Mix / 10 / 10 / 10 / 10 / 10
Dicalcium Phosphate / 13 / 13 / 13 / 13 / 13
Calcium Carbonate / 5.5 / 5.5 / 5.5 / 5.5 / 5.5
Potassium Citrate / 16.5 / 16.5 / 16.5 / 16.5 / 16.5
Vitamin Mix, V10001 / 10 / 10 / 10 / 10 / 10
Biotin / 0 / 0.014 / 0.014 / 0.1 / 0
Choline Bitartrate / 2 / 2 / 2 / 2 / 2
Cholesterol / 0 / 0 / 0.19 / 1.5 / 1.5
Red Wine Extract / 0 / 0.045 / 0 / 0 / 0
Green Tea Extract / 0 / 0 / 0.215 / 0 / 0
FD&C Red Dye #5 / 0 / 0.05 / 0 / 0 / 0.025
FD&C Yellow Dye #40 / 0.05 / 0 / 0.025 / 0 / 0
FD&C Blue Dye #1 / 0 / 0 / 0.025 / 0.05 / 0.025
Total / 1071.0549 / 855.5113 / 1034.6729 / 864.7547 / 609.5533
Supplementary Table 1. Ingredient composition of diets.
Product # / D12052701 / D12052702 / D12052703 / D12052705 / D12052706g / Control / Mediterranean / Japanese / Western / Ketogenic
Protein / 177 / 115.7 / 120.8 / 140.7 / 142.2
Carbohydrate / 570.3 / 406.7 / 689.1 / 455.3 / 3.1
Fat / 72.4 / 172.3 / 44.6 / 139.7 / 339.9
Cholesterol / 0 / 0.21 / 0.23 / 1.81 / 2.09
Fiber / 93.8 / 50.8 / 50 / 22.7 / 46.9
g%
Protein / 16.5 / 13.5 / 11.7 / 16.3 / 23.3
Carbohydrate / 53.2 / 47.5 / 66.6 / 52.6 / 0.5
Fat / 6.8 / 20.1 / 4.3 / 16.2 / 55.8
Cholesterol / 0 / 0.024 / 0.023 / 0.21 / 0.342
Fiber / 8.8 / 5.9 / 4.8 / 2.6 / 7.7
kcal
Protein / 708 / 463 / 483 / 563 / 569
Carbohydrate / 2281 / 1627 / 2757 / 1821 / 13
Fat / 652 / 1551 / 401 / 1257 / 3059
Total / 3641 / 3641 / 3641 / 3641 / 3641
kcal%
Protein / 19 / 12.71 / 13 / 15 / 16
Carbohydrate / 63 / 44.69 / 76 / 50 / 0
Fat / 18 / 42.6 / 11 / 35 / 84
g% contribution to protein sources
Casein / 98.3 / 33.1 / 0 / 23.8 / 97.9
Soy Protein Isolate / 0 / 0 / 11.5 / 0 / 0
Fish Protein Isolate / 0 / 22.2 / 21.2 / 5.7 / 0
Egg White / 0 / 6.3 / 4 / 31.7 / 0
Beef, Cooked / 0 / 35.8 / 3.9 / 36.6 / 0
Rice Starch / 0 / 0 / 56.9 / 0 / 0
L-Cystine / 1.7 / 2.6 / 2.5 / 2.1 / 2.1
g% contribution to carb sources
Corn Starch / 100 / 0 / 0 / 5.9 / 0
Wheat Starch / 0 / 80.1 / 0 / 38.4 / 0
Rice Starch / 0 / 0 / 88.8 / 0 / 0
Potato Starch / 0 / 0 / 0 / 5.3 / 0
Sucrose / 0 / 15.1 / 10.3 / 45.6 / 0
Fructose / 0 / 4.7 / 0.9 / 4.9 / 0
Supplementary Table 2. Nutrient composition of diets.
