Chapter 2. Anti-obese effect of IGBP on differentiated 3T3-L1 cell and high-fat diet-induced C57BL/6J mice
The destruction of energy homeostasis due to fat accumulation leads to serious chronic diseases such as obesity. Obesity is a disease that has been found to affect diseases such as inflammation, high blood pressure, and diabetes, including insulin resistance. We investigated the anti-obese effect of indian gooseberry (Phyllanthus emblica, IG) and barley sprout (BP) mixture on differentiated 3T3-L1 cell and high-fat diet-induced C57BL/6J mice. Natural materials, IG and BP, are active against obesity and diabetes by controlling lipid metabolism, respectively. In vitro study, we differentiated 3T3-L1 cells and performed experiments corresponding to each mechanism, such as adipogenesis, lipogenesis, and lipolysis. We performed Oil red O staining, glycerol release, triglyceride contents and cAMP level measure, and western blot analysis. in vivo study, the mice were randomly divided into eight groups: normal control group, obesity-induced control group (60% high fat diet, HFD), positive control group (60% HFD + metformin 250mg/kg b.w.), IG 400 group (60% HFD + IG 400 mg/kg b.w.), BP 400 group (60% HFD + BP 400 mg/kg b.w.), IGBP groups (60% HFD + IGBP 100, 200, and 400 mg/kg b.w.). After 15 weeks, we performed a micro-CT imaging analysis, and mice were sacrificed. We measured the levels of serum triglyceride, total cholesterol, LDL/HDL cholesterol, fatty acid, and fecal triglyceride, and total cholesterol. We confirmed the cAMP level and lipid size in the white adipose tissue. We confirmed the expression of proteins related to adipogenesis, lipogenesis, lipolysis, and energy metabolism by a Western blot. The results revealed that IGBP mixture inhibited the expression of proteins related to adipogenesis and lipogenesis mechanism and regulated the expression of proteins related to lipolysis in vitro. These results reduced TG production and led to increased cAMP levels and glycerol release. Moreover, the IGBP mixture reduced weight gain, white adipose tissue (WAT) weight, serum lipid profiles, the adipose mass of systemic, subcutaneous, and visceral, and lipid size of WAT in high-fat diet-induced C57BL/6J mice. Furthermore, the IGBP mixture increased the cAMP level of adipose tissue and lipid profiles of feces and regulated the expression of proteins related to adipogenesis, lipogenesis, lipolysis, and energy metabolism in vivo study. Overall, these results suggest that the IGBP mixture may improve obesity by controlling adipogenesis, lipogenesis, lipolysis, and energy metabolism, and may be a useful substance for reducing body fat.
Chapter 3. IGBP ameliorate insulin resistance induced by adipose-derived exosome in C2C12 cells
Skeletal muscle insulin resistance is associated with various metabolic conditions, including obesity and hyperglycemia. In the present study, we investigated the effect of Indian gooseberry and barley sprout mix (IGBP) on insulin resistance induced by adipose-derived exosomes in C2C12 cells. We fed a 60% high-fat diet to the experimental animals for 12-weeks to induce obesity and separated their adipose tissue for exosome isolation. We confirmed exosome biosynthesis by isolating exosomes from adipose tissue-conditioned media, and treated differentiated C2C12 cells with these exosomes to determine their effect on insulin resistance. Our results confirmed the expression of CD63 and Alix, the exosome biosynthesis markers, in the adipose-derived exosomes. Our microRNA expression analysis showed that miR-27a expression increased in the exosomes of the obese control group. Increased expression of miR-27a targeted and suppressed PPARγ. This led to the decreased expression of GLUT4 by controlling IRS1, PI3K, PDK, Akt, and AS160 phosphorylation, thereby increasing insulin resistance. IGBP decreased miR-27a expression and increased PPARγ protein expression. These results influenced the phosphorylation of IRS1, PI3K, PDK, Akt, and AS160. Moreover, IGBP increased intracellular glucose influx by increasing GLUT4 expression. Therefore, our result showed that the IGBP helps inreducing insulin resistance in muscle cells. Significantly, the meaningful aspect of this study that was based on the insulin resistance-inducing effects of adipose tissue-derived exosomes. Based on our results, we suggest that IGBP could protect against obesity-induced muscle insulin resistance and may be used as a useful supplement for preventing hyperglycemia.
Chapter 4. Ellagic acid and saponarin present in IGBP ameliorate insulin resistance developed in response to exposure of C2C12 cells to adipose-derived exosomes
An obesity-induced state increases the incidence of diabetes, and obesity adipose-derived exosomes have been found to increase the muscle insulin resistance by circulating throughout the body. In the present study, we quantitated ellagic acid and saponarin in Indian gooseberry (IG), barley sprout (BP), and IGBP, and we investigated the effect of ellagic acid, saponarin, and a mixture of the two (ES) on insulin resistance developed in response to the exposure of C2C12 cells to adipose-derived exosomes. In IGBP, the concentration of ellagic acid and saponarin was 10 ± 20% mg/g and 2.27 ± 20% mg/g, respectively. Based on the concentration of IGBP (50, 100, and 200 ㎍/mL) in our previous study, the C2C12 cells were treated with ES at a ratio of 4.4:1, followed by immediate stimulation with 30 ㎍ of isolated exosomes for insulin resistance induction for 48 h. microRNA expression analysis revealed that miR-27a expression increased in the exosomes of the obese adipose tissues, thereby reducing the PPARγ expression. This resulted in a decreased glucose transporter 4 (GLUT4) expression via the regulation of IRS1, PI3K, PDK, AKT, and AS160 phosphorylation, finally resulting in increased insulin resistance. ES decreased miR-27a expression and increased PPARγ expression, and both of these phenomenon influenced the phosphorylation of IRS1, PI3K, PDK, AKT, and AS160 and were confirmed to increase the influx of glucose into cells by increasing GLUT4 expression. Therefore, ES helps reduce insulin resistance in skeletal muscle cells. In conclusion, we suggest that the effect of IGBP on insulin resistance in C2C12 cells can be attributed to the presence of ellagic acid and saponarin.