INTRODUCTION
METHODS
Animals and husbandry
Preparation and administration of test substances
Body and organ weight change
Mean daily food consumption
Body fat and abdominal fat density
Serum biochemistry analyses
Measurement of lipid compositions in the feces
Liver lipid peroxidation and antioxidant defense system
Analysis of hepatic glucose-regulating enzyme activity
Real-time RT-PCR (polymerase chain reaction) analysis
Table 1.
Histopathology
Statistical analysis
RESULTS
Changes in organ and tissue weight
Table 2.
Times | Body weight (g) at days after initial test substance treatment | Body weight gain during |
Mean Daily Food Consumption (g) |
||||
---|---|---|---|---|---|---|---|
Groups | 8 days before [A] | 1 day before [B] | 0 day* [C] | 84 days* [D] | Adaptive period [B-A] | Administration period [D-C] | |
Controls | |||||||
Intact | 30.48±1.49 | 30.88±1.55 | 27.86±1.48 | 33.45±1.69 | 0.45±0.20 | 5.59±0.83 | 5.58±0.29 |
HFD | 30.46±1.03 | 34.75±1.04a | 31.79±0.92a | 50.18±1.73a | 4.29±0.25a | 18.39±1.48a | 4.65±0.23a |
Reference | |||||||
Metformin | 30.40±1.35 | 34.78±1.26a | 31.95±1.24a | 43.71±1.25ab | 4.38±0.20a | 11.76±0.97ab | 4.71±0.20a |
Test material - BH | |||||||
400 mg/kg | 30.49±1.49 | 34.70±1.38a | 31.71±1.84a | 40.35±2.18ab | 4.21±0.17a | 8.64±0.85ab | 4.67±0.18a |
200 mg/kg | 30.45±1.50 | 34.74±1.82a | 31.83±1.47a | 43.41±2.20ab | 4.29±0.17a | 11.59±1.59ab | 4.67±0.22a |
100 mg/kg | 30.45±1.31 | 34.68±1.19a | 31.94±1.71a | 45.70±2.37ab | 4.23±0.31a | 13.76±1.22ab | 4.65±0.22a |
Table 3.
Organs | Absolute organ weights (g), Relative organ weights (% of body weights) | |||||
---|---|---|---|---|---|---|
Groups | Organ | Liver | Kidney | Pancreas | Periovarian fat pads | Abdominal wall fat pads |
Controls | ||||||
Intact | Absolute | 1.051±0.057 | 0.214±0.013 | 0.248±0.018 | 0.110±0.015 | 0.044±0.025 |
Relative | 3.151±0.251 | 0.641±0.029 | 0.745±0.079 | 0.329±0.053 | 0.130±0.067 | |
HFD | Absolute | 1.750±0.063a | 0.326±0.012a | 0.241±0.013 | 0.853±0.104c | 0.705±0.147c |
Relative | 3.492±0.181a | 0.650±0.041 | 0.481±0.025c | 1.703±0.221c | 1.402±0.267c | |
Reference | ||||||
Metformin | Absolute | 1.473±0.073ab | 0.271±0.012ab | 0.245±0.006 | 0.485±0.087cd | 0.396±0.078cd |
Relative | 3.369±0.141 | 0.620±0.034 | 0.560±0.017cd | 1.106±0.173cd | 0.906±0.182cd | |
Test material - BH | ||||||
400 mg/kg | Absolute | 1.346±0.050ab | 0.248±0.013ab | 0.245±0.013 | 0.241±0.047cd | 0.229±0.040cd |
Relative | 3.344±0.210 | 0.615±0.039 | 0.607±0.035cd | 0.597±0.111cd | 0.569±0.098cd | |
200 mg/kg | Absolute | 1.472±0.086ab | 0.269±0.007ab | 0.242±0.006 | 0.491±0.078cd | 0.386±0.060cd |
Relative | 3.404±0.336b | 0.621±0.040 | 0.560±0.041cd | 1.138±0.226cd | 0.892±0.148cd | |
100 mg/kg | Absolute | 1.575±0.079ab | 0.292±0.016ab | 0.244±0.014 | 0.653±0.043cd | 0.494±0.064cd |
Relative | 3.456±0.277b | 0.640±0.056 | 0.533±0.024cd | 1.431±0.092cd | 1.081±0.131cd |
Effects on food consumption
Serum biochemical analysis
Table 4.