Control / Mediterranean / Japanese / Western / Ketogenic
Ingredient (g) / gm / gm / gm / gm
Corn Oil / 34.4 / 8
Menhaden Oil, ARBP-F / 13.2 / 5 / 1 / 8
Beef, Cooked / 61.9 / 7 / 76.9
Butter, Anhydrous / 7.4 / 4.2 / 54.1 / 161
Lard / 161
Palm Kernal Oil / 9.9
Olive Oil / 117.7 / 11 / 27.5
Safflower Oil, USP
Flaxseed Oil / 6.1 / 1
Soybean Oil, USP / 70 / 10.5
Total / 70 / 216.2 / 37.7 / 194.9 / 338
C2, Acetic / 0 / 0 / 0 / 0 / 0
C4, Butyric / 0 / 0.2 / 0.1 / 1.7 / 5.2
C6, Caproic / 0 / 0.1 / 0.1 / 1 / 3.1
C8, Caprylic / 0 / 0.6 / 0 / 0.6 / 1.8
C10, Capric / 0 / 0.6 / 0.1 / 1.4 / 4.1
C12, Lauric / 0 / 4.7 / 0.1 / 1.5 / 4.7
C14, Myristic / 0 / 3.6 / 0.8 / 6.1 / 18.5
C14:1, Myristoleic / 0 / 0.3 / 0.1 / 1 / 2.4
C15:0 / 0 / 0.1 / 0 / 0.1 / 0.2
C16, Palmitic / 7.3 / 22.1 / 4.6 / 25.9 / 75.3
C16:1, Palmitoleic / 0 / 3.5 / 0.8 / 2.4 / 6.7
C16:2 / 0 / 0.2 / 0.1 / 0 / 0.1
C16:3 / 0 / 0.2 / 0.1 / 0 / 0.1
C16:4 / 0 / 0.2 / 0.1 / 0 / 0.1
C17:0 / 0 / 0.2 / 0 / 0.2 / 0.6
C17:1, n-9 / 0 / 0.2 / 0 / 0.2 / 0
C18, Stearic / 2.7 / 6.2 / 1.5 / 10.2 / 36.9
C18:1, Oleic, n-9 / 17 / 95.5 / 12.6 / 50.5 / 96.1
C18:1, Vaccenic, n-7 / 0 / 0.7 / 0.1 / 0.9 / 0
C18:2, Linoleic, n-6 / 37.5 / 17.6 / 7.3 / 26.5 / 47.9
C18:3, gamma-Linolenic, n-6 / 0 / 0 / 0 / 0 / 0
C18:3, alpha-Linolenic, n-3 / 5.5 / 4.4 / 1 / 2 / 4.6
C18:4, Stearidonic, n-3 / 0 / 0.4 / 0.2 / 0 / 0.2
C20, Arachidic / 0 / 0.6 / 0.1 / 0.7 / 1.8
C20:1, n-9 / 0 / 0.5 / 0.1 / 0.2 / 1.1
C20:2, Eicosadienoic, n-6 / 0 / 0 / 0 / 0 / 1.3
C20:3, n-6 / 0 / 0.1 / 0 / 0 / 0.2
C20:3, n-3 / 0 / 0 / 0 / 0 / 0
C20:4, Arachidonic, n-6 / 0 / 0.4 / 0.1 / 0.1 / 0.6
C20:4, n-3 / 0 / 0 / 0 / 0 / 0
C20:5, Eicosapentaenoic, n-3 / 0 / 1.9 / 0.7 / 0.1 / 1.1
C21:0 / 0 / 0 / 0 / 0 / 0
C21:5, n-3 / 0 / 0.1 / 0 / 0 / 0.1
C22, Behenic / 0 / 0 / 0 / 0 / 0
C22:1, Erucic / 0 / 0 / 0 / 0 / 0
C22:4, Clupanodonic, n-6 / 0 / 0 / 0 / 0 / 0
C22:5, n-3 / 0 / 0.4 / 0.1 / 0 / 0.4
C22:5, n-6 / 0 / 0 / 0 / 0 / 0
C22:6, Docosahexaenoic, n-3 / 0 / 1.4 / 0.5 / 0.1 / 0.8
C24, Lignoceric / 0 / 0.1 / 0 / 0 / 0
C24:1 / 0 / 0 / 0 / 0 / 0
Total / 69.9 / 167.4 / 31.7 / 133.7 / 316.3
Supplementary Table 3. Lipid composition of diets.
