Effect of black raspberry seed oil on inflammation and lipid metabolism in high-fat diet-induced obese mice and db/db mice = 블랙라즈베리 씨앗 기름이 고지방식이로 유도한 비만 마우스와 당뇨병 모델 마우스의 염증반응과 지질대사에 미치는 영향

다국어 초록 (Multilingual Abstract)

Increased interest in beneficial health effects of raspberries has raised the production of raspberries in Korea in recent years. The processing of black raspberry fruits for juice, wine, and puree typically produces the seeds as a byproduct. Black raspberry seed (BRS) oil, which can be obtained from black raspberry product pomace, contains about 30% α-linolenic acid (ALA), an n-3 fatty acid, and other beneficial phytochemicals such as tocopherols.
The ultimate objective of the study was to evaluate whether BRS oil has beneficial effects on inflammatory status and lipid metabolism in high-fat diet-induced obese mice and db/db (type 2 diabetes) mice.
The objective of the first part of the study was to determine the effect of BRS oil on inflammation and lipid metabolism in high-fat diet-induced obese mice. Five-week old C57BL/6 mice were divided into two groups: 1) mice fed high-fat diet consisting of 50% calories from lard, 5% from soybean oil, and 5% from corn oil (control group), and 2) mice fed high-fat diet consisting of 50% calories from lard and 10% from BRS oil (BRS oil group). The BRS oil used in the study comprised 57.0% linoleic, 29.4% α-linolenic, and 9.80% oleic acids. Mice were fed the experimental diets for 12 weeks ad libitum. The content of ALA was significantly (P<0.001) higher in the BRS oil diet than the control diet. There were no significant (P>0.05) differences in initial body weight, final body weight, weight gain, food intake, and food efficiency between the two groups. The weights of liver, spleen, kidney, and epididymal adipose tissue of the BRS oil group were higher than those of the control group with no significant (P>0.05) differences. After 12 weeks, ALA in the liver of the BRS oil group accounted for 1.84% of the total fatty acids, which was significantly (p<0.01) higher than that of the control group (0.45%). Inflammation-involved proteins such as TLR4, NF-κB, phospho-NF-κB, COX2, I-κBα, and phospho-I-κBα were lower in the liver of the mice fed the BRS oil than those in the control. mRNA levels of pro-inflammatory markers including NF-κB, TNFα, IL-1β, IL-6, iNOS, COX2, and MCP1 in the liver and epipidymal adipose tissue of the BRS oil group were lower than those of the control. On the other hand, mRNA levels of anti-inflammatory markers including IL-10, arginase1, Chi3l3, and Mgl1 were higher in the liver and epipidymal adipose tissue of the BRS oil group than those of the control. Leptin level in serum was lower in the mice fed BRS oil diet than in the control diet without significant (P>0.05) difference. mRNA level of leptin was lower in the epididymal adipose tissue of the mice fed BRS oil than in the control without significant (P>0.05) difference. mRNA level of adiponectin was significantly (P<0.01) higher in the epididymal adipose tissue of the BRS oil group than in the control. Whereas, adiponectin level in serum was higher in the mice fed BRS oil diet than in the control diet with no significant (P>0.05) difference.
Levels of TG in serum and liver of the mice fed BRS oil were 14.2% and 12.1%, respectively, lower than those of the control group. Serum non-esterified fatty acids (NEFA) and total cholesterol levels were 42.1% and 13.0%, respectively, lower (P<0.05) in the mice fed BRS oil diet than in the control. Serum HDL-C level was 4.10% higher in the BRS oil group than in the control group without significant (P>0.05) difference. Total lipid content in the liver of the BRS oil group was 13% lower than that of the control group without significant (P>0.05) difference. NEFA and total cholesterol levels in the liver of the BRS oil group were 25.7% (P<0.05) and 53.2% (P<0.001), respectively, lower than those of the control group. The mRNA levels of lipogenic markers such as CD36, FABP1, SREBP-1c, FAS, and SLC25A1 were lower in the liver of the BRS oil group than those of the control group. Whereas, mRNA levels of fatty acid oxidation markers including CPT1A, ACADL, HADHα, and ACOX were higher in the liver of the mice fed BRS oil than in the control mice. PPARα significantly increased both in mRNA (P<0.001) and protein (P<0.01) levels in the liver of the mice fed BRS oil diet compared with the control group. However, PPARγ mRNA and protein levels showed no significant (P>0.05) differences between the two groups. PPARα mRNA level was significantly (P<0.05) higher in the epididymal adipose tissue of the mice fed BRS oil than in the control. However, there were no significant (P>0.05) differences in PPARα protein and PPARγ mRNA and protein expression levels between the two groups.
