Effects,of,Mogroside,V,on,Glucose,and,Lipid,Metabolism,in,High,Fat,Diet,Mice

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Riming WEI, Haiping LIU

Pharmacology Laboratory of Prevention and Treatment of High Incidence of Disease, Guilin Medical University, Guilin 541199, China

Abstract [Objectives] To explore the effects and mechanism of mogroside V (MV) on glucose and lipid metabolism in high-fat diet (HFD) mice. [Methods] The experiment fed mice with high-fat diet for 8 weeks, and 40 mice with successful modeling were randomly divided into normal group, model group, and MV dose group (100, 200 mg/kg), with 10 mice in each group. From the ninth week, the MV dose group was given intragastric administration, and the normal group and the model group were given an equal volume of distilled water by intragastric administration for 6 weeks, then killed and blood samples and livers were collected. Serum triglycerides (TG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), free fatty acids (FFA), Advanced glycation end products (AGE-P) -peptides (AGE-P) and glycosylated hemoglobin (HbA1c) content, and TG and hepatic glycogen content in liver were detected by biochemical method. Fasting blood glucose (FBG) was measured by glucose oxidase method. The fasting serum insulin (FINS) content was detected by enzyme-linked immunosorbent assay (ELISA), and the insulin resistance index (HOMA-IR) was calculated. Oil red O staining was used to observe the fat deposition in liver tissue. [Results] MV (100, 200 mg/kg) dose groups could significantly down-regulate the contents of TC, TG, LDL-C, FBG, FINS, AGE-P and HbA1c and HOMA-IR, and up-regulate HDL-C and hepatic glycogen content and reduce the fat deposits. [Conclusions] The mechanism of MV regulating glucose and lipid metabolism in mice may be related to the regulation of insulin resistance.

Key words Mogroside V (MV), High fat diet (HFD), Glucose and lipid metabolism, Insulin resistance

Metabolic syndrome is a pathological state in which the metabolism of fat, protein and carbohydrates in the human body is disordered, and it is often accompanied by obesity, hyperglycemia, hypertension, dyslipidemia and hyperinsulinemia[1]. Disorder of glucose and lipid metabolism is an important part of metabolic syndrome, and it will increase the incidence of cardiovascular and cerebrovascular diseases and diabetes[2]. In recent years, due to changes in people’s lifestyles, diseases related to metabolic dysfunction caused by excess nutrition have been increasing significantly, which seriously influences people’s quality of life and health, thus, it is urgent to find effective strategies and drugs to regulate glucose and lipid metabolism disorders[3]. Phytoside is a natural polymer with various biological activities such as antioxidant, dyslipidemia regulation, anti-inflammatory, insulin sensitization,etc., and has been widely used in the field of medical care[4]. Insulin resistance is prevalent in obesity-related diseases such as metabolic syndrome, type 2 diabetes, and atherosclerosis, which are all characterized by a low-grade nonspecific inflammatory state[5]. Although obesity is widely recognized in the field as an important cause of impaired insulin signaling, how obesity contributes to insulin resistance is still not fully understood. Many scholars have proposed a variety of mechanisms for this, such as: endoplasmic reticulum stress, oxidative stress, lipid balance disorders (including FFA disorders), mitochondrial imbalance and hypoxia[6]. However, at present, most evidence suggests that obesity-induced inflammation may also be a key factor in insulin resistance. In view of this, we explored the effects and mechanism of mogroside V (MV) on glucose and lipid metabolism in high fat diet mice from the perspective of insulin.

2.1 Materials

2.1.1Test drugs. Mogroside is marked as a product of Chengdu Biopurify Phytochemicals Co., Ltd.

2.1.2Animals. A total of 46 healthy SPF mice, 8 weeks old, weighing (20±2) g, were provided by the SPF Experimental Animal Center of Guilin Medical University, license number SYXK2013 (Gui)-0001.

2.1.3Reagents. The triglycerides (TG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), free fatty acids (FFA), Advanced glycation end products (AGE-P) -peptides (AGE-P) and glycosylated hemoglobin (HbA1c) content, and liver glycogen kits were provided by Nanjing Jiancheng Bioengineering Institute, and high fat feed was provided by Jiangsu Medison Biomedical Co., Ltd.

2.1.4Instruments. Epoch microplate reader (Bio-Tek, USA), UW-2200H electronic balance (Shimadzu, Japan), TGL-16K desktop high-speed refrigerated centrifuge (Hunan Xiangyi Centrifuge Factory, China), Olympus BX51 microscope (Olympus Corporation, Japan).

2.2 Methods

2.2.1Grouping and treatment. First, the 46 mice were divided into 2 groups, namely the normal group (n=13) and the high fat group (n=33). The normal group was fed with normal mouse diet, and the high fat group was fed with high fat diet for modeling. After 8 weeks, 3 mice were randomly selected from each of the normal group and the high fat group for pathological examination to confirm that the modeling was successful. The remaining 40 mice were randomly divided into normal group, model group and MV dose group (100, 200 mg/kg), 10 mice in each group. Except for the normal group given normal feed, the other groups were fed with high fat diet for 14 weeks. Starting from the ninth week, the MV dose group was given intragastric administration, and the normal group and model group were given an equal volume of distilled water by intragastric administration, once a day, for 6 weeks[7-8]. After the last intragastric administration, the mice were fasted for 16 h, and blood samples and livers were collected. Blood samples were centrifuged for 15 min (4 500 rpm) and stored at -20 ℃. The contents of TG, TC, LDL-C, HDL-C and FFA in serum were detected in accordance with the operating procedures of each kit. Livers were stored in liquid nitrogen.