Control / Mediterranean / Japanese / Western / KetogenicSaturated (g) / 9.9 / 39.0 / 7.6 / 49.1 / 151.3
Monounsaturated (g) / 17.0 / 100.6 / 13.6 / 54.1 / 106.4
Polyunsaturated (g) / 42.9 / 26.5 / 10.0 / 29.0 / 57.3
Saturated (%) / 14.2 / 23.3 / 23.9 / 36.7 / 47.9
Monounsaturated (%) / 24.3 / 60.1 / 43.0 / 40.5 / 33.6
Polyunsaturated (%) / 61.4 / 15.9 / 31.6 / 21.7 / 18.1
Total Omega-6 (g) / 37.5 / 18.1 / 7.5 / 26.7 / 50.1
Total Omega-6 (%) / 53.6 / 10.8 / 23.6 / 20.0 / 15.9
Total Omega-3 (g) / 5.5 / 8.5 / 2.6 / 2.4 / 7.2
Total Omega-3 (%) / 7.8 / 5.1 / 8.1 / 1.8 / 2.3
n6 : n3 Ratio / 6.9 / 2.1 / 2.9 / 11.3 / 6.9
Supplementary Table 4. Lipid profiles of diets.
Body Weight / 0.100 / **** / 0.055 / **** / 0.034 / *** / 0.486 / **** / 0.003 / * / 0.322
Lean Weight / 0.184 / **** / 0.015 / *** / 0.013 / 0.538 / **** / 0.015 / **** / 0.236
Fat Weight / 0.083 / **** / 0.119 / **** / 0.056 / *** / 0.124 / **** / 0.075 / **** / 0.544
LDL cholesterol / 0.148 / **** / 0.355 / **** / 0.100 / ** / 0.004 / NA / 0.394
HDL Cholesterol / 0.133 / **** / 0.303 / **** / 0.124 / *** / 0.076 / **** / NA / 0.364
Total Cholesterol / 0.209 / **** / 0.292 / **** / 0.119 / *** / 0.054 / **** / NA / 0.326
Serum Triglycides / 0.186 / **** / 0.076 / ** / 0.098 / * / 0.041 / ** / NA / 0.599
Fasted Insulin / 0.051 / ** / 0.076 / *** / 0.147 / **** / 0.024 / ** / 0.022 / ** / 0.679
Fasted Leptin / 0.287 / **** / 0.103 / **** / 0.055 / * / 0.001 / 0.011 / * / 0.543
Fasted Glucose / 0.266 / **** / 0.075 / **** / 0.043 / ** / 0.136 / **** / 0.033 / ** / 0.447
Glucose Tolerance / 0.205 / **** / 0.119 / **** / 0.057 / **** / 0.154 / **** / 0.056 / **** / 0.410
ALT / 0.087 / *** / 0.059 / ** / 0.090 / * / 0.001 / 0.014 / * / 0.757
Liver Triglycerides / 0.057 / **** / 0.330 / **** / 0.108 / **** / 0.037 / **** / 0.003 / 0.464
Metabolic Rate (O2) / 0.081 / **** / 0.287 / **** / 0.091 / **** / 0.337 / **** / NA / 0.204
Metabolic Rate (heat) / 0.092 / **** / 0.199 / **** / 0.096 / **** / 0.423 / **** / NA / 0.190
Food Intake / 0.105 / **** / 0.113 / ** / 0.133 / ** / 0.030 / ** / NA / 0.619
Water Intake / 0.032 / ** / 0.212 / **** / 0.153 / **** / 0.224 / **** / NA / 0.380
Activity / 0.154 / **** / 0.033 / 0.016 / 0.152 / **** / NA / 0.646
RER / 0.009 / * / 0.835 / **** / 0.009 / 0.005 / * / NA / 0.142
Supplementary Table 5. Proportion of phenotypic variance explained across metabolic traits. Significance is indicated by * p <0.05, ** p <0.01, *** p <0.001, p <0.0001. Glucose tolerance test area under the curve (GTT AUC), alanine aminotranferase (ALT), respiratory exchange ratio (RER), not applicable (NA) for traits measured in a single cohort.