Items |
AST (IU/l) |
ALT (IU/l) |
ALP (IU/l) |
LDH (IU/l) |
GGT (IU/l) |
BUN (mg/dl) |
Creatinine (mg/dl) |
---|---|---|---|---|---|---|---|
Groups | |||||||
Controls | |||||||
Intact | 58.63±14.69 | 33.50±11.64 | 88.63±20.37 | 298.25±125.20 | 1.75±0.71 | 33.13±10.36 | 0.64±0.30 |
HFD | 185.13±25.77a | 146.25±24.07a | 219.38±31.95a | 2139.50±376.26a | 10.63±1.60a | 99.38±15.90a | 2.38±0.41d |
Reference | |||||||
Metformin | 128.63±20.63ac | 99.00±15.45ac | 150.75±26.08ac | 1466.13±233.26ac | 6.88±1.25ac | 66.50±12.55ac | 1.60±0.14de |
Test material - BH | |||||||
400 mg/kg | 80.50±11.95bc | 66.38±14.89ac | 109.00±14.96c | 990.88±159.80ac | 4.75±1.39ac | 47.13±10.80bc | 1.14±0.22de |
200 mg/kg | 127.00±14.67ac | 98.75±10.94ac | 145.75±23.85ac | 1439.50±252.60ac | 7.00±1.20ac | 66.63±12.18ac | 1.61±0.27de |
100 mg/kg | 146.13±10.53ac | 113.25±17.41ac | 173.38±16.65ac | 1652.50±228.50ac | 7.88±1.36ac | 76.13±9.31ac | 1.88±0.16de |
Serum biochemical analysis
Table 5.
Items |
Glucose (mg/dl) |
Total cholesterol (mg/dl) |
Triglyceride (mg/dl) |
Low density lipoprotein (mg/dl) |
High density lipoprotein (mg/dl) |
---|---|---|---|---|---|
Groups | |||||
Controls | |||||
Intact | 91.75±16.24 | 97.25±14.15 | 72.59014.96 | 17.63±3.81 | 89.25±18.87 |
HFD | 268.38±35.62e | 263.00±33.09e | 245.75±27.69a | 79.38±10.20a | 16.75±2.96a |
Reference | |||||
Metformin | 179.63±21.31ef | 178.13±19.66ef | 166.75±29.51ac | 53.88±12.09ac | 41.25±14.20ac |
Test material - BH | |||||
400 mg/kg | 128.25±10.78ef | 123.88±13.25ef | 117.75±20.32ac | 38.00±11.43ac | 70.63±17.42bc |
200 mg/kg | 177.25±26.69ef | 179.88±26.82ef | 164.00±21.11ac | 52.13±11.47ac | 41.38±12.37ac |
100 mg/kg | 210.38±37.28eg | 209.38±19.92ef | 194.88±12.57ac | 60.50±13.86ac | 30.38±10.20ad |
Effects on lipid peroxidation and the antioxidant defense system
Table 6.
Items | Lipid peroxidation | Antioxidant defense system | ||
---|---|---|---|---|
Groups |
Malondialdehyde (nM/mg tissue) |
Glutathione (μM/mg tissue) |
Catalase (U/mg tissue) |
SOD (U/mg tissue) |
Controls | ||||
Intact | 6.08±1.91 | 69.95±13.05 | 67.48±18.37 | 7.57±1.05 |
HFD | 76.25±10.33d | 11.48±3.76a | 11.34±2.97d | 1.24±0.47a |
Reference | ||||
Metformin | 50.95±10.97de | 30.19±10.65ab | 31.51±13.58de | 3.39±0.97ab |
Test material - BH | ||||
400 mg/kg | 31.55±10.98de | 50.39±10.79ab | 48.15±12.16e | 5.03±1.37ab |
200 mg/kg | 49.98±12.40de | 31.77±11.17ab | 31.34±8.02de | 3.43±0.65ab |
100 mg/kg | 58.53±12.66de | 24.56±9.04ac | 23.36±8.84de | 2.57±0.97ab |
Effects on body fat density and total and abdominal fat mass
Effects on adipocyte histopathology analysis
Table 7.
Items | Lipid peroxidation | Antioxidant defense system | ||
---|---|---|---|---|
Groups |
Thickness (mm) |
Adipocyte diameters (μm) |
Thickness (mm) |
Adipocyte diameters (μm) |
Controls | ||||
Intact | 1.27±0.48 | 33.23±5.80 | 1.61±0.77 | 36.12±3.86 |
HFD | 4.53±0.77a | 101.82±13.22c | 5.92±0.60a | 110.08±9.07a |
Reference | ||||
Metformin | 3.14±0.64ab | 60.14±7.07cd | 4.52±0.56ab | 76.30±16.79ab |
Test material - BH | ||||
400 mg/kg | 2.63±0.47ab | 48.82±7.52cd | 3.83±0.73ab | 60.45±11.41ab |
200 mg/kg | 3.12±0.47ab | 59.03±7.35cd | 4.53±0.51ab | 73.91±8.46ab |
100 mg/kg | 3.50±0.56ab | 73.55±4.10cd | 4.75±0.79ab | 88.26±8.06ab |
Effects on the exocrine pancreas zymogen granule content
Table 8.