The objective of the second part of the study was to evaluate the status of the markers related to inflammation and lipid metabolism in db/db mice fed diets containing different concentrations of BRS oil. Mice were divided into four groups: 1) C57BL/6 mice fed 16% calories from soybean oil (normal CON); 2) C57BL/KsJ-db/db mice fed 16% calories from soybean oil (CON); 3) C57BL/KsJ-db/db mice fed 8% calories from soybean and 8% calories from BRS oil (BRS 50%); and 4) C57BL/KsJ-db/db mice fed 16% calories from BRS oil (BRS 100%). Mice were fed the experimental diets for 10 weeks ad libitum. There were no significant (P>0.05) differences in the food intake and initial body weight among the three db/db groups. The food intake and body weight of the normal CON were significantly (P<0.05) lower than those of the db/db groups. The final body weight of the BRS 50% was significantly (P<0.05) lower than that of the CON. Final body weight of the BRS 100% was lower than that of the CON without significant (P>0.05) difference. The weights of the liver, epididymal adipose tissue, spleen, and kidney were not significantly (P>0.05) different among the db/db mice. There was no significant (P>0.05) difference in blood glucose level among the db/db mice. Insulin level was significantly (P<0.05) lower in the serum of the BRS 50% and BRS 100% than in the CON. After 10 weeks, n-6 to n-3 fatty acid ratios were significantly (P<0.05) lower in the livers and epididymal adipose tissues of the BRS 50% and BRS 100% mice than in the CON. Whereas, ALA and total n-3 fatty acids contents were significantly (P<0.05) higher in the livers and epididymal adipose tissues of the BRS oil-treated mice than in the normal CON and CON. Serum TNFα and IL-6 were significantly (P<0.05) lower in the BRS 50% and BRS 100% than in the CON. Serum IL-10 was significantly (P < 0.05) higher in the BRS 100% than in the CON. Protein expression levels involved in inflammation such as TLR4, NF-κB, and COX2 were lower in the epididymal adipose tissues of the BRS 50% and BRS 100% than those of the CON. In the liver and epididymal adipose tissue, mRNA levels of pro-inflammatory markers including TLR4, TNFα, IL-1β, IL-6, iNOS, COX2, MCP1, and CCR2 in the BRS 50% and BRS 100% were lower than in the CON. On the other hand, anti-inflammatory markers including IL-10, arginase1, Chi3l3, and Mgl1 were higher in the epididymal adipose tissues of the BRS 50% and BRS 100% than in the CON. In the epididymal adipose tissue, macrophage infiltration markers (F4/80 and CD68) and leptin mRNA were significantly (P<0.05) lower in the BRS 50% and BRS 100% than in the CON.
Levels of TG, NEFA, and total cholesterol in the serum were significantly (P<0.05) lower in the BRS oil-treated groups than in the CON. Serum HDL-cholesterol level was significantly (P<0.05) higher in the BRS 50% than in the CON. Levels of total lipid, TG, NEFA, and total cholesterol in the liver were significantly (P<0.05) lower in the BRS oil-treated groups than in the CON. The mRNA levels of lipogenesis markers including CD36, FABP1, SREBP-1c, FAS, and SLC25A1 in the livers of the BRS oil groups were significantly (p<0.05) lower than in the CON. On the other hand, fatty acid oxidation markers were significantly (CPT1A and ACOX; P<0.05) or without significance (ACADL and HADHα; P>0.05) higher in the BRS oil groups than in the CON. PPARα mRNA level was significantly (P<0.05) higher in the liver of the mice fed BRS 50% diet than in the CON, whereas PPARα protein expression was significantly (P<0.05) higher in the liver of the mice fed BRS 100% diet than in the CON. PPARγ mRNA level was significantly (P<0.05) lower in the liver of the mice fed BRS 50% diet than in the CON, whereas PPARγ protein expression was significantly (P<0.05) lower in the liver of the mice fed BRS 100% diet than in the CON. These results are in agreement with the PPARα results. PPARα mRNA level was significantly (P<0.05) higher in the epididymal adipose tissue of the BRS 50% and BRS 100% than in the normal CON and CON. PPARα protein was significantly (P<0.05) higher in the epididymal adipose tissue of the BRS 100% than in the normal CON and CON. There were no significant (P>005) differences in PPARγ mRNA levels between the BRS oil groups and CON. To detect the effect of BRS oil on hepatic lipid accumulation, Oil Red O staining was performed. The Oil Red O staining showed that the lipid droplets in the livers of the BRS oil-treated mice were smaller and fewer than those of the CON, indicating reduced hepatic lipid accumulation in the BRS oil groups.
This study demonstrated that dietary BRS oil treatment reduced pro-inflammatory markers and promoted anti-inflammatory markers in liver and epididymal adipose tissue of high-fat diet-induced obese mice and db/db mice. BRS oil also lowered lipogenesis markers and raised fatty acid oxidation markers in the livers of high-fat diet-induced obese mice and db/db mice.