Strain / Response / Diet / Control / Mean / SE / n / pA / Macrovesicular steatosis / Control / N/A / 0.091 / 0.191 / 11 / N/A
A / Macrovesicular steatosis / American / Control / 1.182 / 0.191 / 11 / 0.0009
A / Microvesicular steatosis / Control / N/A / 0.091 / 0.231 / 11 / <.N/A
A / Microvesicular steatosis / American / Control / 2.182 / 0.231 / 11 / <.0001
A / Inflammation / Control / N/A / 0.182 / 0.138 / 11 / N/A
A / Inflammation / American / Control / 0.273 / 0.138 / 11 / 0.6423
A / Hypertrophy / Control / N/A / 0.364 / 0.213 / 11 / N/A
A / Hypertrophy / American / Control / 1.000 / 0.213 / 11 / 0.0412
B6 / Macrovesicular steatosis / Control / N/A / 0.000 / 0.364 / 6 / N/A
B6 / Macrovesicular steatosis / American / Control / 1.714 / 0.250 / 7 / 0.0407
B6 / Microvesicular steatosis / Control / N/A / 0.000 / 0.153 / 6 / N/A
B6 / Microvesicular steatosis / American / Control / 2.857 / 0.105 / 7 / <.0001
B6 / Inflammation / Control / N/A / 0.500 / 0.614 / 6 / N/A
B6 / Inflammation / American / Control / 1.000 / 0.421 / 7 / 0.4972
B6 / Hypertrophy / Control / N/A / 0.000 / 0.153 / 6 / N/A
B6 / Hypertrophy / American / Control / 1.000 / 0.105 / 7 / 0.1898
FVB / Macrovesicular steatosis / Control / N/A / 0.250 / 0.243 / 9 / N/A
FVB / Macrovesicular steatosis / American / Control / 1.500 / 0.252 / 8 / 0.0044
FVB / Microvesicular steatosis / Control / N/A / 1.375 / 0.299 / 9 / N/A
FVB / Microvesicular steatosis / American / Control / 2.750 / 0.309 / 8 / 0.0061
FVB / Inflammation / Control / N/A / 1.125 / 0.330 / 9 / N/A
FVB / Inflammation / American / Control / 0.875 / 0.342 / 8 / 0.8544
FVB / Hypertrophy / Control / N/A / 0.500 / 0.338 / 9 / N/A
FVB / Hypertrophy / American / Control / 1.250 / 0.350 / 8 / 0.1657
NOD / Macrovesicular steatosis / Control / N/A / 0.143 / 0.160 / 7 / N/A
NOD / Macrovesicular steatosis / American / Control / 1.500 / 0.137 / 10 / <.0001
NOD / Microvesicular steatosis / Control / N/A / 0.286 / 0.362 / 7 / N/A
NOD / Microvesicular steatosis / American / Control / 0.900 / 0.312 / 10 / 0.2092
NOD / Inflammation / Control / N/A / 0.000 / 0.263 / 7 / N/A
NOD / Inflammation / American / Control / 1.100 / 0.226 / 10 / 0.0071
NOD / Hypertrophy / Control / N/A / 0.000 / 0.208 / 7 / N/A
NOD / Hypertrophy / American / Control / 0.900 / 0.179 / 10 / 0.0039
Supplementary Table 6. Histological analysis of liver compared to controlmouse diet. Histological analysis of mice eating American diet relative to control mouse diet was performed across four categories: macrovesicular steatosis, microvesicular steatosis, inflammation, and hypertrophy. The scoring system for macrovesicular steatosis, microvesicular steatosis, and cellular hypertrophy was based on the percentage of hepatocytes within the stained section. These parameters utilized the following categories: 0 (< 5% of hepatocytes), 1 (5-33%), 2 (34- 66%), and 3 (>66%). The level of inflammation was assigned using the following categories: 0 (normal, < 0.5), 1 (slight, 0.5-1.0), 2 (moderate, 1.0-2.0), and 3 (severe, >2). Two-way ANOVA was performed and p-values were corrected by Dunnett’s test.