2.2.2Detection of serum lipid content. Pipetted the centrifuged serum in accordance with the kit instructions, and added the corresponding reagents in turn on the ice bath. After color development, placed on a microplate reader, and measured the serum TG, TC, LDL-C, HDL-C and FFA content in sequence at each specific wavelength. Took a certain weight of liver tissue and added 9 times the volume of cold normal saline.

2.2.3Detection of TG content in liver tissue. The indicators were measured in accordance with the kit instructions. The homogenate was manually homogenized in an ice bath, centrifuged, and the supernatant was taken for the determination of TG and liver glycogen content.

3.1 Effects of MV on serum lipid and liver TG content

Compared with the normal group, the serum levels of TC, TG and LDL-C in the model group were significantly increased (P<0.01), while the HDL-C content was significantly decreased (P<0.01). Compared with the model group, except for the MV (100 mg/kg) dose group, which had no significant difference in LDL-C, the other MV dose groups could reduce the levels of TC, TG and LDL-C in the serum of mice (P<0.05 orP<0.01), significantly increased HDL-C content (P<0.01). Compared with the normal group, the contents of TG and FFA in the liver of the mice in the model group were significantly increased (P<0.01); compared with the model group, the MV dose groups could significantly reduce the content of TG and FFA (P<0.05 orP<0.01), as shown in Table 1 and 2.

Table 1 Effects of MV on lipid content in serum (n=10,

Table 2 Effects of MV on liver tissue TG and serum FFA (n=10,

3.2 Effects of MV on insulin resistanceCompared with the normal group, the serum FBG, FINS and HOMA-IR of mice in the model group were increased (P<0.01). Compared with the model group, the MV dose groups could effectively reduce the serum FBG, FINS and HOMA-IR in high fat diet mice (P<0.01 orP<0.05), indicating that MV intervention can improve insulin resistance (Table 3).

Table 3 Effects of MV on insulin resistance (n=10,

3.3 Effects of MV on serum AGE-P, HbAlc and liver glycogenCompared with the normal group, the serum levels of AGE-P and HbAlc in the model group were significantly increased (P<0.01), while the liver glycogen content was significantly decreased (P<0.01). Compared with the model group, the MV dose groups significantly decreased the serum AGE-P and HbAlc contents (P<0.01), and increased the liver glycogen content (P<0.01 orP<0.05), as shown in Table 4.

Table 4 Effects of MV on serum AGE-P, HbAlc and liver glycogen (n=10,

3.4 Effects of MV on the pathological morphology of mouse liver tissueThe liver tissue was stained with Oil Red O, and there was no red-stained fat vacuole and no inflammatory cell infiltration in the normal mice. The model group presented a large area of red fat droplets with inflammatory infiltration around them. The number of red fatty deposits and inflammatory cells was significantly reduced in the liver of mice treated with MV, especially at 200 mg/kg (Fig.1).

Note: A: normal group; B: model group; C: MV dose group (100 mg/kg); D: MV dose group (200 mg/kg).Fig.1 Effects of MV on the pathological morphology of mouse liver tissue (400×)

Lipid disorder (dyslipidemia) is one of the manifestations of abnormal lipid metabolism, mainly including the increase of serum TC, TG, LDL-C and the decrease of HDL-C[9]. When the peripheral tissue is less sensitive to insulin, the level of glucagon in the blood increases, the lipolysis increases, and a large amount of free fatty acids are released, some of which enter the liver, and the excess enters the systemic circulation, which promotes the increase of lipid synthesis in the liver and the increase of TG synthesis[10]. Excessive FFA can be converted into TG and stored in hepatocytes, leading to the accumulation of TG, the decrease of HDL-C level, and the decrease of the binding force of LDL-C to its receptor, which promotes the increase of LDL-C level[11-12]. Lipid metabolism is closely related to glucose metabolism. After the level of FFA increases, it is often enriched in insulin target organs such as fat and liver, resulting in decreased organ sensitivity to insulin, and the decline in the ability of these organs to absorb glucose will increase gluconeogenesis and lead to the occurrence of insulin resistance[13]. The increase in FFA caused by excess nutrition promotes the accumulation of TG in the liver, reduces the ability of insulin to promote glycogen synthesis and inhibits gluconeogenesis, and leads to the formation of insulin resistance, which is often accompanied by dyslipidemia and increases the risk of atherosclerotic diseases such as coronary heart disease[14]. AGE-P can form second-generation AGEs with target tissues or proteins (such as low-density lipoprotein), and this chain reaction often aggravates uremia, diabetes, and vascular disease[15]. Clinically, HbAlc is often used to assess the degree of glycemic control in patients with diabetes, and high concentrations of blood glucose can combine with hemoglobin to increase glycosylated hemoglobin[16]. Liver cell damage leads to the destruction of various enzymes in hepatocytes, especially the glycogen synthase system, which leads to the blockage of hepatic glycogen synthesis and the decrease of hepatic glycogen content[17], which is consistent with the decrease in liver cell inflammation and liver glycogen test data in the pathological examination in the model group of this study. MV can effectively reduce the content of TC, TG, LDL-C, AGE-P and HbAlc in the serum of mice, increase the content of HDL-C and liver glycogen, and reduce the levels of FBG, FINS and HOMA-IR, indicating that MV could effectively regulate the blood lipid metabolism disorder in mice caused by high fat diet and improve insulin resistance. It can be speculated that the mechanism by which MV regulates glucose and lipid metabolism disorders in high fat diet mice may be related to mediating insulin-related pathways.

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