Results of this study suggest that BRS oil may be a good source of ALA, an n-3 fatty acid, with anti-inflammatory effects on high-fat diet-induced obese mice and obese diabetic mice by ameliorating inflammatory responses. Also, BRS oil may improve lipid metabolism by inhibiting lipogenesis and promoting fatty acid oxidation in high-fat diet-induced obese and db/db mice. These results would promote utilization of BRS, a byproduct of beverage processing of black raspberries, and ultimately help black raspberry producers and manufacturers.

목차 (Table of Contents)

• Chapter 1 Introduction and Literature Review 1
• 1.1 Background 2
• 1.2 Objectives 3
• 1.3 Literature Review 4
• 1.3.1 Black raspberry seed oil 4
• 1.3.2 ALA as an n-3 PUFA 8
• 1.3.3 Fruit seed oils as a source of health beneficial compounds 9
• 1.3.4 Obesity, inflammation, and diabetes 11
• 1.3.5 Anti-inflammatory effect of n-3 PUFA 12
• 1.3.6 Effect of n-3 PUFA on lipid metabolism 19
• Chapter 2 Effect of Black Raspberry Seed Oil on Inflammation and Lipid Metabolism in High-Fat Diet-Induced Obese Mice (Study 1) 25
• 2.1 Introduction 26
• 2.2 Materials and methods 31
• 2.2.1 Sample preparation 31
• 2.2.2 Animals 32
• 2.2.3 Experimental diets 34
• 2.2.4 Tissue collection 36
• 2.2.5 Lipid extraction from diets 36
• 2.2.6 Lipid extraction from livers 36
• 2.2.7 Fatty acid composition analysis 37
• 2.2.8 Protein extraction from liver and western blotting 38
• 2.2.9 Total RNA extraction, cDNA synthesis, and real-time polymerase chain reaction (PCR) 39
• 2.2.10 Serum analysis 43
• 2.2.11 Lipid extraction from liver and analysis of liver lipid content 45
• 2.2.12 Hepatic lipid analysis 45
• 2.2.13 Statistical analysis 46
• 2.3 Results 46
• 2.3.1 Fatty acid compositions of BRS oil and experimental diets 46
• 2.3.2 Body weight, food intake, and organ weight of experimental animals 49
• 2.3.3 Fatty acid compositions of livers of experimental animals 49
• 2.3.4 Protein expressions involved in inflammation in liver 52
• 2.3.5 mRNA levels involved in pro-inflammation in liver and epididymal adipose tissue 55
• 2.3.6 mRNA levels involved in anti-inflammation in liver and epididymal adipose tissue 60
• 2.3.7 Leptin and adiponectin levels in serum and epididymal adipose tissue 63
• 2.3.8 Lipid profiles of serum and liver 65
• 2.3.9 mRNA levels involved in lipogenesis in liver 68
• 2.3.10 mRNA levels involved in fatty acid oxidation in liver 70
• 2.3.11 mRNA and protein levels of PPAR in liver and epididymal adipose tissue 72
• 2.4 Discussion 75
• Chapter 3 Effect of Black Raspberry Seed Oil on Inflammation and Lipid Metabolism in db/db Mice (Study 2) 80
• 3.1 Introduction 81
• 3.2 Materials and methods 84
• 3.2.1 Sample preparation and BRS oil extraction 84
• 3.2.2 Experimental diets 84
• 3.2.3 Animals 85
• 3.2.4 Tissue collection and blood glucose analysis 88
• 3.2.5 Fatty acid composition analysis 88
• 3.2.6 Serum analysis 88
• 3.2.7 Protein extraction and western blotting 89
• 3.2.8 Total RNA extraction, cDNA synthesis, and PCR 89
• 3.2.9 Liver lipid content and hepatic lipid analysis 89
• 3.2.10 Statistical analysis 89
• 3.3 Results 91
• 3.3.1 Fatty acid composition of the diets, and body weight, food intake, and organ weight of the animals 91
• 3.3.2 Blood glucose level and insulin level in serum 91
• 3.3.3 Fatty acid compositions of the livers and epididymal adipose tissues of experimental animals 95
• 3.3.4 Inflammatory markers in serums of the mice 99
• 3.3.5 Leptin and adiponectin levels in serum 99
• 3.3.6 Protein and mRNA levels involved in pro-inflammation in liver and epididymal adipose tissue 102
• 3.3.7 mRNA levels involved in anti-inflammation in liver and epididymal adipose tissue 110
• 3.3.8 Macrophage markers, leptin, and adiponectin mRNA levels in epididymal adipose tissue 113
• 3.3.9 Lipid profiles of serum and liver 116
• 3.3.10 mRNA levels involved in lipogenesis in liver 119
• 3.3.11 mRNA levels involved in fatty acid oxidation in liver 122
• 3.3.12 mRNA and protein levels of PPAR in liver and epididymal adipose tissue 124
• 3.3.13 Improvement of hepatic lipid accumulation 127
• 3.4 Discussion 129
• Chapter 4 Summary and Conclusion 135
• References 140
• 국문 초록 163