Strain / Response / Diet / Control / Mean / SE / n / pA / Macrovesicular steatosis / Japanese / American / 1.200 / 0.344 / 5 / 0.4157
A / Macrovesicular steatosis / Ketogenic / American / 0.500 / 0.201 / 12 / 0.0697
A / Macrovesicular steatosis / American / N/A / 1.182 / 0.211 / 11 / N/A
A / Microvesicular steatosis / Japanese / American / 0.200 / 0.407 / 5 / 0.0002
A / Microvesicular steatosis / Ketogenic / American / 0.750 / 0.237 / 12 / 0.0008
A / Microvesicular steatosis / American / N/A / 2.182 / 0.249 / 11 / N/A
A / Inflammation / Japanese / American / 0.400 / 0.395 / 5 / 0.7504
A / Inflammation / Ketogenic / American / 0.667 / 0.231 / 12 / 0.4116
A / Inflammation / American / N/A / 0.273 / 0.242 / 11 / N/A
A / Hypertrophy / Japanese / American / 0.600 / 0.342 / 5 / 0.2439
A / Hypertrophy / Ketogenic / American / 0.417 / 0.199 / 12 / 0.1136
A / Hypertrophy / American / N/A / 1.000 / 0.209 / 11 / N/A
FVB / Macrovesicular steatosis / Ketogenic / American / 0.800 / 0.514 / 5 / 0.053
FVB / Macrovesicular steatosis / American / N/A / 1.500 / 0.314 / 8 / N/A
FVB / Microvesicular steatosis / Ketogenic / American / 1.400 / 0.242 / 5 / 0.0007
FVB / Microvesicular steatosis / American / N/A / 2.750 / 0.148 / 8 / N/A
FVB / Inflammation / Ketogenic / American / 0.800 / 0.646 / 5 / 0.8062
FVB / Inflammation / American / N/A / 0.875 / 0.396 / 8 / N/A
FVB / Hypertrophy / Ketogenic / American / 0.000 / 0.455 / 5 / 0.0137
FVB / Hypertrophy / American / N/A / 1.250 / 0.278 / 8 / N/A
NOD / Macrovesicular steatosis / Ketogenic / American / 2.167 / 0.287 / 6 / 0.217
NOD / Macrovesicular steatosis / American / N/A / 1.500 / 0.228 / 10 / N/A
NOD / Microvesicular steatosis / Ketogenic / American / 1.167 / 0.446 / 6 / 0.728
NOD / Microvesicular steatosis / American / N/A / 0.900 / 0.354 / 10 / N/A
NOD / Inflammation / Ketogenic / American / 1.167 / 0.467 / 6 / 0.7722
NOD / Inflammation / American / N/A / 1.100 / 0.370 / 10 / N/A
NOD / Hypertrophy / Ketogenic / American / 1.333 / 0.323 / 6 / 0.6702
NOD / Hypertrophy / American / N/A / 0.900 / 0.256 / 10 / N/A
Supplementary Table 7. Histological analysis of liver compared to American diet. Groups of interest were further investigated by histological examination across four categories: macrovesicular steatosis, microvesicular steatosis, inflammation, and hypertrophy. The scoring system for macrovesicular steatosis, microvesicular steatosis, and cellular hypertrophy was based on the percentage of hepatocytes within the stained section. These parameters utilized the following categories: 0 (< 5% of hepatocytes), 1 (5-33%), 2 (34- 66%), and 3 (>66%). The level of inflammation was assigned using the following categories: 0 (normal, < 0.5), 1 (slight, 0.5-1.0), 2 (moderate, 1.0-2.0), and 3 (severe, >2). Two-way ANOVA was performed and p-values were corrected by Dunnett’s test.