Items |
Zymogen granules (%/mm2 of exocrine) |
Mean islet numbers (numbers/10 mm2) |
Mean islet diameter (μm/islet) |
Insulin-IR cells (cells/ mm2) [A] |
Glucagon-IR cells (cells/ mm2) [B] |
Insulin/glucagon ratio [A/B] |
---|---|---|---|---|---|---|
Groups | ||||||
Controls | ||||||
Intact | 45.75±4.23 | 11.13±2.53 | 96.70±15.38 | 82.38±15.57 | 24.13±5.11 | 3.44±0.24 |
HFD | 16.61±5.36a | 29.25±3.54a | 319.59±50.14a | 923.38±112.49d | 141.50±8.82a | 6.51±0.46a |
Reference | ||||||
Metformin | 30.46±4.78ac | 21.88±3.87ac | 216.11±30.10ac | 392.13±68.17de | 82.00±12.41ac | 4.79±0.54ac |
Test material - BH | ||||||
400 mg/kg | 39.16±5.40ac | 17.50±2.62ac | 158.47±26.39ac | 180.63±33.33de | 45.75±9.71ac | 3.99±0.40bc |
200 mg/kg | 30.94±3.27ac | 21.63±2.00ac | 212.73±38.47ac | 412.13±39.41de | 86.88±3.94ac | 4.75±0.41ac |
100 mg/kg | 26.34±4.46ac | 24.13±2.17ac | 244.31±34.20ac | 653.00±94.68de | 122.63±10.68ac | 5.30±0.36ac |
Effects on hepatocyte hypertrophy
Table 9.
Items |
Liver steatosis (%/mm2 of hepatic tissues) |
Mean hepatocyte diameters (μm/cell) |
Degenerative renal tubule numbers (%) |
---|---|---|---|
Groups | |||
Controls | |||
Intact | 9.80±4.57 | 17.33±2.11 | 4.50±2.20 |
HFD | 80.05±10.65a | 33.56±2.05a | 73.50±11.84a |
Reference | |||
Metformin | 53.66±13.38ab | 26.13±2.62ab | 48.38±11.25ab |
Test material - BH | |||
400 mg/kg | 34.57±10.38ab | 23.58±2.13ab | 30.00±11.11ab |
200 mg/kg | 53.94±10.77ab | 26.02±1.44ab | 48.13±14.47ab |
100 mg/kg | 62.86±10.58ab | 28.37±1.98ab | 57.00±10.34ab |
Effects on hepatic enzyme activity
Table 10.
Effects on expression of lipid metabolism-associated gens
Table 11.
Items | Controls | Reference | Test material - BH | |||
---|---|---|---|---|---|---|
Groups | Intact | HFD | Metformin | 400 mg/kg | 200 mg/kg | 100 mg/kg |
Hepatic tissue | ||||||
ACC1 | 1.00±0.08 | 4.64±0.94e | 2.93±0.53ef | 1.98±0.55ef | 2.92±0.62ef | 3.53±0.44ef |
AMPKα1 | 1.06±0.09 | 0.25±0.08a | 0.47±0.09ac | 0.75±0.17ac | 0.47±0.12ac | 0.40±0.10ad |
AMPKα2 | 0.99±0.06 | 0.34±0.11a | 0.63±0.12ac | 0.82±0.15ac | 0.63±0.11ac | 0.53±0.07ac |
Adipose tissue | ||||||
Leptin | 1.00±0.07 | 6.70±0.99e | 4.37±0.59ef | 2.69±0.96ef | 4.23±0.79ef | 5.04±0.90ef |
UCP2 | 1.00±0.06 | 0.13±0.05a | 0.36±0.10ac | 0.54±0.16ac | 0.35±0.08ac | 0.24±0.04ad |
Adiponectin | 0.98±0.08 | 0.14±0.04a | 0.40±0.08ac | 0.60±0.10ac | 0.41±0.12ac | 0.26±0.07ac |
C/EBPα | 1.03±0.10 | 3.28±0.82a | 2.05±0.36ac | 1.46±0.20bc | 2.04±0.20ac | 2.34±0.28ac |
C/EBPβ | 1.03±0.07 | 4.28±1.00e | 2.74±0.54ef | 2.00±0.43ef | 2.77±0.52ef | 3.12±0.24ef |
SREBP1c | 1.02±0.09 | 3.79±0.76e | 2.39±0.43ef | 1.81±0.33ef | 2.42±0.43ef | 2.75±0.50ef |
NFD = Normal pellet diet; HFD = 45% kcal high fat diet; RT-PCR = reverse transcription polymerase chain reaction; UCP = Mitochondrial uncoupling protein; C/EBP = CCAAT-enhancer-binding protein; SREBP = Sterol regulatory element-binding protein; ACC1 = Acetyl-CoA carboxylase 1; AMPK = 5' adenosine monophosphate-activated protein kinase; GAPDH = Glyceraldehyde 3-phosphate dehydrogenase