Supplemental Figure 1. Study design and timeline. The study timeline indicates the age of the mice during experimental procedures. The colored lines below the timeline indicate the diets studied. All mice were fed a typical mouse chow (PicoLab Mouse Diet 20 5058) before the study began (black line). In order to put the novel American diet used in this study into perspective with a more typical mouse diet, we first compared the responses on the two diets (Comparison A). Then, we evaluated a precision dietetics paradigm, comparing the American diet to three other human-relevant diets in Comparison B. Both comparisons A and B were evaluated in each of the four mouse strains.
Supplemental Figure 2. Comparison of additional metabolic phenotypes in each strain for mice fed American relative to control mouse diet. Effect of diets relative to American diet in each strain for (a) heat expenditure (n = 9-10), (b) respiratory exchange rate (n = 9-10), (c) fat mass (n = 19-20), (d) lean mass (n = 19-20), (e) total cholesterol (n = 4-10), (f) plasma triglyceride concentration (n = 5 -10), (g) fasted glucose (n = 12–20), (h) fasted insulin (n = 8-16), (i) alanine aminotransferase concentration (ALT) (n = 9-14). Data are mean +/- standard error. * p < 0.05, ** p < 0.01, *** p < 0.001 by ANOVA between means.
Supplemental Figure 3. Comparison of body weight and composition of B6 mice fed isocaloric quantities of control mouse diet and American diet for 14 weeks. Male mice were each fed 11.5 kcal/day of control mouse diet or American diet, n=10 per diet. (a) The American diet group weighed less at the beginning of the study but were significantly heavier at 14 weeks on diet (p < 0.001.) (b) Lean mass did not differ by diet. (c) Mice fed American diet had significantly more body fat from 4 weeks (p =0.013) onward. Two-tailed Student’s t-test was used for statistical analysis. Error is shown as standard error.
Supplemental Figure 4. Comparison of additional metabolic phenotypes in each strain for mice fed Mediterranean, Japanese, or Ketogenic diets relative to American diet. Effects of Mediterranean, Japanese and ketogenic diets relative to American diet in each strain for (a) heat expenditure (n = 8-10), (b) respiratory exchange rate (n = 8-10), (c) fat mass (n = 17 - 20), (d) lean mass (n = 17-20), (e) total cholesterol (n = 4-10), (f) plasma triglyceride concentration (n = 6 – 10, except NOD ketogenic (n = 4)), (g) fasted glucose (n = 9-20) (h) fasted insulin (n = 5-15), (i) alanine aminotransferase (n = 8-15). Data are mean +/- standard error. * p < 0.05, ** p < 0.01, *** p < 0.001 by ANOVA between means.
A. / B. / C.R-Square / 0.0146 / R-Square / 0.0071 / R-Square / <.0001
R-Square Adj / 0.0109 / R-Square Adj / 0.0033 / R-Square Adj / <.0001
p-value / .0486 / p-value / 0.1702 / p-value / .9488
Supplemental Figure 5. Correlation of calorie intake with fat mass, lean mass, and total body weight. Each dot represents the relationship between average food consumption per day of a mouse with fat mass (panel A), lean mass (panel B), and total body weight (panel C). A fit line is shown in red with corresponding Pearson correlation statistics shown below. All diet-strain groups are included.